Appendixes I Spatial Filtration for Video Line Removal
GORDON W. ELLIS
The growing popularity in microscopy of video recording and image-processing techniques presents users with a problem that is inherent in the method-horizontal scan lines. These lines on the monitor screen can be an obtrusive distraction in photographs of the final video image. Care and understanding in making the original photograph can minimize the contrast of these lines. Two simple, but essential, rules for photography of video images are: (l) Use exposures that are multiples of the video frame time (l/30 sec in the USA). An exposure time less than this value will not record a completely interlaced image.* (2) Adjust the v HOLD control on the monitor so that the two fields that make up the frame are evenly interlaced. Alternate scan lines should be centered with respect to their neighbors (a magnifier is helpful here). t Following these rules will often result in pictures in which the scan lines are acceptably unobtrusive without recourse to further processing. If the subject matter is such that the remaining line contrast is disturbing, Inoue (l981b) has described a simple technique that can often yield satisfactory results using a Ronchi grating. However, on occasion, when important image details are near the dimensions of the scan lines, the slight loss in vertical resolution resulting from this diffraction-smoothing method may make it worth the effort to remove the lines by spatial filtration. The technique of spatial filtration, pioneered by Marechal, is described in many current optics texts. A good practical discussion of these techniques is found in Shulman (1970). To produce the pictures for the present article, I used a somewhpt simpler apparatus than the optical correlator illustrated in Shulman (see also Walker, 1982). In the present instance, the apparatus consists of: a monochromatic light source; two surplus aerial camera lenses (Kodak Aero-Ektar 12-inch f/~.5); an enlarger's negative carrier; and a 35-mm single-lens reflex camera with an extension bellows and a long-focal-length lens (in this case an ancient 7-inch Kodak Rapid-Rectilinear objective borrowed from a Kodak Model 3A Folding Pocket Camera, ca. 1910). The camera must have a plain ground-glass focusing screen with a clear central disk bearing a cross for parallax checking. I use a Nikon F with a type "C" screen. The light source for the optical processor is a vibrating light-fiber conveying light from an argon ion laser (Ellis, 1979). The source does not need to be a laser, but the requirements are easily met by the laser source described and it was available. The requirements are: high intensity, monochromaticity, and small size (but not too small). A 100-watt HBO 100 superpressure mercury arc has about the right source size. If fitted with a heat filter and a 546-nm interference filter and
*See also page 426 regarding minimum suggested exposure time. tSee footnote *on page 256.
GORDON W. ELLIS • Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
463 464 APPENDIX I housed in a suitable box (for safety as well as stray light control) without a lens, it would provide a more economical source for a dedicated optical processor. The two Aero-Ektars are mounted on a common axis with the light source and the camera. The light source is at the entrance pupil (film plane) of the first Aero-Ektar, which is face to face with its mate and about 13 em away. The negative carrier is mounted between them about 10 em from the second lens. Ideally, these lenses would be mounted with their front focal planes coinciding with each other and the film carrier, but this is not a necessary condition for this purpose. The closer spacing was dictated by space limitations. The spatial filter is mounted on axis at the back focal plane of the second Aero-Ektar and is carried on the barrel of the 35-mm camera's lens. The camera is focused on the object transparency in the negative carrier. The action of the spatial filter is selectively to prevent the light diffracted by the scan lines in the transparency from reaching the camera film plane where they would produce line images by interference with the undiffracted zeroth order. The simplest spatial filter that would eliminate the lines (as in the classic Abbe-Porter experiments) is an iris diaphragm closed down to the point that the first orders of the line diffraction pattern are just blocked. At first glance, this might seem the optimum filter for this purpose; however, video camera modulation transfer functions are not necessarily the same for vertical and horizontal detail. In fact, today's instrumentation cameras typically offer higher resolution in the horizontal direction (parallel to the scan lines) than can be achieved vertically within the constraints of the standard (USA) 525-line format. Consequently, in front of the camera )ens , I use a glass plate bearing opaque spots about 50% larger than the light source image and spaced to occlude all the diffracted orders from the scan lines while leaving the zeroth order and most of the aperture unobstructed. Then, to reduce stray light, I close the lens iris down to the level of the second orders of the diffraction pattern. Two accessories simplify adjusting the processor. They are an auxiliary focusing magnifier (actually a telescope), used to aid parallax focusing, and an aperture-viewing magnifier. The latter is
FIGURE 1-1 . Original. SPATIAL FILTRATION FOR VIDEO LINE REMOVAL 465 used to examine the exit pupil of the camera's viewfinder, which provides a clear and magnified image of the filter and the diffracted light that it must be positioned to block. Photographically, there are several options in going from the original lined photograph to the filtered negative for the final print. One could, in principle, treat the optical processor as a special giant enlarger and project unlined prints from lined negatives. Unfortunately, this would move the printing easel well into the next room, in addition to requiring impractically long exposures. Otherwise, the shortest path in steps, though not necessarily in time, is to photograph the spatially filtered image on S0-185 Rapid Processing Copy film (EKC). Because this a self-reversing film, the developed S0-185 image is a filtered negative that can be used in an ordinary enlarger. I have found that this works well using the 488-nm line of the argon ion laser, but is about 10,000 times slower using the 546-nm light from a 100-W mercury arc, and will not work at all with a helium• neon laser. An equally direct alternative I have not yet tried is to use S0-185 to make the original photograph of the video monitor. The resulting positive transparency could then be filtered in the optical processor and photographed on Panatomic-X, Plus-X, or other panchromatic film to produce the filtered negative. Using a CRT with a highly actinic phosphor might make this a practical approach. Conventional monitors would probably require excessively long exposures. For the photographs shown, I have followed a more complex path, but one that allows use of panchromatic film and short exposures. The original negative was exposed on Pan-X at 1/30 sec. This negative was copied onto Kodak Technical pan and developed in I : 3 Microdoi-X to produce a long-scale positive transparency for use in the processor. The filtered negative was then made on Pan-X. Here the exposure was 1/ 125 sec using 0.5 W from the 514-nm argon laser. Figure I-1 is a print from the original negative. The photograph does not show optimal adjustment of the interlace, but instead represents a less favorable case to demonstrate the power of the filtering technique. The result, the spatially filtered print in Fig. I-2, is entirely free of the video scan lines. Note the subjective impression that the delined print looks sharper than the original.
FIGURE 1-2. Spatially filtered reproduction. 466 APPENDIX I
The subject of the video micrograph is a diatom frustule that is one of 50 on an arranged slide from Turtox (No. Bl.434). It is viewed in polarized light through a 43/0.65-NA Leitz stress-free achromat and displayed on a 9-inch Panasonic monochrome monitor by a Dage-MTI Model 65 camera equipped with a Newvicon camera tube. Magnification of the image on the monitor was 1980X and covers 93 IJ.m horizontally, edge to edge. II Modulation Transfer Function Analysis in Video Microscopy
ERIC W. HANSEN
11.1. INTRODUCTION
The modulation transfer function (MTF) is a powerful tool for the analysis of imaging systems. It describes in quantitative terms the relationship of object and image, and provides a convenient framework for analyzing the performance of complex imaging systems, which may combine optical and electronic components. This appendix first presents a brief tutorial introduction to the MTF, and then illustrates its use with an analysis of a typical video microscope system.
11.2. MODULATION TRANSFER
To begin, let us consider the imaging system shown schematically in Fig. II-1. An object with a sinusoidal spatial variation of intensity, / 0 (x0 ) along the X0 axis, is imaged with unity magnifica• tion, producinga sinusoidal image intensity 1/x). (The subscripts o and i denote object and image, respectively.) The peak-to-peak distance along the sinusoid* is its spatial period; the reciprocal of the period is the spatial frequency,Jx, expressed in units of cycles per unit length (e.g., cycles/mm). In general, we may observe three things about the image. First, the spatial frequency of the image is the same as that of the object. Second, the contrast, or modulation, defined as
(11-1)
is typically less in the image than in the object. Third, the object is centered about X0 = 0, but the image is displaced by an amount LU 1 • If we image several such test objects with different spatial frequencies, we will find that the image modulation and position shift will vary as a function of spatial frequency. The modulation transfer function. M(f), is defined
M(f) = image modulation/object modulation (11-2)
The MTF expresses the way the imaging process alters the contrast of a sinusoidal object, as a function of spatial frequency. The position shift .:lxi is equivalent to a phase shift .:li of the
*The sinusoid is a sine or cosine function.
ERIC W. HANSEN • Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire 03755
467 468 APPENDIX II
IMAGING SYSTEM
AXj
FIGURE 11-1. Imaging a sinusoidal test object.
sinusoid; we relate the two by noting that one period of the sinusoid (a distance Xi l!f) corresponds to 2'1T radians of phase. Hence, we have the proportion
from which it follows that ~
(11-3)
In mathematical terms, the intensity of the sinusoidal test object as a function of position is
and that of its image is
(11-4)
For notational simplicity, we shall consider one-dimensional systems whenever possible; it should be remembered, however, that in an actual imaging system the MTF and phase response depend on spatial frequencies in both the horizontal and vertical directions,Jx andfy- This is especially true in video, where the raster scanning process causes the horizontal and vertical MTFs to differ. An alternative definition of modulation transfer, used in the television industry to evaluate video components, is based on the response to square-wave (bar) patterns, rather than sinusoids. Using the Fourier series, whereby a square wave is expressed as a sum of sinusoids, the square• wave response C(f) (also called the contrast transfer function, CTF) is approximately related to the MTF M(f) by the equation (Schade, 1975, p. 6)
M(f) = ('IT/4) [C(f) + C(3f)!3 - C(5f)!5 + C(1!J17 + C(llf)/11 -C(13f)/13] (11-5)
It can be shown that, except at very low spatial frequencies, the square-wave response and the MTF are virtually identical, and we shall use them interchangeably. The MTF and phase response are sometimes combined into a single mathematical quantity, called the optical transfer function (OTF), denoted H(j):
H(f) = M(f) expUif)] where j is the (imaginary) square root of -1, and exp[.] is the exponential function. The MTF and phase response are the modulus (or magnitude) and phase, respectively, of this complex-valued function. MODULATION TRANSFER FUNCTION ANALYSIS 469
We proceed from the simple sinusoidal object to real objects via Fourier theory, which says that an arbitrary object may be expressed as a combination of sinusoids of various frequencies, amplitudes, and phases. The imaging system is understood as weighting and shifting the sinusoidal components of the object according to their spatial frequencies. A good example is the ''softening'' of sharp edges by a low-NA system. The sharpness of the edge is due to the presence of high-spatial• frequency components. These are attenuated relative to the lower-frequency components by the MTF, which decreases in value to zero as spatial frequency is increased. The loss of sharpness (reduced resolution) reflects the general "low pass" nature of the MTF. The phase response of an ideal imaging system is linearly proportional to frequency. Thus:
(11-6)
The relationship between phase and position (11-3) shows that a linear phase results in a position shift that is independent of frequency:
All the sinusoidal components of the image are displaced the same amount, hence the image merely undergoes a position shift, without degradation of image quality. If the phase deviates from this ideal linear characteristic, then some sinusoidal components will be shifted more than others, and a degradation of the image will result. Interestingly, phase is often more important than magnitude in preserving image fidelity (Shack, 1974; Oppenheim and Lim, 1981). In an aberration-free optical system with a symmetric aperture (e.g., round and centered on the optical axis), the phase transfer function is identically zero for all spatial frequencies in all directions, and only the MTF is important. However, the electronics in a video system possess less ideal phase characteristics, and can noticeably degrade image quality. The MTF model of imaging is particularly valuable for analyzing multicomponent systems, e.g., a microscope followed by a video camera and a monitor. The system MTF is simply the product of the MTFs of the individual components:
(11-7)
The system phase response is the sum of the individual phases:
(11-8)
It is important to note, however, that (square wave) contrast transfer functions do not multiply in this way, except for those spatial frequencies where the MTF and CTF are essentially identical. The optical transfer function is essentially determined by two things: the entrance and exit pupils of the optics (e.g., the condenser and objective NAs in a microscope), and aberrations in the optical elements, which are considered to introduce phase errors in the optical fields. Even in the absence of aberrations, diffraction at the pupils will limit the spatial frequency response, and hence the spatial resolution of the instrument. [Figures 5-24, 5-25; see also pp. 318-324 of Smith (1966) for graphs of MTFs under various conditions.] Since the effects of diffraction establish the ultimate limits to resolution, a perfectly aberration-free instrument is termed diffraction limited. The MTF of a diffraction-limited microscope with circular pupil, under conditions of incoherent illumination (NAcond = NAob}• is shown in Fig. 11-2. Image contrast is highest for low spatial frequencies, and decreases to zero as spatial frequency increases. The frequency at which contrast finally goes to zero is called cutoff, and is related to system parameters by the formula
where A is the wavelength of light. This expresses, in spatial frequency terms, the well-known fact that resolution increases with larger NAs and shorter wavelengths. 470 APPENDIX II
M(f)
100 %CONTRAST 50
FIGURE 11-2. Modulation transfer function of a diffraction-limited microscope with circular pupil, incoherent illumination.
11.3. THE MTF AND THE DIFFRACTION PATTERN
The MTF is closely related to a second quantity, the point-spread function (PSF). The PSF is simply the image of a point object (Fig. 11-3). Due to aberrations and diffraction at apertures, a point object is never perfectly imaged, in the geometric optics sense, but is spread into a finite-sized distribution of intensity. The image of an arbitrary object is a superposition of PSFs, and appears blurred in proportion to the spatial extent of the PSF. In microscopy, the PSF is more commonly referred to as the "diffraction pattern" (Section 5.5); for a diffraction-limited instrument, the PSF is the well-known Airy pattern. To completely understand the connection between the MTF and PSF requires Fourier theory. [The mathematically inclined reader is referred to the excellent books by Bracewell (1978), Good• man (1968), and Castleman (1979) for details.] However, an intuitive appreciation of this signifi• cant relationship is easily obtained. In Abbe's theory of the microscope, the back aperture deter• mines the highest spatial frequency passed by the system; as we saw in the previous section, this is the cutoff frequency of the MTF, which is proportional to NA. The other well-known measure of resolution-Rayleigh's two-point criterion-is based on the narrowness of the PSF. Two adjacent points in an object are spread by the optics into two PSFs. The narrower the PSF, the closer the points can be to one another and still be resolved by the unaided eye. Taken together, the Abbe and Rayleigh statements imply that having a wide MTF (high-spatial-frequency "bandwidth") is the same as having a narrow PSF (fine spatial resolution). In the language of Fourier theory, the MTF (more correctly, the OTF) and the PSF are each other's Fourier transform. A fundamental property
Yo
I·I
FIGURE 11-3. The point-spread function is the image of a point object. MODULATION TRANSFER FUNCTION ANALYSIS 471
PSF M(fl 100%
~Ifc ro = .61>. f = 2 NA NA >.
FIGURE 11-4. Relationship between diffraction-limited MTF and point-spread function.
of Fourier transforms is that the width of a function is inversely proportional to the width of its transform (Section 10.12). This relationship is illustrated, for a diffraction-limited incoherent system with circular aper• ture, in Fig. II-4. The cutoff frequency of the MTF is
fc = 2NA/A. and the radius of the first dark ring of the PSF (Airy disk) is
r0 = 0.61!../NA
We observe thatfc and r0 are indeed inversely proportional. An alternative definition of resolution is provided by the Sparrow criterion. Unlike the Rayleigh criterion, two points separated by the Sparrow distance are barely resolved, if at all; however, any separation beyond the Sparrow distance produces a minute dip in intensity between the two points, which may be visualized using contrast enhancement techniques. With video contrast enhancement, resolution beyond the Rayleigh limit and close to the Sparrow limit has been achieved in practice. The Sparrow distance for a diffraction-limited system is 0.5!../NA, precisely the inverse of the MTF cutoff frequency. This same reciprocal relationship between spatial resolution and spatial frequency response carries over to analog electronic systems, where the PSF is replaced by the time response to a very short electrical impulse, and the OTF is replaced by the system's response, in magnitude and phase, to a sinusoidal electrical signal. The shapes of the impulse response and transfer function are quite different from PSFs and OTFs; in particular, the impulse response lacks the symmetry of an optical PSF, so there will be nonnegligible phase effects, which are seldom linear. However, the underlying concepts are the same, and this permits combined optical-electronic systems to be analyzed within a common framework.
11.4. SAMPLING AND DIGITIZED VIDEO
In video image processing, sampling comes into play in two ways. First, the television raster scan samples the image vertically, converting the two-dimensional scene into discrete rows-the raster lines. Second, when a video image is converted to digital form for computer processing, each row of the video image-each horizontal scan-is converted to a set of discrete sample values, called picture elements, or pixels. The sampling process does not alter the MTF of the imaging system per se. However, the question of how many samples are necessary to accurately represent an object imposes a limit on the spatial frequency content of the image, and so it is appropriate to discuss it here. Figure 11-5 shows several sampling situations. The objects consist of alternating light and dark bars of various spatial frequencies. The samples are regularly spaced in position, and are indicated 472 APPENDIX II
A -~~-·--·--·--·~~~~~~~·--·. 1 ••••. __ · __ ·-L._._._~I_· 1 ••••. __· __·_·_J1-~~~-~~- 1 ••• __ -_·__ -_
B D.D.D.D.D.D.D.D.D.D.D.D.D.C
c ooonoonnoonnnoonnoonnonnonnn
D nn illl nQn oun on.n ollil oun oun on.n oDJJ oQn oQn o FIGURE 11-5. Sampling a bar pattern.
by the heavy dots. In Fig. II-5a there are four samples per bar; the original pattern is easily reconstructed from the samples. In Fig. II-5b the spatial frequency of the pattern has increased, so there is only one sample per bar, but the original pattern will still be reconstructable from the samples. In Fig. ll-5c the spatial frequency has increased further, to where the dark bars fall between the samples, and all the samples are light. Based on the samples, one would be led to conclude that the object was uniform in intensity rather than a fine bar pattern. Evidently the spatial frequency information necessary to reconstruct the pattern has been lost in the sampling process. Finally, Fig. II-5d shows the case of one s~ple every third bar. The samples alternate light and dark, just as in Fig. ll-5b, and would be recontructed as a bar pattern of lower spatial frequency. This is the phenomenon of aliasing, so called because a higher spatial frequency masquerades as a lower one. It can be a serious source of error in digitized images. It is evident from this example that sampling establishes an upper limit to the spatial frequency content of an image. From Fig. 11-5, it may be concluded that this limit is one sample per bar, or two per cycle. This same limit may be established by more rigorous mathematical arguments based on Fourier theory and sinusoidal rather than bar patterns. The fundamental conclusion, however, is the same: to recover an image from its samples, the sample density must be at least two per cycle of the highest spatial frequency present in the image. Turning this around, if a digital image memory consists of 512 x 512 pixels, the image must be limited in both directions to 256 cycles (line pairs), or 512 lines of resolution. Likewise, the raster scan of a video camera imposes a vertical spatial frequency limit around 240 cycles, or 480 lines. For a variety of practical reasons, however, it is advisable to stay below the upper limit and "oversample" by a factor of two or so (see Figures 6- 15, 6-16).
11.5. SYSTEMS ANALYSIS OF A VIDEO MICROSCOPE
To illustrate the power of MTF methods in systems analysis, we consider a practical exam• ple-a microscope with video camera and digital frame memory. We assume the microscope is diffraction limited, and incoherent (NAcond = NAob)· The MTF, shown in Fig. 11-2, has the mathematical description:
for f :5fc (11-9) for f> fc MODULATION TRANSFER FUNCTION ANALYSIS 473
The cutoff frequency, fc, is given by
fc = 2NAob/A
For a 40X /0.95-NA objective in green light (546 run), the spatial frequency cutoff is 3480 cy• cles/mm; the Rayleigh resolution distance is 0.35 Jl.m. Resolution for the video camera is considered separately for the vertical and horizontal direc• tions. The limit to vertical resolution is the number of lines in the raster scan, which we denote Nv. For conventional television, Nv is around 480. Horizontal resolution is limited by the dimensions and profile of the scanning electron beam. It is expressed as a number (here denotedNH) of TV lines per picture height (TVL PPH), which is interpreted as the number of resolvable lines in a horizontal distance equal to the height of the picture. Since a conventional television image has a 4 : 3 aspect ratio (width : height), the actual number of resolvable lines in the horizontal direction is 4/3 NH. (Recall that in video terminology, two "lines" comprise one "cycle" or "line pair" in optical terminology.) MTF curves for vidicon tubes are approximately Gaussian. They are measured with square• wave test objects, with the response to a fully modulated 15-Iine object taken to be maximum contrast (unity MTF). Two critical frequencies typically appear in camera specs-the limiting resolution, and a midrange value, which may be either the point of 50% contrast or the percent contrast at 400 lines. The Fourier transform of a Gaussian function is itself a Gaussian, so the (horizontal) MTF may be modeled by a Gaussian function:
Mcam(f) = exp( -0. 1f2/f1,) (11-10)
where fo = 50% contrast frequency (TVL PPH). Note that when f = / 0 , we have Mcamifo) = exp( -0. 7) = 0.496, approximately 50%. This model is a very good fit to the published MTF curves for most common vidicon-type tubes. The MTF of a type S4076 l-inch Newvicon tube (Panasonic) is shown in Fig. 11-6. Typical limiting resolution is 800 lines, with 50% contrast at 370 lines. There are about 480 active lines vertically, and o/3 x 800 = 1067 (barely) resolvable lines across the full width. It should be kept in mind, though, that the camera tube is only one component of the camera system. The camera MTF is significantly affected by the operating voltages supplied to the tube, the characteristics of the magnets that help scan and focus the electron beam, and the frequency response of the electronic circuits that amplify the video signal from the tube. In some high• performance cameras, the resolution may be much better than that predicted by the tube specifica• tions. In addition, tube-to-tube variability may cause two "identical" cameras to differ in perfor• mance. Nevertheless, the data provide helpful indicators of average behavior, and are useful for the illustrative example that follows.
100
%CONTRAST
TV LINES
FIGURE 11-6. MTF of Panasonic S4076 l-inch Newvicon. (See also Fig. 7-9C.) Information courtesy of Matsushita Electronics Corp., Takatsuki, Japan. 474 APPENDIX II
The simple model shown in Fig. II -7 is used to evaluate the combined MTF of the microscope• camera system. The specimen is imaged via the microscope optics (magnification Mobj) to the primary image plane, and perfectly transmitted to the vidicon camera face, with magnification Mrei• by the relay optics, which may include an ocular, a low-magnification relay such as a Zeiss Optovar, and/ or a zoom lens. The microscope MTF is described in terms of cycles per millimeter of resolution at the specimen plane, while the camera MTF is expressed in terms of lines per picture height at the camera face. The two planes are related by the net magnification, MobJMreJ· A spatial frequency fsp at the specimen plane appears as a lower frequency fsp/MobJMre1 at the camera target. The active scanned area of the vidicon target has a 4 : 3 aspect ratio with a diagonal measure• ment D. therefore its dimensions are 0.8D x 0.6D. In a "l-inch" tube, D = 0.625 inch= 15.875 mm. The camera field has Nv lines of resolution vertically and o/3 NH lines horizontally, thus there are Nyi0.6D lines/mm vertically and o/3 NH/0.8D = NH/0.6D lines/mm horizontally. (The funda• mental relationship is the same for horizontal and vertical.) Dividing by 2 to convert from lines to cycles (line pairs) gives the key expression relating spatial frequency at the specimen plane, fsp• to lines of resolution, N (in either direction), at the camera face:
(11.11)
Substituting the Newvicon resolution limits, NH = 800 and Nv = 480, into this expression, and assuming a 40X, 0.95-NA objective with unity magnification relay, we find that the maximum (specimen) spatial frequencies that will be passed by the tube arefH = 1680 cycles/mm andfv = 1009 cycles/mm. This is considerably less than the cutoff of 3480 cycles/mm computed earlier for the microscope, and demonstrates that significant resolution is lost in the video camera.
The solution to this problem is to magnify the image (Mre1 > 1), reducing the effective field size and increasing the "density" of TV lines in the field. Twofold magnification with increase fH to 3360, which nearly matches the microscope's cutoff. The field diameter is reduced twofold. However, this will still not recover all the microscope's resolution, since the system MTF, which is the product of the microscope and camera MTFs (equations 11-9 and 11-10), is depressed below the diffraction-limited curve by the camera response. Figure 11-8 shows the system MTF plotted for various magnification factors. The graphs show that Mre1 of between 4 and 8 is required for the microscope-camera system to approach the diffraction-limited performance of the microscope alone. A similar analysis may be applied to digitization of the video signal. A typical frame memory is 512 x 512 pixels, and if aliasing is to be avoided, spatial frequencies in the image cannot exceed the equivalent of 512lines. This will always be met in the vertical direction, sinceNv = 480, but it may
Mobj
SPECIMEN
MICROSCOPE
IMAGE
CAMERA TARGET
FIGURE 11-7. Model for analysis of microscope-camera system. MODULATION TRANSFER FUNCTION ANALYSIS 475
System MTF
~ \" \ ' c \ ' 0 :;::; \ ',""" IU \ ' " :; \ ' '~ 1J 50 \ ' '~ 0 \ ', '~~ E 40 \ ', ' ::::... diffraction hm1ted ..c 30 \ ' ...... Ql \ ' 4 ..... -.: ...u 20 ' 2 '...... ~~~ Ql Mrel =1',, '...., '..._,..;;:: D.. 10 .... 0 500 1000 1500 2000 2500 3000 Spatial frequency (cycles/mm)
FIGURE 11-8. MTF curves of a microscope-camera system, for various magnifications in the relay optics. constrain the horizontal direction. To determine the horizontal limits, we use equation (11-11); lettingfsp = 3480, the spatial cutoff of the microscope optics, and N = 512, we obtain a value Mre1 = 5. Since Mre1 2:: 8 is required to achieve full resolution, we are not constrained by the digitizer in this example. Likewise, we may include the effects of a video tape recorder on the system MTF. The (horizontal) resolution limit of a monochrome unit is typically 320 lines PPH. (Color recorders are limited to 240 lines, and many have this limit even when operating monochrome, if the color circuits are not bypassed.) Again using equation (11-11), at the specimen plane, the 320-line cutoff corresponds to
_ 40 X 320 X Mrel _ /H - 1.2 X 15.875 - 672Mrel cycles/mm
A 5 x magnification will match the cutoff of the VTR to the optics, but more will probably be required to completely overcome the recorder's limitations depending on its MTF curve. A further consideration is image brightness, which is proportional to the area of the field. If the field diameter is reduced by Mre1 to improve resolution, the image brightness will go down by a factor of M2 rei· In some situations this may be unacceptable, and the microscopist will be forced to trade resolution for brightness. This example is illustrative of the insight into system behavior that can be gained by MTF analysis. The method, expressions, and plots are sufficiently general to allow other microscope• camera combinations to be analyzed. In any actual experiment, the "best" system configuration will depend on the specimen and the goals of the experiment, as well as the instrumental charac• teristics. The results of these analyses, e.g., the relay magnification, should therefore be regarded as nominal design points about which the microscopist may search for the best overall imaging performance. Ill An Introduction to Biological Polarization Microscopy*
111.1. INTRODUCTION
Electron micrographs show fine structural detail and organization in thin sections of fixed cells. Time-lapsed motion pictures show the continually changing shape and distribution of organelles in living cells. How can we combine these observations and follow the changing fine structure in a single living cell as it undergoes physiological activities or experimental alterations? The relevant dimensions are too small for regular light microscopy since many of the changes of interest take place in the 0.1 to 100 nm range, i.e., the size range of atoms to macromolecular aggregates. From wave optics, the resolution of a light microscope is limited by its NA and the wavelength of light according to equation (5-9, p. 115). t On the other hand, the electron microscope with its very high resolution, practically 1-2 nm for many biological specimens, does not usually permit nondestructive study of cells. One effective way of closing this gap is to take advantage of anisotropic optical properties, which, albeit at a rather low level, are often exhibited by cellular fine structures. Anisotropic optical properties, such as birefringence and dichroism, measure molecular and fine structural anisotropy as we shall soon see. These properties can be studied nondestructively in individual living cells with a sensitive polarizing microscope, so that the time course of fine structural changes can be followed in small parts of a single cell. From equation (5-9), the smallest distance resolvable with any microscope with a 1.40-NA objective (with a nearly matched NA condenser at>-.. = 550 nm) is = 0.2 !J.m, or 200 nm. With a sensitive polarizing microscope, we can determine molecular alignment or changes in fine structure taking place within each resolvable unit area that is smaller than 0.2 X 2 !J.m, or less than 1 !J.m2.
*Revision of a manuscript originally prepared for a NATO Advanced Summer Institute held at Stresa, Italy, in 1969, but which was never published. Lectures related to this were delivered to the Physiology course and the short course on Analytical and Quantitative Light Microscopy in Biology, Medicine, and the Materials Sciences at the Marine Biological Laboratory, Woods Hole, Massachusetts, and the University of Pennsylva• nia. The active discussions and willing cooperation of my students and colleagues guided me in developing these lectures and demonstrations. Several of the illustrations in this appendix were initially prepared by Richard Markley, Dr. and Mrs. Hidemi Sato, and Hiroshi Takenaka. They have been refined and finished by Bob and Linda Golder and Ed Hom of the MBL. Their efforts, and support by NIH Grant SROI-GM-31617 and NSF Grant PCM 8216301, are gratefully acknowledged. tThis does not mean that we cannot form an image of individual organelles, filaments, or particles that are smaller than half the wavelength of light. If they provide enough contrast, and are separated from other comparable structures by a distance greater than the resolution limit, they can be visualized (by their image expanded to the width of their diffraction pattern; Figs. 1-7, 11-3, 11-5, 11-8).
477 478 APPENDIX Ill
111.2. ANISOTROPY
Anisotropy relates to those properties of matter that have different values when measurements are made in different directions within the same material. It is the opposite of isotropy, where the property is the same regardless of the direction of measurement. A common example of anisotropy is found in a piece of wood. Wood is much stronger parallel to the grain than across. As the humidity changes, wood shrinks and swells across the grain much more than it does along the grain. Also, wood splits more easily along the grain. In these examples, we might have anticipated the anisotropic properties from the grain structure of wood, but there are other anisotropic properties that are not quite so obvious. Related to the fact that it is more difficult to stretch wood along the grain than across the grain-that is, the coefficient of elasticity is greater parallel to the grain than across-sound waves travel faster parallel to the grain than across the grain. Tyndall pointed out many years ago that, depending on the type of wood, the velocity of sound may be three times as great when the sound is traveling parallel to the grain than across the grain. We can demonstrate this phenomenon by taking two blocks of wood cut from a single plank (Fig. III-1). The two blocks are made to the same dimensions but one (P) is cut parallel to the grain and the other (S) across. When we tap on the end of a block, a standing wave is formed. The compressive sound wave travels back and forth quickly, and since the ends are open, a longitudinal standing wave is formed with its loops at the ends of the block and a node in the middle. This standing wave takes on a particular pitch or tone. The tone tells us how quickly the wave is traveling in the block. Parallel to the grain we get one tone. Across the grain we get another. In fact, the velocity of sound across the grain is much lower, so that the tone is quite a bit lower (by an octave in one example; see legend to Fig. III-1). This is a dramatic demonstration of acoustic anisotropy, or anisotropic propagation of sound waves. The demonstration tells us how structural anisotropy can relate to wave propagation. In this case, the grain structure of the wood was obviously visible, and it is not difficult to grasp the reason for the anisotropy; but anisotropy can exist even if we cannot see heterogeneous struc• tures. The anisotropic properties can lie in the material that appears homogeneous at the microscopic level but which is anisotropic at the atomic or molecular levels.
111.3. SOME BASIC FEATURES OF LIGHT WAVES
Before discussing anisotropy further, we will review some basic physical optics needed in the subsequent discussions. Light is a form of electromagnetic wave. Assume a capacitor that is charged and let it discharge as a spark through two electrodes as shown in Fig. III-2. In the spark, a current flows
t----£=6-8"'--..j
I" ~ ' d =1-1- ~4 "'''' s'""" ,., d !'... r ; I FIGURE 111-1. Wooden blocks for demonstrating acoustic aniso• I I tropy. Blocks P and S are finished to the same dimensions, but Pis I I I I cut parallel to the grain while S is cut across grain. The frequency I IP 1I. of the standing wave, generated by gently holding onto the middle I I of the block and tapping (with a steel ball with a flexible handle) I I I from an end, measures the acoustic velocity for the compressive wave that travels through the block. In one example (8-inch-long ~~-~---~, l~ blocks made of hard oak), P resonated at 4500Hz and S at 2500 ~ ~ Hz. In this case, the compressive sound wave traveled nearly two times faster parallel to the grain than across. INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 479
FIGURE 111-2. Propagation of electromagnetic wave. The wave is generated by the discharging spark (or oscillating molecular dipole) to the left. The spark current oscillates at a frequency v char• acteristic of the circuit. The resultant electromag• netic disturbance is propagated with the electric (E) and magnetic (H) vectors vibrating perpen• dicular to each other and to the direction of propa• gation Z. v is determined by the oscillator, and the wavelength (A.) is given by A = v/v , where the velocity (v) is given by equation (III-I) or(III-2).* z
down for a short time, slows down, but because of the inductance of the circuit, flows back upwards, recharging the capacitor again. The current thus oscillates back and forth at a frequency (v) that is characteristic of the circuit. The electric current oscillating up and down in the spark gap creates a magnetic field that oscillates in a horizontal plane. A changing magnetic field in tum induces an electric field so that we end up with a series of electrical and magnetic oscillations that propagate as an electromagnetic wave. As seen in Fig. III-2, the electric field in an electromagnetic wave vibrates with its vectorial force growing stronger and then weaker, pointing in one direction, and then in the other direction, alternating in a sinusoidal fashion. The magnetic field oscillates perpendicular to the electric field, at the same frequency. The electric and the magnetic vectors, which express the amplitude and vibration directions of the two waves, are oriented perpendicular to each other and to the direction of propagation. From the relationships defining the interaction of the electric and magnetic fields, one can deduce the velocity of the resulting electromagnetic wave. The equations of Maxwell show that the velocity (v) is exactly c (the velocity of light in vacuum = 3 X 1010 em/sec) divided by the square root of the dielectric constant mof the medium times the magnetic permeability ([J.) of the medium. Thus,
v=c / ~ (III-I)
For most materials that occur in living cells, namely for nonconducting material, fL = I, so that
v = cl\l'f (III-2)
Empirically, we also know that the velocity of light is inversely proportional to the refractive index (n) of the material through which it propagates, i.e.,
v = c!n (III-3)
Equations (III-2) and (III-3) tell us that the refractive index is equal to the square root of the dielectric constant of that material (if the measurements are both made at the same frequency, v). Thus,
(III-4)
This tells us that optical measurements are, in fact, measurements of electrical properties of the material. The dielectric properties in tum directly reflect the arrangement of atoms and molecules in a substance. The direction of interaction between an electromagnetic field and a substance can be consid-
*In the equations, ~ = 11 = I for a vacuum, and very close to I for air. 480 APPENDIX Ill ered to lie in the direction of the electric vector. That is so whether we consider the electric or the magnetic vectors, since what matters is the effect of the electric or magnetic fields on the electrons in the material medium. (The magnetic field affects those electrons which move in a plane perpen• dicular to the magnetic field.) From here on, then, we will represent the vibrations of an electromag• netic wave by indicating the direction of the electric field alone. (The magnetic field is, of course, still present perpendicular to the electric field, but we will not show it in our vector diagrams, in order to avoid confusion.)
111.4. OPTICAL ANISOTROPY
The dielectric constant is anisotropic in many substances. Take a cube of such a substance and place it on a Cartesian coordinate, with the sides of the cube parallel to the X, Y, Z axes. The dielectric constant for an electric field measured along the X axis-that is, ~ measured by placing the plates of a capacitor on the faces of the cube parallel to the Y-Z plane (the plane that includes the Y and Z axes, or perpendicular to the X axis)-would have one value, ~x· Along the Y axis, it would have another value, ~Y' and along the Z axis, a third value, ~z· We now take the square roots of these three dielectric constants and make the lengths of the X, Y, and Z axes proportional to these values. Then we draw an ellipsoid whose radii coincide with the X, Y, and Z axes as just defined (Fig. III-3). What we obtain is known as the Fresnel ellipsoid. This ellipsoid describes the dielectric properties measured in all directions in the material. Each radius provides the ~value for an electromagnetic wave whose electric vector lies in the direction of that radius. Since v'f = n from equation (III-4), the Fresnel ellipsoid is, in fact, a refractive index ellipsoid. ~is the refractive index for waves whose electric fields are vibrating in the X-axis direction, ~ for waves with electric fields vibrating along the Y axis, and Vf. for waves with electric fields vibrating along the Z-axis direction. The value of the refractive index given by the radius of the Fresnel ellipsoid is valid for all waves whose electric vector vibrates in the direction of that radius regardless of the wave's direction ofpropagation. For example, waves with their electric vector in the X-axis direction may be propagating along the Y- or Z-axis direction. Therefore, given the direction of the electric vector
z
FIGURE 111-3. The refractive index, or the Fresnel, ellip• soid. The radius of the Fresnel ellipsoid gives the refractive index (n), or the square root of the dielectric constant(~, for waves whose electric displacement vectors lie in the direction of the radius of the ellipsoid within an anisotropic medium. A cross section through the center of the ellipsoid gives the refractive index ellipse for waves traveling normal to that section. The major and minor axes of the ellipse denote the refractive indices encountered by the slow and fast waves, which vibrate with their electric displacement vec• tors along those two axes. INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 481
(or more properly, the electric displacement vector in the material medium), the refractive index suffered by a wave is defined regardless of its direction of propagation.
111.5. BIREFRINGENCE
How do we use this ellipsoid? If light is traveling along the X-axis direction, for example, then the refractive indices for the waves vibrating in the material are given by an index ellipse, which is the cross section of the three-dimensional ellipsoid cut perpendicular to the direction ofpropagation (the X-axis direction in this example). For light traveling in any direction, we obtain an index ellipse by making a major cross section of the ellipsoid perpendicular to that direction of travel. The index ellipse gives us the refractive indices and shows the vibration direction (of the electric vector) of the light waves, because the waves vibrate with the electric vectors along the major and minar axes of the index ellipse. The refractive indices that the two waves suffer are given by the major and minor radii of the ellipse. In other words, for each direction of travel in an anisotropic medium, there are two plane-polarized light waves that vibrate perpendicular to each other and travel at different velocities. This is, in fact, the phenomenon of birefringence. To restate what we have just seen, as light travels through an anisotropic material, the wave gets split into two vibrations. The two vibrations (remember, we are just considering the electric vector directions and not the magnetic vectors) are mutually perpendicular to each other and perpendicular to the direction that the wave travels. The wave whose electric vector vibrates along the major axis of the index ellipse is called the slow wave, because the refractive index for this wave is greater than the refractive index for the other wave.* The wave vibrating perpendicular to the slow wave is the fast wave. Each index ellipse then provides the slow and fast vibration axes for waves traveling perpen• dicular to the plane of the ellipse. The index ellipse (the cross section of the index ellipsoid) is commonly used to designate the birefringence of a material observed with light propagating perpen• dicular to that plane (e.g., Figs. III-14, III-17). We shall now examine another manifestation of birefringence, taking advantage of another demonstration. We place a piece of cardboard with a small hole in front of a light source and on top of the hole, a crystal of calcite. Calcite is a form of calcium carbonate, whose rhombohedral shape is given in Fig. III-4. When we place the calcite crystal on top of the hole, we find that the image seen through the crystal is doubled. As we turn the crystal on the cardboard, we find that one of the images is stationary, while the other one precesses around the first. This observation is explained by the birefringence of calcite. The birefringence is so strong that we not only get two waves, but even the directions of travel of the two waves become separated (Fig. III-5). One of the waves is traveling straight through-its image remains stationary when the crystal is turned. That is called the ordinary ray, or the o-ray, because it behaves (is refracted) in an ordinary fashion. The other wave, the precessing one, refracts in an extraordinary fashion, and is called the extraordinary ray, or the e-ray. Not only is the light split into two, but as we argued before, each must be vibrating in a unique direction. The two must also be vibrating with their electric vectors perpendicular to each other. It turns out that the e-ray always vibrates in the plane that joins it and the o-ray. The o-ray always vibrates at right angles to this, as we will now verify. We orient the crystal so that thee-ray image (which is the one that precesses) appears on top (Fig. III-4). The direction of vibration of the e-ray then should be in the direction joining it and the o-ray image, namely up and down. The o-ray should have its electric vector horizontally. If we use a polarizer that transmits waves with horizontally oscillating electric fields, the top image should disappear; if we use one that transmits electric fields with vertical vibration, the bottom image should disappear. One handy, inexpensive standard polarizer, or polar, is a pair of Polaroid sunglasses. It shows no apparent anisotropy if we examine an ordinary source of light. No difference is apparent as we
*Equation (III-3) shows the relation of the refractive index to the (phase) velocity of travel. 482 APPENDIX Ill
FIGURE 111-4. Double image of a single light spot seen through a calcite crystal. As the crystal is turned, the e-ray image pre• cesses around the a-ray image. The e-wave vibrates in the plane that includes the c-axis (the principal section). The a-wave vibrates perpendicular to it. The c-, or optic-axis, of the crystal is indicated by c. In calcite, it is the axis of threefold symmetry. It makes an equal angle with all three of the crystal faces that join at the two corners, where all edges lie at 103° angles with each other. The c-axis lies in the direction of the semi-ionic bond that links the planar C03 groups and Ca atoms in the calcite (CaC03) lattice. (Evans, 1976; Inoue and Okazaki, 1978.)
tum the sunglasses in different directions. But if we look at the surface of water, or at the glare on the road or painted surfaces, the reflected light is cut out as the Polaroid glasses are intended to do. Light that is reflected from the surface of water or any nonconducting material is plane polarized, and especially strongly so at a particular angle of incidence-the Brewster angle. It is polarized so that the electric vector is vibrating parallel to the surface from which it is reflected, and not vibrating perpendicular to the surface. This behavior can be explained from a series of equations of Fresnel, but an easier way is to use the stick model proposed by Robert W. Wood. Consider a stick of wood in place of the electric vector. If it hits the water surface at an angle, the stick goes into the water and is not reflected. If the stick comes in parallel to the surface, it can bounce back. Since in nature we are dealing with horizontal surfaces, it will be a horizontal vibration that is reflected. We want to cut the glare, so Polaroid sunglasses are made to remove the horizontal vibrations and transmit the
FIGURE 111-5. Path of light rays through a calcite crystal. The crystal is shown through a principal section-that is, through a plane including the optic axis, c. The a-ray is refracted as though it were traveling through an isotropic medium; hence, it proceeds without deviation after normal incidence. The e-ray direction is deviated (in the principal sec• tion) even for nonnal incidence, hence the name extraordinary ray. The c-axis is the axis of symme• try of the index ellipsoid. Calcite is a negatively birefringent crystal so that the a-ray is the slow wave. The a-wave always vibrates in a plane per• pendicular to the principal section while the e-wave e-ray o-ray vibrates in the principal section. INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 483
FIGURE 111-6. Determining the electric vector directions for the e- and o-ray in calcite with Polaroid sun• glasses. Polaroid sunglasses are made to absorb and remove the horizontal electric vectors and transmit the vertical vibrations, PP'. On the left side, the e-ray image from the calcite crystal is transmitted but the o-ray image is absorbed. On the right side, the calcite crystal is turned 90° and the e-ray image is absorbed. At an intermediate angle, both images are partially transmitted following the cosine squared law.
electric vector that is vibrating vertically. In these glasses, then, we have a standard for transmission of the electric vector (Fig. III-6). Returning to the calcite crystal (Fig. III-4), the top image has its e-vector vertically, so that when we look through our sunglasses, the bottom image should disappear (Fig. III-6, left). We find that is exactly the case. If we turn the sunglasses or the crystal slowly, two images appear for a while, until at 90° the "top" image becomes extinguished (Fig. III-6, right). Thus, with birefrin• gence a light beam is split into two waves, each of the waves vibrates with its electric vector perpendicular to the other, and they travel at different velocities. (Close inspection will reveal that the e-ray image appears farther through the crystal than the o-ray image, indicating that the o-ray image suffered greater refraction, or that ne < n0 in calcite.) Calcite crystals can be used as very effective polarizers; in fact, Nicol, Glan-Thompson, Ahrens, and other prisms are made of calcite to isolate and transmit one of the polarized waves.* For UV microbeam work, it is much less expensive and more effective to use a simple cleaved piece of calcite crystal that has been optically polished on two opposite faces. Placed in front of the microbeam source, the crystal splits the beam into two and gives two equally intense images of the microbeam source (Fig. III-7). One can choose an image with known vibration (or use both) because the e-ray, whose electric wave vibration joins the two images, can be identified by its precession when the crystal is turned around the axis of the microscope. The o-ray vibration is perpendicular to it. One now has two polarized microbeam sources with equal intensities, which can be used for analyzing e.g., the arrangement of DNA bases as discussed later. Down to which wavelength will calcite transmit? Generally, about 240 nm, but this depends on the calcite. One can get calcite that transmits to somewhat shorter wavelengths, depending on the source and purity of the crystal. In material such as calcite, the two beams were visibly split into two, but for most material, especially the kind of biological material making up cytoplasmic or nuclear structures in a living cell, the birefringence is so weak that the two beams are not visibly split. Even when they are not visibly split, the extraordinary and the ordinary waves are present, with their individual vibration planes and separate velocities. Before we look into the properties of these two waves and how they can be used to detect birefringence in living cells, let us briefly consider the molecular and atomic meaning of dichroism, another form of optical anisotropy.
*It is unfortunate that the wave transmitted by these otherwise superior polars is thee-wave. The velocity of the e-wave, or refractive index of the e-ray, varies with direction of propagation, so that these calcite prisms introduce astigmatism unless all the beams travel parallel to each other through the crystal. 484 APPENDIX Ill
LIGHT SOURCE
POLARIZER
UV LAMP UV MIRROR HB0-200
UV. POLARIZER
COMPENSATOR= REFLECTING CONDENSER RECTIFIED ~ CONDENSER ~ STAGE
RECTIFIED OBJECTIVE
ANALYZER
I \ I I \ I ~ IMAGE
FIGURE 111-7. Schematic diagram of polarized UV microbeam apparatus. Compare with schematics of invert• ed polarizing microscope (Fig. III-21) and photograph of rectified instrument (Fig. 111-22). Each ray from the UV source (a small, first-surface mirror mounted on a dovetail slide beneath the polarizer for visible light illumination), as well as from the visible light source diaphragm (which lies between the polarizer and the UV mirror), is split into e- and o-rays by the UV polarizer. The UV polarizer is a cleaved crystal of calcite optically polished on the top and bottom faces. The reflecting condenser focuses the double image of the UV mirror and the visible light source diaphragm onto the specimen plane, each image with its electric vector as defined in Figs. 111-4 to 111-6. The image of the UV mirror with the desired polarization is superimposed on the specimen to be microbeamed. (From Inoue and Sato, 1966b.)
111.6. DICHROISM
Again, we start with a demonstration. A small source of light, say a flashlight bulb, is placed behind a crystal of tourmaline. Tourmaline is a prismatic crystal with a threefold rotational symme• try, and the type we use for this demonstration is a clear green crystal. Tourmaline crystals are dichroic and transmit in the green (or blue) as long as the axis of symmetry is oriented perpendicular to the light beam (Fig. lli-8). But if we turn the crystal and look down the axis of symmetry, no light, or very little light with a brownish tint, is transmitted. We can understand the wave-optical basis of these observations by returning to an analysis similar to the one used for birefringence. Dichroic crystals are also birefringent, so that light is again split into two waves in the crystal. As shown in Fig. lli-9, for each direction of travel, the light wave is split into two waves, whose electric vectors are oriented perpendicular to each other and to the direction of travel. For light traveling along the Z axis, we have one wave (Y) with its electric vector vibrating in the Y-Z plane and another (X) with its electric vector vibrating in the X -Z plane. Both of these must be absorbed because light does not come through the Z-axis direction. This means that the electronic disturbances caused by both the y electric vector and the x electric vector result in INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 485
FIGURE 111-8. Transmission of light through a dichroic crys• y tal-in this illustration, a clear green crystal of tourmaline. Viewed along the X and Y axes, the crystal is clear green. Viewed along the Z axis, no light is transmitted and the crystal is black or dark brown, even when the crystal is considerably shorter along X the Z axis than along the X andY axes.
absorption of the energy. The chromophores in the crystal are oriented such that they absorb light whose electric fields are vibrating in the X-Y plane. If we examine the light traveling along the X direction, we again know that light must vibrate in two directions perpendicular to that-the Z direction and theY direction. We already know that the Y vibration is absorbed, and therefore the green light we see must have its electric vector in the Z direction. Likewise for the Y propagation, the x electric vectors are absorbed and only the z vectors must make up the transmitted green light. We thus deduce that the light corning through the tourmaline crystal is plane polarized with its electric vector parallel to the crystalline (Z) axis of symmetry. This is readily confirmed with our standard Polaroid sunglasses.* This is the basis of dichroism. As we saw, both dichroism and birefringence can be manifested as macroscopically recognizable optical properties, which vary with the direction of propagation of light, but they are at the same time a reflection of fine-structural, molecular, or atomic anisotropies. For dichroism it is the orientation of chromophores that determines anisotropic absorptions. For birefringence it is the anisotropy of dielectric polarizabilities. The anisotropic polarizability can originate in the fine structure or within the molecules.
111.7. DETERMINATION OF CRYSTALLINE AXES
We are now almost ready to apply these principles to the polarizing microscope to study biological material. Let us place two pieces of plastic sheet Polaroid in front of a diffuse light source and orient them at right angles to each other so that the light is extinguished. In between the "crossed polars" we insert our samples, just as we do with a polarizing microscope. When, for example, a piece of cellophane is placed between crossed polars, we observe light through that area (Fig. Ill-10). The phenomenon is due to birefringence, as explained below, even though we do not see a double image. As in Fig. Ill-11, let the light (with an amplitude of OP) from the polarizer (PP') vibrate vertically. Upon entering the specimen. OP is vectorially split into two vibrations, OS and OF. These vibration directions are uniquely defined by the crystalline structure of the specimen since
*Polaroid sunglasses and filters are themselves made from a dichroic material. One common form uses a stretched film of polyvinyl alcohol (PV A) impregnated with polyiodide crystals. In the stretched PV A film, the micelles and molecular backbones are oriented parallel to the direction of stretch (Fig. 111-17). The needle• shaped, dichroic polyiodide crystals are deposited in the interstices, parallel to the PV A molecules. All of the polyiodide crystals, therefore, become oriented parallel to each other, and one obtains a large sheet of dichroic material (Land, 1951; Land and West, 1946). 486 APPENDIX Ill z
z
y
FIGURE 111-9. Explanation of dichroic ab• sorption in tourmaline. The x and y electric vectors are absorbed regardless of the direc• tion of propagation. Only the z vectors are not absorbed, so that the transmitted light is plane polarized parallel to the Z axis. they lie in the directions of the major and minor axes of the index ellipse. In the crystal (cellophane), these two waves travel at different velocities so that when they come out of the crystal, they (O'S' and O'F') are out of phase (d) relative to each other.* The combination of these two waves, which remain out of phase and continue to oscillate in planes perpendicular to each other in air, yields an elliptically polarized wave (O"P" -0"A"). Light that is elliptically polarized no longer vibrates only along the PP' axis, as it has a component that exists along the AA' axis. That component (O"A") of the elliptically polarized light can pass through the second polar (the analyzer) and give rise to the light that we observe. For the birefringent specimen between crossed polars, the elliptically polarized wave is pro• duced by the splitting of the original plane-polarized wave into two vectors. Therefore, we should not get elliptical polarization if, for some reason, one of the two vectors were missing. This happens when the slow or fast specimen axes become oriented parallel to the polarizer axis. At that orientation of the crystal, OP' cannot be split into two vectors, so OP' emerges from the crystal unaltered. This wave is then absorbed completely by the analyzer and no light comes through. Therefore, by rotating the specimen between crossed polars and observing the orientation where the specimen turns dark (Fig. III-10), we can determine its crystalline axes. When the specimen turns dark between crossed polars, the orientations of the major and minor axes of the index ellipse are parallel to the axes of the polars. (Note that these axes may or may not coincide with the geo• metrical axes or cleavage planes of the specimen.) We have now established the directions of the two orthogonal axes in the crystal, but we still do not know which is the fast axis and which is the slow axis. In order to determine this, we
*A, expressed in fraction of wavelength (of the monochromatic light wave in air; Ao), is proportional to the thickness of the crystal (d) and to its "coefficient of birefringence" (ne - no). Thus,
(III-5)
where n0 is the refractive index for the extraordinary ray, and n0 that of the ordinary ray. The e-ray may either be the slow or fast wave depending on the crystal type. If the e-ray is the slow wave (as it is in quartz and cellophane), the crystal is said to be positively birefringent; if thee-ray is the fast wave (as it is in calcite), the crystal is said to be negatively birefringent. The Fresnel ellipsoid for a positively birefringent material is prolate, while the one for a negatively birefringent material is oblate. INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 487
FIGURE 111-10. Appearance of a birefringent crystal between crossed polars. Polarizer transmission PP' and analyzer transmis• sion AA' are at 90° to each other and extinguish the background light. The crystal is viewed in various orientations. When the optical axes of the crystal are parallel to PP' or AA', the crystal is dark. When the axes are at any other orientation, the crystal is A.-A' brighter.
superimpose a crystalline material whose slow and fast axis directions are already known. Such a crystal is known as a compensator. Let us assume in our next demonstration that a particular piece of plastic-the compensator• has its slow and fast axes oriented as shown in Fig. III-12. We superimpose this on an unknown sample; both are placed between a pair of crossed polars. If the slow axis of the unknown sample and that of the compensator lie perpendicular to each other, then the two effects cancel each other out. Where the compensator and the sample are superimposed on each other, we observe that the light is extinguished; the specimen appears dark (Fig. III-12, the crystal in the lower right position). What has happened is that the elliptically polarized light produced by the specimen was restored by the compensator to the original plane-polarized light (Fig. III-13). Therefore, no vectorial compo• nent is present in the analyzer transmission direction, so that all the light is absorbed by the analyzer. On the other hand, if the specimen and the compensator slow axes were parallel to each other, the two retardations would add up together, and we would get more light through the analyzer than before (Fig. III-12, the crystal oriented as in the top). By finding the orientation of the compensator
FIGURE 111-11. Explanation of the phenomenon in Fig. Ill-10. So long as the angle 9 between the slow axis (OS) of the crystal and OP (the electric vector from the polarizer) is not 0 or 90°, OP is split into vectors OS and OF by the birefringent crystal. Passing through the crystal, they acquire a phase difference, 6., which remains constant once in air (an isotropic medium) up to the analyzer. Viewed from the analyzer direction, the O'S' and O'F' vibrations combine to describe an elliptically polarized wave;-·O"P"-O"A". The O"P" component is absorbed by the analyzer, but the O"A" component is transmitted, and hence the crystal appears bright on a dark background. Where 9 is 0 or 90°, the OF or OS vector is missing, so that O"P" = OP and O"A" ~ 0. Hence, no light passes the analyzer. 488 APPENDIX Ill
FIGURE 111-12. Birefringent crystal between crossed polars, in the presence of a compensator. The compensator (large circle) introduces some light between crossed polars similar to the crystal in Figs. III-10 and III-11. On this gray background, the crystal appears darker (lower right orientation) when its s axis is in the opposite quadrant from the s axis of the compensator (subtractive orientation; see Fig. III-13). It is completely extinguished when the conditions in equation (III -6) or (III -7) are satisfied. When the crystal and compensators axes are in the same quadrant (top orien• tation), the crystal appears considerably brighter than the gray background. The contrast of the crystal against the gray back• ground disappears when the axes of the crystal coincide with PP' A-A' and AA'.
that extinguishes or increases light from a birefringent specimen, we can then determine the slow and fast axis directions of the specimen.* At this point I would like to point out that birefringence is not to be confused with optical rotation. Optical rotation is a phenomenon that we would encounter, for exmaple, if we observed a solution of sugar instead of our crystalline specimen (or if we observed quartz along its optical axis, the axis of symmetry). A solution of sugar has no directionality, but it can twist polarized light (in a left-handed or right-handed direction depending on its chemical nature). The result is as though the polarizer PP' were turned; so the light can be extinguished by turning the analyzer AA' by that same amount. With birefringence, you can generally not extinguish the light by turning the analyzer, but with optical rotation you can (for monochromatic light). Optical rotation reflects the three-dimen• sional asymmetry of the individual molecules, which themselves can be randomly oriented (as in the solution of sugar). The dispersion (variation with different wavelengths of light) is usually very substantial for optical rotation but rather small (paretically negligible) for birefringence.
111.8. MOLECULAR STRUCTURE AND BIREFRINGENCE
The next demonstration will let us correlate molecular and micellar structure with optical and mechanical anisotropy. We take a sheet of polyvinyl alcohol (PVA) produced by casting a hot water sol of the polymer onto a clean piece of glass. The basic structure of PVA is shown in Fig. III-14. In a cast (dried gel) sheet of PVA viewed normal to the surface, the polymers and their micelles would
*Quantitatively, we obtain extinction when
.!1.28 .Rc·za sm 2 sm = -sm 2 sm c (III-6)
where ll and Rc are the retardances of the specimen and the compensator, respectively, expressed in degrees of arc (lA = 360°), and 8 and Be are respectively the angle between the slow axes of the specimen and of the compensator relative to OP, the polarizer transmission direction (Fig. III-13). We use this formula extensively for the photographic photometric analysis of retardance and azimuth angles of DNA microcrystalline domains in a cave cricket sperm (Figs. III-24, lll-25; Inoue and Sato, 1966b). Equation (III-6) can be simplified as follows. The specimen is generally oriented so that 8 = 45 ± 5°. When, in addition, ll and Rc are both not greater than 20°, or less than approximately A/20, then to a very close approximation, equation (Ill-6) can be written:
(III-7)
This is the principle by which specimen retardance is measured with the Brace-Koehler compensator (see also Salmon and Ellis, 1976). INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 489
FIGURE 111-13. Compensation. The left half of the figure is identical to most of Fig. III-II. However, after the light becomes elliptically polarized O'P'-O'A') by the specimen, a compensator with a retardance, Rc, is added to the light path, with OSc, the compensator slow axis, lying in the subtractive orientation, i.e., in the quadrant opposite to OS, the specimen slow axis. Rc is adjusted so as to restore the phase difference, a, between O'S' and O'F' that was introduced by the specimen. Then, the phase difference, -a', between O"S" and O''F' in the waves emerging from the compensator becomes zero and O"S" + O''F' = O"P". The conditions in equation (III-6) or (III-7) are now satisfied-that is, we have achieved compensation. At compensation, O"A"--+ 0 and O"P'' = OP, and hence the specimen is extinguished (Fig. III-12, lower right orientation). If -a' is not 0, O"A" is also not 0, and light whose amplitude is O"A" passes the analyzer. The sine squared relation in equation (111-9) explains why the crystal is much brighter than the background when OS and OSc are parallel. [Substitute (a + Rc) for a in equation (Ill-9).]
be expected to be arranged helter-skelter as shown in Fig. III-15. Such a structure would be optically isotropic, as we can see by the lack of birefringence-no light passes the cast PVA sheet placed between crossed polars at any orientation. Such a structure should also be mechanically isotropic and difficult to tear in any direction, since covalent molecular backbones traverse in every direction. Indeed, cast sheets of PVA are very tough. The micelles and molecular chains of the PVA sheet can be aligned by stretching a sheet that has been softened by mild heating with an electric iron. Figure III-16 shows the film before (A) and after (B) stretching. The arrangement of the micelles and molecular backbones after stretch is shown in Fig. III-17. The stretched film is highly birefringent and produces several wavelengths of
H H H H H H H H \/ \/ \1 \1 c c c c /"c / '\. c/ '\ c/" c I 1\ /\ /\ 1\ H OH H OH H OH H OH
EB 0.25nm FIGURE 111-14. Chemical structure of PVA. PVA is a long, linear, polymeric chain of -(CH2-CH·OH)-. In the absence of external constraints, rotation around the C-C bonds allows the chain to fold into random-shaped strands, except where adjacent chains lie in close proximity parallel to each other and form a minute crystalline domain or a "micelle" (Fig. 111-15). FIGURE 111-15. "Fringed micelle" structure of a cast gel of PVA viewed from its face. The circle to the right indicates a micelle where several PV A molecules run parallel and form a minute crystalline region. The micelles, and the polymer chains of PV A in between, are randomly oriented. The structure is isotropic as indicated by the index ellipse at the bottom.
A
FIGURE 111-16. Stretching PVA film. (A) A thin rectangular sheet of isotropic PVA is supported on both ends, taped and ! stapled onto pieces of cardboard. (B) The sheet is mildly heated, B for example with a tacking iron in the middle, and then stretched. The stretched portion becomes optically anisotropic (birefringent) with the slow axis in the stretch direction. It is also mechanically anisotropic and tends to split parallel to the stretch direction.
ffis f FIGURE 111-17. Fringed micelles in stretched PVA. As manifested in the optical and mechanical anisotropy (Fig. III-16), the fringed micelles and the PVA molecules in general have become aligned with their long axes parallel to the stretch direction. INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 491 retardation as seen from its color* between crossed polars. In spite of the strong birefringence, light is extinguished when the film is oriented with its stretch direction parallel to the axis of the crossed polars. Compensation reveals that it is the slow vibration axis that lies parallel to the stretch direction. This agrees with the molecular polarizability expected in PYA. In a polymer, with a covalently linked backbone and with small side groupst as in PYA, the polarizability (hence, the dielectric constant) is considerably greater parallel to the covalent chain backbone than across it. A striking mechanical anisotropy of the stretched film of PYA also reflects the micellar and molecular arrangements. The film is even tougher than before when one attempts to tear across the stretch direction. However, in response to a rap on the tautly held film, it readily splits into strips parallel to the stretch direction as shown in Fig. III-16B. The mechanical and optical anisotropies and the directions of their axes reflect the arrange• ments and anisotropic properties of the underlying molecules. In some biological samples, the molecules may take on a more complex arrangement than the homogeneous distribution seen in this PYA model. Nevertheless, the dielectric and optical properties specific to the particular molecular species provide important clues regarding the biological fine structure. For example, in a protein molecule with extended chains in the 13-form, or with a collagen helix, the slow axis lies parallel to the long axis of the polypeptide chain; the molecular polar• izability is considerably greater in that direction than across the chain. In B-form DNA, the slow axis is perpendicular to the backbone of the Watson-Crick helix. The conjugated purine and pyrimidine bases exhibit a greater UY absorbance and electrical polar• izability in their plane than perpendicular to the planes. Becausethe base planes in B-form DNA are oriented at right angles to the molecular backbone, they show a characteristic UY negative di• chroism and strong negative birefringence. In lipids, the slow birefringence axis of the molecule lies parallel to its backbone. But since they tend to make layered sheets with the molecular axes lying perpendicular to the plane of the sheets, the layered structure also introduces form birefringence.+ In form birefringence of platelets, the slow axis is parallel to the plane of the plates. The axis of symmetry is perpendicular to the plates, and hence platelet-form birefringence always has a negative sign (greater refractive index perpendicular to the axis of symmetry, orne< n0 ). Lipid bilayers (or multilayers in the Schwann sheath of myelinated nerve) show a combination of intrinsic positive and form negative birefrin• gence. Depending on the refractive index of the imbibing medium, the negative-form birefringence of the plates may become so strong as to overcome the intrinsic positive birefringence of the lipid molecules.
*See equation (III-5). In white light that is composed of various wavelengths (A0 's), the thickness (d) of the
crystal that gives rise to a full-wavelength retardation [(n0 - n0 )d = N A0 , where N = 1, 2, 3 ...] varies with Ao. Where the retardation equals N A0 , the light exiting the crystal returns to a plane-polarized wave identical to the one before it entered the crystal. Here it is extinguished by the analyzer and that wavelength is missing from the light transmitted through the analyzer. Thus, we get white minus that wavelength, or a series of "inter• ference colors," from strongly birefringent specimens observed between crossed polars in white light. t As the side groups become larger, or if there are side groups with greater polarizabilities-often associated with light-absorbing conjugated bonds-the birefringence of the polymer becomes weaker, and can even reverse in sign. For example, the degree of nitration of nitrocellulose is monitored by observing its birefringence, which changes from positive to zero to negative with increased nitration. The birefringence of DNA is strongly negative as discussed later. +Form birefringence, also known as textural birefringence, arises when platelets or rodlets of submicroscopic dimensions are stacked. The platelets or rodlets must be regularly aligned, with spacings that are considerably smaller than the wavelength of light. As shown by Wiener (1912) and by Bragg and Pippard (1953), such bodies generally exhibit anisotropy of electrical polarizability and therefore of refractive index. The anisotropy is stronger, the greater the difference of refractive index between the medium lying between the rodlets or platelets and the rodlets or platelets themselves. The anisotropy disappears or becomes least strong when the refractive index of the medium matches that of the rodlet or platelet. Form birefringence is due to the shape and orientation of molecules or molecular aggregates, and is independent of the birefringence that is intrinsic to the constituent molecules themselves. In contrast to form birefringence the value of intrinsic birefringence usually does not change with the refractive index of the imbibing medium. 492 APPENDIX Ill
In contrast to platelet-form birefringence, form birefringence of rodlets is positive. Positive• form birefringence is observed in microtubules, which are thin, elongated structures approximately 24 nm in diameter, made up of rows of globular protein molecules approximately 5 nm in diameter, or in actin, a twisted double cable of globular protein molecules, which themselves possess low molecular asymmetry.
111.9. THE POLARIZING MICROSCOPE
All the principles discussed so far provide the basis for analyzing molecular arrangements and cellular fine structure with the polarizing microscope. A polarizing microscope is nothing more than an ordinary microscope equipped with a polarizer underneath the condenser, an analyzer above the objective, and somewhere in the path, in a convenient place between the polarizer and analyzer, a compensator (Fig. ill-18). The compensator can come before or after the specimen. The specimen is supported on a rotatable stage. In principle, that is all there is to a polarizing microscope. However, if one decides to study cytoplasmic or nuclear structure with a polarizing microscope and tries with one borrowed from a friend in crystallography or mineralogy, chances are that it will be a quite disappointing experience. Other than a few crystalline inclusions, little of interest is seen in the living cell. One reason is that the amount of light that strays through an oridinary polarizing microscope between crossed polars often cannot be reduced enough to see the weak birefringence exhibited by the filaments and membranes of interest to us. The degree to which we can darken the field is expressed quantitatively as the extinction factor (EF), which is defined as
(ill-8) where IP is the intensity of light that comes through a polarizing device when the polarizer and analyzer transmission directions are parallel, and I. the minimum intensity that can be obtained when the polarizer and analyzer are crossed. For an ordinary polarizing microscope, the EF is often of the order of 103 or even lower with high-NA lenses. What we need for cell study is an EF of at least 1()4. In order to achieve this high an EF, we must attain a number of conditions concurrently. The polarizer and analyzer themselves obviously must be of good quality. They can be of selected sheet Polaroids, as far as the extinction goes (but their transmittance is low, and the rippled surface can deteriorate the image). We cannot use optical elements between the polars that are strained. As seen by stressing a piece of glass or plastic between crossed polars, the strain introduces birefringence. This must not take place because that birefringence can be much higher than the specimen birefrin• gence. So we must use lenses, slides, and coverslips that are free from strain. A little speck of airborne lint (e.g., from our clothing or lens tissue) can be highly birefringent, so we must keep our system meticulously clean. Also, since almost all the light is extinguished by the analyzer, the light source must be very bright (yet harmless to our specimen), and we should work in a darkened area to improve the sensitivity of our eye. Alignment of the optical components is critical, and Koehler illumination must be used to gain minimum stray light coupled with maximum brightness of the field. The reason for all of this care is that the light that comes from the specimen is only a minute fraction of the original light. If ~ is the retardance of the specimen and 6 its azimuth orientation, both expressed in degrees, the luminance (I) due to specimen birefringence (~) is given by
I= I sin2 .! (III-9) p 2 where IP is the luminance of the field with the polarizer and analyzer transmission parallel. I turns out to be of the order of IQ-4 to IQ-6 X IP for the range of specimen retardations of interest to us. So we are trying to see very dim light from the specimen-as though we were trying to see starlight INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 493
EYE
OCULAR
BERTRAND LENS
ANALYZER
COMPENSATOR
OBJECTIVE LENS
SPECIMEN FIGURE 111-18. Arrangement of conventional polarizing mi• croscope. The polarizer was commonly oriented with its trans• CONDENSER LENS mission axis PP' north-south to the observer. AA' was oriented east-west.* The Bertrand lens converts the ocular into a tele- scope and allows one to view an image of the objective lens back POLARIZER aperture. This provides a "conoscopic" image of the specimen, showing the interference pattern of convergent polarized light LIGHT SOURCE passing through the specimen at different angles. The Fraunhofer diffraction pattern produced by the specimen is also seen there. With the Bertrand lens out of the light path, the polarizing micro- MI R R0 R scope gives a regular "orthoscopic" image. during the daytime. The light is there, but there is so much more light around our specimen that we cannot see it. We have to somehow make the background dark enough so we can see the object. We must increase the EF and improve the specimen contrast. The situation is improved by good use of a compensator. This point can be demonstrated by placing, between crossed polars, a weakly birefringent specimen that we can just barely see. The birefringent region is barely brighter than the background (Fig. III-10). When we introduce the compensator, the background is now gray and the specimen is darker or much brighter than the background (Fig. III -12). In the presence of the compensator, the image brightness and the contrast can be vastly improved as discussed in Chapter 11 (Fig. 11-48), and images of weakly birefringent objects can now be displayed clearly (Figs. III-19, III-20, III-25).
111.10. RECTIFICATION
There is yet one more improvement required for detecting weakly birefringent specimens at high resolution in polarization microscopy. Following equation (5-9), we require high objective and condenser NAto obtain high resolving power. But each time the NA is increased by 0.2, stray light increases tenfold even when we use strain-free lenses. With objective and condenser NAs at 1.25, the EF may drop to nearly 102. This lowered extinction (increased stray light) at high NA is due to the rotation of the plane of polarization at every oblique-incidence interface between the polarizer and the analyzer. It is an inherent physical optical phenomenon that had been considered uncorrecta• ble until we developed the polarization rectifier (Figs. III-21; Inoue and Hyde, 1957; see also Huxley, 1960). With the rectifier the extinction is very good up to high NAs, and we are finally able
*More recently, the orientations of PP' and AA' have been reversed in most commercial instruments. FIGURE 111 -19. Living pollen mother cell of Easter lily (Lilium longiflorum). Spindle fibers (sp.f.) show stronger birefringence adjacent to kinetochores (spindle fiber attachment point) of helical chromosomes (chr) which show little birefringence. The maximum retardance of the spindle is less than ;1./100. (From Inoue, 1953.)
FIGURE 111-20. Birefringent spindle fibers in living oocyte of marine worm (Chaetopterus pergamentaceus). Chromosomal spindle fibers, white; astral rays, oriented at right angles to spindle axis, dark. The maximum retardance of these spindle fibers is about 3.5 nm. (From Inoue , 1953.) POLARIZER
COMPENSATOR
}RECT.} CONDENSER
+ .. ~--SPECIMEN ::R
} OBJECTIVE
1 E St } ANALYZER
St2
I I I I I I I I I I I r ' EM
+
FIGURE 111-21. Schematic optical path of transilluminating, universal polarizing microscope designed to provide maximum sensitivity and superior image quality. Inverted system with light source (S) on top and detectors (EM and E) at the bottom. Light from a high-pressure arc lamp is filtered (to remove infrared and provide monochromatic illumination) and focused by L1 and L2 onto a fiber-optic light scrambler at A2. The fiber scrambles the image of concentrated mercury arc (footnote p. 127) and provides a uniform circular patch of light that acts as the effective light source at A3. This source, projected by the zoom lens (L3), is made to just fill the condenser aperture diaphragm (A7) . Illuminance of the field can be regulated without affecting the cone angle of illumination (or disturbing the color temperature in white light) by adjusting the iris (A 1). The polarizing Glan-Thompson prism is placed behind stop A5 away from the condenser to prevent light scattered by the polarizer from entering the condenser. Half-shade and other special plates are placed at level A6 , and compensators above the condenser. Depolarization by rotation of polarized light in the condenser and objective lenses, slide, and coverslips are corrected by the rectifier (RECT.). The image of the field diaphragm (A4 or A6) is focused on the specimen plane by the condenser, whose NA can be made equal to that of the objective. Stigmatizing lenses (St., St2), which minimize the astigmatism that is otherwise introduced by the calcite analyzer, are low-reflection coated on their exterior face and cemented directly onto the analyzing Glan•
Thompson prism to protect the delicate surfaces of the calcite prism. Stops (A 1-A 11 ), placed at critical points, minimize scattered light from entering the image-forming system. The final image is directed by OC 1 onto a photographic, video, or other sensor (e.g., photomultiplier; EM), or to the eye (E) via mirror (M) and ocular (OC2) . Components A3 through EM are aligned on a single optical axis to minimize degradation of the image. 496 APPENDIX Ill INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 497
l.OlJm lOlJm
FIGURE 111-23. The effect of rectification on image reliability in the polarizing microscope. (I) The Airy disk diffraction pattern of a pinhole viewed through crossed polars with a rectified polarizing microscope of NAobJ = NAcond = 1.25. The pattern is identical with that obtained with a microscope in nonpolarized light. (2) The same as I, but with nonrectified lenses between crossed polars. The diffraction pattern is anomalous; each bright point in the specimen is represented in the image by a bright four-leaf clover pattern surrounding a dark cross. (3) A Siemens Test Star pattern viewed between crossed polars in the absence of rectification. Contrast is reversed and spurious toward the center of the image. (4) The same test pattern viewed with the same optics after rectification. The aperture function of the lens is now uniform and the- diffraction anomaly has disappeared. (From Inoue and Kubota, 1958.)
FIGURE 111-22. Rectified, universal polarizing microscope designed by author, G. W. Ellis, and Ed Hom (see also footnote on p. 145). Optical layout as shown schematically in Fig. III-21. The components, in eight units, are mounted on slides that ride on a II IO,OOOth-inch-precision, 3-inch-wide dovetail, (4V + !H) feet long. The well-aged cast iron (Mehanite) dovetail bench is mounted on a horizontal axis and can be used vertically, horizontally, or at angles in between. Two smaller dovetails, built into the same casting. run precisely parallel to the central dovetail and provide added flexibility (micromanipulators, UV microbeam source, etc. are mounted on these dovetails). Supported on the sturdily built wooden bench, by the horizontal axis near the center of gravity of the massive optical bench, and designed for semikinematic support of the components wherever practical, the microscope is quite immune to vibration. 498 APPENDIX Ill
\ \ \ \ \ \ M \ j \ '\ ~ \ ~I \ y "" \ ~~~I '
tolJm
FIGURE 111-24. Photograph of living cave cricket sperm in rectified polarizing microscope, with densitometer trace. The broad dark band in the middle of the sperm (where the densitometer trace dips below background) is where the birefringence of the DNA bases were selectively perturbed by irradiation with polarized UV micro• beam. The compensator is in the subtractive orientation so that the sperm head is darker than the background except where the specimen retardation is greater than the compensator (especially at the small dumbbell-shaped white patches, which give rise to theM shapes on the densitometer trace). In these regions, the birefringence and optical axes of the "microcrystalline domains" of the sperm DNA are such as to overcome the subtractive effect of the compensator. (From Inoue and Sato, 1966b.) to use the polarizing microscope to study weakly retarding specimens at the theoretical limit of microscopic resolution. Diffraction image errors, which can be present without rectification, are also corrected with the rectifier (Fig. III-23). *
*As shown in Fig. III-23, rectification corrects for the diffraction error that is introduced by conventional lenses when we observe low-retardation objects between crossed polars. However, the sensitivity for detecting weak retardations at high resolution is so improved by rectification that another hitherto unnoticed optical phe• nomenon becomes apparent. At the edges of any specimen, including isotropic materials, light is diffracted as though each edge were covered with a double layer of extremely thin, birefringent material; the slow axis on the high index side lying parallel, and on the low index side lying perpendicular, to the edge. We have named this phenomenon edge birefringence. It is found at all sharp boundaries (edges) whether the two sides of the boundary are solid, liquid, or gas, so long as there exists a refractive index difference on the two sides of the boun• dary (e .g., see Fig. l-7D,F). It is clearly not based on the presence of a membrane at the optical interface, but is a basic diffraction phenomenon taking place at every edge. The electric vectors parallel and perpendicular to the edges must contribute to diffraction in a slightly asymmetric way on both sides of the edge and give rise to edge birefringence. Edge birefringence disappears when the refractive indices on both sides of the boundary are matched. It reverses in sign when the relative magnitude of refractive indices on the two sides of the boundary are reversed. This behavior of edge birefringence is distinct from form birefringence. Form birefringence becomes zero (reaches a minimum value for rodlets, or maximum absolute value for platelets) when the refractive index of the immersion medium matches that of the rodlets or platelets, but then rises again (parabolically) when the refractive index of the immersion medium exceeds the match point (Ambronn and Frey, 1926; Sato et al., 1975). INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 499
l-- lOlJm --l
FIGURE 111 -25. Sperm head of a cave cricket (Ceunthophilus nigricans) observed with the rectified polarizing microscope at three different settings of a mica compensator. The detailed distribution of intrinsic birefringence in these chromosomes is shown with great clarity in the specimen immersed in dimethylsulfoxide (N0 20 = 1.475). Horizontal white bars: positions of helix "breaks" that correspond to the ends of chromosomes. (From Inoue and Sato, 1966b.) Prior to these studies, few chromosomes had been seen in mature sperm of any species.
The improvements achieved with the rectified polarizing microscope have made possible a determination of the complex alignment of DNA molecules in each unit diffraction area of a live insect sperm (Figs. III-24, III-25). The birefringence and azimuth orientation of each DNA micro• domain were measured (both to a precision of 0.1°) from microdensitometer traces (of the type shown in Fig. III-24) by correlating the traces taken at several compensator settings and stage orientations. The changes in birefringence and azimuth angles, following polarized UV microbeam irradiation (which selectively abolishes the birefringence contribution by those bases that absorbed the UV), reveal the helical packing arrangement of the DNA molecules within the microdomains (Inoue and Sato, l966b). The rectified polarizing microscope offers many unique opportunities for studying molecular organization, and its changes, in a single living cell undergoing physiological activities and devel• opmental changes. 500 APPENDIX Ill
ANNOTATED BIBLIOGRAPHY*
PHYSICAL OPTICS General introductory texts 23, 119, 223, 264 Electromagnetic waves 9, 23, 43, 253 Polarized light 84, 120, 121, 178, 182, 208, 216a, 217, 246, 263, 264 Crystal optics 27, 52, 79, 80, 144, 178, 251, 263 MICROSCOPY Wave optics and diffraction 66, 104, 129, 147, 152a, 218, 266 Practices of microscopy 16, 33, 78, 152a, 216 UV microbeam techniques 58, 219, 247 POLARIZING MICROSCOPE General 21, 76, 79, 122, 175, 183, 208, 265 Polarizers 75, 122, 136, 137, 138, 142, 216a, 256 Compensators, general4, 41, 70, 82, 84,120, 121, 127, 175, 182, 183, 193, 216a, 217, 227,235,246 Compensators, special application, etc. 3, 4, 18, 24, 45, 83, 87, 100, 103, 199, 227 Limits and refinements 4, 5, 90, 93, 100, 103, 108, 109, 227, 265; see also Section 11.3 Rectification 86, 93, 101, 104, 129 MOLECULAR STRUCTURE AND PHYSICAL PROPERTIES General47, 79, 118, 249 Orbital electrons and optical properties 177, 213 Polymer structure and physical properties, 71, 128, 130, 146, 181, 245, 255 Liquid crystals 22, 39, 74 Birefringence, intrinsic and general 31, 33, 79, 202, 248 Flow 14, 32, 125, 229, 229a, 240, 250 Form 6, 17, 25, 32, 161, 195, 199, 241 (especially see footnote on p. 417 for correction of error), 258, 259 Stress/strain 6, 26, 27, 128, 130, 202, 255 Electric 125, 126, 229a Dichroism, visible 8, 77a, 138, 144, 166, 169, 185, 206, 247a, 256 Infrared and ultraviolet 7, 81, 107, 108, 109, 140, 142, 166, 168, 185, 214, 215, 224 Form and flow 69, 81, 268 Optical Rotation 257 BIOLOGICAL FINE STRUCTURE AND MoLECULAR ORGANIZATION Generaltextsandarticles2,6,45a,53,68, 72, 72a, 79, 98, Ill, 152,164,165,171,181, 184a, 185,202, 208, 210, 220, 243a, 248, 26la, 262 Biocrystals 105, 162, 163, 201, 209 MITOSIS General review on mitosis 15, 59, 97, 148, 149, 150, 160, 176a, 212, 261 Spindle birefringence and chromosome movement 19a, 19b, 42, 56, 57, 60, 83, 85, 91, 92 (time-lapse movie), 94, 95, 99, 100,106,110, 115, 116,124,131, 132,133,134,145,174,184,186, 196,202, 203, 225, 226, 244, 254 Experimental alterations of spindle birefringence Temperature 30, 89, 94, 95, %, 113,114, 191, 197,221, 222 Pressure 62, 114, 187, 188, 189, 190, 192 Mechanical deformation 19a, 19b, 48, 88, 100, 110, 160, 160a, 170 UV microbeam 29, 56, 57, 58, 94, 95, 116b, 116c, 138a, 138b, 247, 266a Ca2+ 102, 123a D20, etc. 28, 30, 110, 116a, 180, 199a Colchicine, etc. 13, 83a, 88, 143, 194, 217a, 217b, 228, 239 Antimetabolites 110, 200 Isolated spindle 28, 29a, 61, 63, 64, 73, 112, 148a, 179, 180a, 195, 199, 254 Form birefringence 174, 179, 195, 199 BIREFRINGENCE AND DICHROISM IN LIVING CELLS General review 50, 68, 166, 202, 205, 207, 209, 210 Ground cytoplasm and endoplasmic reticulum I, 2, 65, 123, 157, 158, 159, 171, 202, 218a, 233, 234, 237, 238 Cell membrane and cortex 17, 40, 153, 154, 155, 157, 158, 202, 207 209, 211, 218a, 228
*Italicized reference numbers indicate material that may be of special interest to the reader. INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 501
Nerve and receptor membranes, change with activity 19, 34, 35, 36, 37, 77, 77a, 117, 206, 211, 230 Axoplasm 139, 202, 242 Mitochondria, golgi, chloroplasts 151, 156, 202 Nuclear envelope 202, 207 Chromosomes 67, 109, 172, 173, 202, 204 Sperm head 20, 107, 108, 109, 167, 198, 203, 260, 267 DNA 108, 109, 141, 202, 214, 215 Mitotic spindle: See Mitosis Cilia, flagella, axostyle 53, 97a, 135, 202, Axopod 205, 243 Hemoglobin 169 Amyloid 44, 236, 26la Muscle Birefringence and contraction 12, 24, 38, 46, 49, 50, 51, 54, 55, 161, 202, 219, 231, 232, 252 Developmental and other changes 10, 11, 202 UV microbeam 219 Myosin, actin 14, 32, 81, 125, 165, 250, 252 Insect scale in development 176
BIBLIOGRAPHY
l. Allen, R. D. (1972) Pattern of birefringence in the giant amoeba, Chaos carolinensis. Exp. Cell Res. 72:34-45. 2. Allen, R. D., and N. Karniya, eds. (1964) Primitive Motile Systems in Cell Biology. Academic Press, New York. 3. Allen, R. D., and L. I. Rebhun (1962) Photoelectric measurement of small fluctuating retardations in weakly birefringent, light-scattering biological objects. I. The revolving tilted compensator method. Exp. Cell Res. 29:583-592. 4. Allen, R. D., I. Brault, and R. D. Moore (1963) A new method of polarizing microscopic analysis. I. Scanning with a birefringence detector system. J. Cell Bioi. 18:223-235. 5. Allen, R. D., J. W. Brault, and R. Zeh (1966) Image contrast and phase modulation light methods in polarization and interference microscopy. Adv. Opt. Electron Microsc. 1:77-114. 6. Ambronn, H., and A. Frey, (1926) Das Polarisationsmikroskop, seine Anwedung in der Kolloid• forschung in der Farbarei. Akademische Verlag, Leipzig. 7. Ambrose, J., A. Elliot, and R. B. Temple (1949) New evidence on the structure of some proteins from measurements with polarized infra-red radiation. Nature 163:859-862. 8. Anderson, S. (1949) Orientation of methylene blue molecules absorbed on solids. J. Opt. Soc. Am. 39:49-56. 9. Andrews, C. L. (1960) Optics of the Electromagnetic Spectrum. Prentice-Hall, Ep.glewood Cliffs, N.J. 10. Aronson, J. F. (1961) Sarcomere size in developing muscles of a tarsonemid mite. J. Biophys. Biochem. Cytol. 11:147-156. II. Aronson, J. F. (l%3) Observations on the variations in size of the A regions of arthropod muscle. J. Cell Bioi. 19:359-367. 12. Aronson, J. F. (l%7) Polarized light observations on striated muscle contraction in a mite. J. Cell Bioi. 32:169-179. 13. Aronson, J., and S. Inoue (1970) Reversal by light of the action of N-methyl N-desacetyl colchicine on mitosis. J. Cell Bioi. 45:470-477. 14. Asakura, S., M. Kasai, and F. Oosawa (1960) The effect of temperature on the equilibrium state of actin solutions. J. Poly. Sci. 44:35-49. 15. Bajer, A., and J. Mole-Bajer (1972) Spindle dynamics and chromosome movements.lntntl. Rev. Cytol. suppl. 3: 1-271. 16. Barer, R., and V. E. Cosslett, eds. (1966) Advances in Optical and Electron Microscopy, Volume l. Academic Press, New York, pp. 77-114. 17. Bear, R. S., and F. 0. Schmitt (1936) Optical properties of the axon sheaths of crustacean nerves. J. Cell Physiol. 9:275-288. 18. Bear, R. S., and F. 0. Schmitt (1936). The measurement of small retardation with the polarizing microscope. J. Opt. Soc. Am. 26:363-364. 19. Bear, R. S., F. 0. Schmitt, and J. Z. Young (1937) The sheath components of the giant nerve fibres of the squid. Proc. R. Soc. London 123:496. 502 APPENDIX Ill
19a. Begg, D. A., and G. W. Ellis (1979) Micromanipulation studies of chromosome movement. I. Chromo• some-spindle attachment and the mechanical properties of chromosomal spindle fibers. J. Cell Bioi. 82:528-541. 19b. Begg, D. A., and G. W. Ellis (1979) Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle. J. Cell Bioi. 82:542-554. 20. Bendet, I. J., and J. Bearden, Jr. (1972) Birefringence of spermatozoa. II. Form birefringence of bull sperm. J. Cell Bioi. 55:501-510. 21. Bennett, H. S. (1950) The microscopical investigation of biological materials with polarized light. In: Handbook of Microscopical Technique (C. E. McClung, ed.). Harper & Row (Hoeber), New York, pp. 591-677. 22. Bernal, J.D., and I. Fankuchen (1941) X-ray and crystallographic studies of plant virus preparations. I. Introduction and preparation of specimens. II. Modes of aggregation of the virus particles. J. Gen. Physiol. 25:111-146 and 4 plates. 23. Born, M., and E. Wolf (1980) Principles of Optics, 6th ed. Pergamon Press, Elmsford, N.Y. 24. Bozler, E. (1937) The birefringence of muscle and its variation during contraction. J. Cell. Comp. Physiol. 10:165-182. 25. Bragg, W. L., and A. B. Pippard (1953) The form birefringence of macromolecules. Acta Crystallogr. Sect. B 6:865-867. 26. Brewster, D. (1916) On new properties of heat, as exhibited in its propagation along plates of glass. Philos. Trans. 106:46-114 and Plates II-V. 27. Brewster, D. (1818) On the laws of polarization and double refraction in regularly crystallized bodies. Philos. Trans. 108:199-273 and Plates XV-XVI. 28. Bryan, J., and H. Sato ( 1970) The isolation of the meiosis I spindle from the mature oocyte of Pisaster ochraceus. Exp. Cell Res. 59:371-378. 29. Campbell, R. D., and S. Inoue (1965) Reoorganization of spindle components following UV micro• irradiation. Bioi. Bull. 129:401. 29a. Cande, W. Z., and K. L. McDonald (1985) In vitro reactivation of anaphase spindle elongation using isolated diatom spindles. Nature 316:168-170. 30. Carolan, R. M., H. Sato, and S. Inoue (1965) A thermodynamic analysis of the effect of D20 and H20 on the mitotic spindle. Bioi. Bull. 129:402; 131:385. 31. Cassim, J. Y., and E. W. Taylor (1965) Intrinsic birefringence of poly--y-benzyl-L-glutamate, a helical polypeptide, and the theory of birefringence. Biophys. J. 5:531-551. 32. Cassim, J. Y., P. S. Tobias, and E. W. Taylor (1968) Birefringence of muscle proteins and the problem of structural birefringence. Biochim. Biophys. Acta 168:463-471. 33. Chamot, E. M., and C. W. Mason (1958) Handbook of Chemical Microscopy, 2nd ed. Wiley, New York. 34. Chinn, P., and F. 0. Schmitt (1937) On the birefringence of nerve sheaths as studied in cross section. J. Cell. Comp. Physiol. 9:289-296. 35. Cohen, L. B., B. Hille, and R. D. Keynes (1969) Light scattering and birefringence changes during activity in the electric organ of Electrophorus electricus. J. Physiol. (London) 203:489-509. 36. Cohen, L. B., and R. D. Keynes (1969) Optical changes in the voltage-clamped squid axon. J. Physiol. (London) 204:100-101. 37. Cohen, L. B., R. D. Keynes, and B. Hille (1968) Light scattering and birefringence changes during nerve activity. Nature 218:438-441. 38. Colby, R. H. (1971) Intrinsic birefringence of glycerinated myofibrils. J. Cell Bioi. 51:763-771. 39. Configurations and Interactions of Macro-Molecules and Liquid Crystals (1958) Discuss. Faraday Soc. 25:1-235. 40. Dan, K., and K. Okazaki (1951) Change in the birefringence of the cortical layer of sea-urchin eggs • induced by stretching. J. Cell. Comp. Physiol. 38:427-435. 41. deSenarmont, H. (1840) Surles modifications que Ia reflexion speculaire a Ia surface des corps metalli• ques imprime a un rayon de lumiere polarisee. Ann. Chim. Phys. 73(Ser. 2):337-362. 42. Dietz, R. (1963) Polarisationsmikroskopische Befunde zur chromosomeninduzierten Spindelbildung bei der Tipulide. Zoo/. Anz. Suppl. 26:131-138. 43. Ditchburn, R. W. (1963) Light. Blaclde, London. 44. Dreizel, P. B., and A. Pfleidener (1959) Histochemische und Polarisationsoptische untersuchungen am Amyloid. Arch. Pathol. Anat. Pathol. Physiol. 332:552. 45. Dvorak, J. A., T. R. Clem, and W. F. Stotler (1972) The design and construction of a computer• compatible system to measure and record optical retardation with a polarizing or interference microscope. J. Microsc. 96:109. INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 503
45a. Dustin, P. (1978) Microtubules. Springer-Verlag, New York. 46. Eberstein, A., and A. Rosenfalck (1963) Birefringence of isolated muscle fibres in twitch and tetanus. Acta Physiol. scand. 57:144-166. 47. Eisenberg, D., and W. Kauzmann (1969) The Structure and Properties of Water. Oxford University Press (Clarendon), London. 48. Ellis, G. W., and D. A. Begg (1981) Chromosome micromanipulation studies. In: Mitosis/Cytokinesis. (A. M. Zimmerman and A. Forer eds.). Academic Press, New York, pp. 155-179. 49. Engelmann, T. W. (1873) Mikroskopische Untersuchungen iiber die quergestreifte Muskelsubstanz. I, II. Pfluegers Arch. 7:33-71, 155-188. 50. Engelmann, T. W. (1875) Contractilitat and Doppelbrechung. Pfluegers Arch. 11:432-464. 51. Engelmann, T. W. (1906) Zur Theorie der Contractilitat. Sitzungsber. K. Preuss. Akad. Wiss. 39:694- 724. 52. Evans, R. C. (1976) An Introduction to Crystal Chemistry. Cambridge University Press, London. 53. Faure-Fremiet, E. (1970) Microtubules et Mecanmismes Morphopoietiques. Ann. Bioi. 9:1-61. 54. Fischer, E. (1936) The submicroscopic structure of muscle and its changes during contraction and stretch. Cold Spring Harbor Symp. Quant. Bioi. 4:214-221. 55. Fischer, E. (1938) The birefringence of smooth muscle (Phascolosoma and Thyone) as related to muscle length, tension and tone. J. Cell. Camp. Physiol. 12:85-101. 56. Forer, A. (1965) .Local reduction in spindle fiber birefringence in living Nephrotoma suturalis (Loew) spermatocytes induced by ultra-violet microbeam irradiation. J. Cell Bioi. 25(Mitosis Suppl.):95-117. 57. Forer, A. (1966) Characterization of the mitotic traction system, and evidence that birefringent spindle fibers neither produce nor transmit force for chromosome movement. Chromosoma 19:44-98. 58. Forer, A. ( 1966) A simple conversion of reflecting lenses into phase-contrast condensers for ultraviolet light irradiations. Exp. Cell Res. 43:688-691. 59. Forer, A. (1969) Chromosome movements during cell division. In: Handbook ofMolecular Cytology (A. Lima-de Faria, ed.). North-Holland, Amsterdam, pp. 553-601. 60. Forer, A. (1976) Actin filaments and birefringent spindle fibers during chromosome movement. In: Cell Motility, Volume 3 (R. D. Goldman, T. D. Pollard, and J. L. Rosenbaum, eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 1273-1293. 61. Forer, A., and R. D. Goldman (1972) The concentrations of dry matter in mitotic apparatuses in vivo and after isolation from sea-urchin zygotes. J. Cell Sci. 10:387-418. 62. Forer, A., and A. M. Zimmerman (1976) Spindle birefringence of isolated mitotic apparatus analysed by pressure treatment. J. Cell Sci. 20:309-327. 63. Forer, A., and A. M. Zimmerman (1976) Spindle birefringence of isolated mitotic apparatus analyzed by treatments with cold, pressure, and diluted isolation medium. J. Cell Sci. 20:329-339. 64. Forer, A., V. I. Kalnins, and A. M. Zimmerman (1976) Spindle birefringence and isolated mitotic apparatus: Further evidence for two birefringent spindle components. J. Cell Sci. 22:115-131. 65. Francis, D. W., and R. D. Allen (1971) Induced birefringence as evidence of endoplasmic viscoelasticity in Chaos carolinensis. J. Mechanochem. Cell Motil. 1:1-6. 66. Franc 77a. Harosi, F. (1981) Microspectrophotometry and optical phenomena: Birefringence, dichroism, and anom• alous dispersion. In: Springer Series in Optical Sciences, Volume 23 (J. M. Enoch and F. L. Tobey, Jr., eds.). Springer-Verlag, Berlin, pp. 337-399. 78. Hart, R., R. Zeh, and R. D. Allen (1977) Phase-randomized laser illumination for microscopy. J. Cell Sci. 23:335-343. 79. Hartshorne, N. H., and A. Stuart (1960) Crystals and the Polarising Microscope: A Handbook for Chemists and Others, 3rd ed. Arnold, London. 80. Hartshorne, N. H., and A. Stuart (1964) Practical Optical Crystallography. American Elsevier, New York. 81. Higashi, S., M. Kasai, F. Oosawa, and A. Wada (1963) Ultraviolet dichroism ofF-actin oriented by flow. J. Mol. Bioi. 7:421-430. 82. Hiramoto, Y., Y. Hamaguchi, Y. Shoji, and S. Shimoda (1981) Quantitative studies on the polarization optical properties of living cells. I. Microphotometric birefringence detection system. J. Cell Bioi. 89:115-120. 83. Hiramoto, Y., Y. Hamaguchi, Y. Shoji, T. E. Schroeder, S. Shimoda, and S. Nakamura (1981) Quantitative studies on the polarization optical properties of living cells. II. The role of microtubules in birefringence of the spindle of the sea urchin egg. J. Cell Bioi. 89:121-130. 83a. Hiramoto, Y., andY. Shoji (1982) Location of the motive force for chromosome movement in sand• dollar eggs. In: Biological Functions ofMicrotubules and Related Structures (H. Sakai, H. Mohri, and G. G. Borisy, eds.). Academic Press, New York, pp. 247-249. 84. Hsien Y. H., M. Richartz, and K. L. Yung (1947) A generalized intensity formula for a system of retardation plates. J. Opt. Soc. Am. 37:99-106. 85. Hughes, A. F., and M. M. Swann (1948) Anaphase movements in the living cell: A study with phase contrast and polarized light on chick tissue culture. J. Exp. Bioi. 25:45-70. 86. Huxley, A. F. (1960) British patent specification 856,621. Improvements in or relating to polarizing microscopes (applied July 20, 1956). 87. Inoue, S. (1951) A method for measuring small retardations of structures in living cells. Exp. Cell Res. 2:513-517. 88. Inoue, S. (1952) The effect of colchicine on the microscopic and sub-microscopic structure of the mitotic spindle. Exp. Cell Res. Suppl. 2:305-318. 89. Inoue, S. (1952) Effect of temperature on the birefringence of the mitotic spindle. Bioi. Bull. 103:316. 90. Inoue, S. (1952) Studies on depolarization of light at microscope lens surfaces. I. The origin of stray light by rotation at the lens surfaces. Exp. Cell Res. 3:199-208. 91. Inoue, S. (1953) Polarization optical studies of the mitotic spindle. I. The demonstration of spindle fibers in living cells. Chromosoma 5:487-500. 92. Inoue, S. (1960) Birefringence in Dividing Cells. Time-lapse motion picture. 93. Inoue, S. (1961) Polarizing microscope: Design for maximum sensitivity. In: The Encyclopedia of Microscopy (G. L. Clarke, ed.). Reinhold, New York, pp. 480-485. 94. Inoue, S. (1964) Organization and function of the mitotic spindle. In: Primitive Motile Systems in Cell Biology (R. D. Allen and N. Kamiya, eds.). Academic Press, New York, pp. 549-598. 95. Inoue, S. (1969) The physics of structural organization in living cells. In: Biology and the Physical Sciences (S. Devons, ed.). Columbia University Press, New York, pp. 139-171. 96. Inoue, S. (1976) Chromosome movement by reversible assembly of microtubules. In: Cell Motility, Volume 3 (R. D. Goldman, T. D. Pollard, and J. L. Rosenbaum, eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 1317-1328. 97. Inoue, S. (1981) Cell division and the mitotic spindle. J. Cell Bioi. 91(Part 2):131s-147s. 97a. Inoue, S. (1981) Video image processing greatly enhances contrast, quality, and speed in polarization• based microscopy. J. Cell Bioi. 89:346-356. 98. Inoue, S. (1982) The role of self-assembly in the generation of biologic form. In: Developmental Order: Its Origin and Regulation (P. B. Green, ed.). Liss, New York, pp. 35-76. 99. Inoue, S., and A. Bajer (1961) Birefringence in endosperm mitosis. Chromosoma 12:48-63. 100. Inoue, S., and K. Dan (1951) Birefringence of the dividing cell. J. Morpho/. 89:423-456. 101. Inoue, S., and W. L. Hyde (1957) Studies on depolarization of light at microscope lens surfaces. II. The simultaneous realization of high resolution and high sensitivity with the polarizing microscope. J. Biophys. Biochem. Cytol. 3:831-838. 102. Inoue, S., and D. P. Kiehart (1978) In vivo analysis of mitotic spindle dynamics. In: Cell Reproduction: In Honor of Daniel Mazia (E. Dirksen, D. Prescott, and C. F. Fox, eds.). Academic Press, New York, pp. 433-444. 103. Inoue, S., and C. Koester (1959) Optimum half shade angle in polarizing instruments. J. Opt. Soc. Am. 49:556-559. INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 505 104. Inoue, S., and H. Kubota (1958) Diffraction anomaly in polarizing microscopes. Nature 182:1725- 1726. 105. Inoue, S., and K. Okazaki (1978) Biocrystals. Sci. Am. 236(4):82-92. 106. Inoue, S., and H. Ritter, Jr. (1975) Dynamics of mitotic spindle organization and function. In: Molecules and Cell Movement (S. Inoue and R. E. Stephens, eds.). Raven Press, New York, pp. 3-30. 107. Inoue, S., and H. Sato (1962) Arrangement of DNA in living sperm: A biophysical analysis. Science 136:1122-1124. 108. Inoue, S., and H. Sato (1966) Arrangement of DNA molecules in the sperm nucleus: An optical approach to the analysis of biological fine structure. In: Biophysical Science Series 3, Progress in Genetics III (Japanese Biophysical Society ed.). Yoshioka, Kyoto, pp. 151-220. 109. Inoue, S., and H. Sato (1966) Deoxyribonucleic acid arrangements in living sperm. In: Molecular Architecture in Cell Physiology (T. Hayashi and A. G. Szent-Gyorgyi, eds.). Prentice-Hall, Englewood Cliffs, N.J., pp. 209-248. 110. Inoue, S., and H. Sato (1967) Cell motility by labile association of molecules: The nature of mitotic spindle fibers and their role in chromosome movement. J. Gen. Physiol. 50:259-292. Ill. Inoue, S., and L. G. Tilney (1982) The acrosomal reaction of Thyone sperm. I. Changes in the sperm head visualized by high resolution video microscopy. J. Cell Bioi. 93:812-819. 112. Inoue, S., G. G. Borisy, and D.P. Kiehart (1974) Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin. J. Cell Bioi. 62:175-184. 113. Inoue, S., G. W. Ellis, E. D. Salmon, and J. W. Fuseler (1970) Rapid measurement of spindle birefringence during controlled temperature shifts. J. Cell Bioi. 47:95a. 114. Inoue, S., J. Fuseler, E. D. Salmon, and G. W. Ellis (1975) Functional organization of mitotic micro• tubules-Physical chemistry of the in vivo equilibrium system. Biophys. J. 15:725-744. 115. Inoue, S., D. P. Kiehart, I. Mabuchi, and G. W. Ellis (1979) Molecular mechanism of mitotic chromo• some movement. In: Motility in Cell Function, First John M. Marshall Symposium in Cell Biology (F. A. Pepe, J. W. Sanger, and V. T. Nachmias, eds.). Academic Press, New York, pp. 301-311. 116. Inoue, S., H. Ritter and D. Kubai (1978) Mitosis in Barbulanympha. II. Dynamics of a two-stage anaphase, nuclear morphogenesis, and cytokinesis. J. Cell Bioi. 77:655-684. 116a. Itoh, T. J., and H. Sato (1984) The effects of deuterium oxide (2H20) on the polymerization of tubulin in vitro. Biochim. Biophys. Acta 800:21-27. ll6b. Izutsu, K. (1961) Effects of ultraviolet microbeam irradiation upon division in grasshopper sper• matocytes. I. Results of irradiation during prophase and prometaphase I. Mie Med. J. 11(2):199-212. ll6c. Izutsu, K. (1961) Effects of ultraviolet microbeam irradiation upon division in grasshopper sper• matocytes. II. Results of irradiation during metaphase and anaphase I. Mie Med. J. 11(2):213-232. 117. Jagger, W. S., and P. A. Liebman (1970) Birefringent transients in photoreceptor outer segments. Biophys. Soc. Abstr. 14th Annu. Meet. p. 59 A. 118. Jelley, E. E. (1945) Microscopy. In: Physical Methods of Organic Chemistry (A. Weissberg, ed.). Interscience, New York, pp. 451-524. 119. Jenkins, F. A., and H. White (1957) Fundamentals of Optics, 3rd ed. McGraw-Hill, New York. 120. Jerrard, H. G. (1948) Optical compensators for measurement of elliptical polarization. J. Opt. Soc. Am. 38:35-59. 121. Jerrard, H. G. (1954) Transmission oflight through birefringent and optically active media: The Poincare sphere. J. Opt. Soc. Am. 44:634-640. 122. Johansen, A. (1918) Manual of Petrographic Methods, 2nd ed. McGraw-Hill, New York. 123. Kautz, J., Q. B. De Marsh, and W. Thornburg (1957) A polarizing and electron microscope study of plasma cells. Exp. Cell Res. 13:596-599. 123a. Kiehart, D. P. ( 1981) Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system. J. Cell Bioi. 88:604-617. 124. Kiehart, D. P., I. Mabuchi, and S. Inoue (1982) Evidence that myosin does not contribute or force production in chomosome movement. J. Cell Bioi. 94:165-178. 125. Kobayasi, S. (1964) Effect of electric field on F-actin orientated by flow. Biochim. Biophys. Acta 88:541-552. 126. Kobayasi, S., H. Asai, and F. Oosawa (1964) Electric birefringence of actin. Biochim. Biophys. Acta 88:528-540. 127. Kohler, A. (1921) Ein Glimmerplattchen Grau. I. Ordnung zur Untersuchung sehr schwach dop• pelbrechender Praparate. Z. Wiss. Mikrosk. 38:29-42. 128. Kubo, R. (1947) Statistical theory of rubber-like substances. J. Colloid Sci. 2:527-535. 129. Kubota, H., and S. Inoue (1959) Diffraction images in the polarizing microscope. J. Opt. Soc. Am. 49:191-198. 506 APPENDIX Ill 130. Kunitz, M. (1930) Elasticity, double refraction and swelling of isoelectric gelatin. J. Gen. Physiol. 13:565-606. 131. LaFountain, J. R. (1972) Changes in the patterns of birefringence and filament deployment in the meiotic spindle of Neophrotoma suturalis during the first meiotic division. Protoplasma 75:1-17. 132. LaFountain, J. R. (1974) Birefringence and fine structure of spindles in spermatocytes of Nephrotoma suturalis at metaphase of first meiotic division. J. Ultrastruct. Res. 46:268-278. 133. LaFountain, J. R. (1976) Analysis of birefringence and ultrastructure of spindles in primary sper• matocytes of Nephrotoma suturalis during anaphase. J. Ultrastruct. Res. 54:333-346. 134. LaFountain, J. R., and L.A. Davidson (1979) An analysis of spindle ultrastructure during prometaphase and metaphase of micronuclear division of Tetrahymena. Chromosoma 75:293-308. 135. Langford, G. M., and S. Inoue (1979) Motility of the microtubular axostyle in Pyrsonympha. J. Cell Bioi. 80:521-538. 136. Lambrecht, K. See catalog "Polarizing Optics," 4204 N. Lincoln Ave., Chicago, Ill. 60618. 137. Land, E. H. (1951) Some aspects of the development of sheet polarizers. J. Opt. Soc. Am. 41:957-963. 138. Land, E. H., and C. D. West (1946) Dichroism and dichroic polarizers. In: Colloid Chemistry, Vol. 6 (1. Alexander, ed.). Reinhold, New York, pp. 160-190. 138a. Leslie, R. J., and J. D. Pickett-Heaps (1983) Ultraviolet microbeam irradiations of mitotic diatoms: Investigation of spindle elongation. J. Cell Bioi. 96:548-561. 138b. Leslie, R. J., and J.D. Pickett-Heaps (1984) Spindle microtubule dynamics following ultraviolet micro• beam irradiations of mitotic diatom. Cell36:717-727. 139. Luthy, H. (1946) Dichroismus der einzelnen Nervenfaser. Helv. Physiol. Pharmacol. Acta 4:50. 140 Macinnes, J. W., and R. B. Uretz (1967) Thermal depolarization of fluorescence from polytene chromo• somes stained with acridine orange. J. Cell Bioi. 33:597-604. 141. Maestre, M. F., and R. Kilkson (1965) Intrinsic birefringence of multiple coiled DNA, theory and applications. Biophys. J. 5:275-287. 142. Makas, A. S. (1962) Film polarizer for visible and ultraviolet radiation. J. Opt. Soc. Am. 52:43-44. 143. Malawista, S. E., K. G. Bensch, and H. Sato (1968) Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle. Science 160:770-772. 144. Mandarino, J. A. (1959) Absorption and pleochroism: Two much-neglected optical properties of crystals. Am. Mineral. 4:65-76. 145. Marek, L. F. (1978) Control of spindle form and function in grasshopper spermatocytes. Chromosoma 68:367-398. 146. Mark, H. F. (1957) Giant molecules. Sci. Am. 197(Sept.):80-89. 147. Martin, L. C. ( 1966) The Theory of the Microscope. Blackie, London. 148. Mazia, D. (1961) Mitosis and the physiology of cell division. In: The Cell (J. Brachet and A. Mirsky, eds.). Academic Press, New York, pp. 77-394. 148a. Mazia, D., and K. Dan (1952) The isolation and biochemical characterization of the mitotic apparatus of dividing cells. Proc. Nat/. Acad. Sci. USA 38:826-838. 149. Mcintosh, J. R., W. Z. Cande, and J. A. Snyder (1975) Structure and physiology of the mammalian mitotic spindle. In: Molecules and Cell Movement (S. Inoue and R. E. Stephens, Raven Press, New York, pp. 31-76. 150. Mcintosh, J. R., P. K. Hepler, and D. G. van Wie (1969) Model for mitosis. Nature 224:659-663. 151. Menke, W. (1943) Dichroismus and Doppelbrechung der Plastiden. Bioi. Zentralbl. 63:326-349. 152. Mercer, E. H. (1952) The biosynthesis of fibers. Sci. Mon. 75:280-287. 152a. Michael, K. (1981) Die Grundziige der Theorie des Mikroskops in elementarer Darstellung. Wissenschaftliche Verlag, Stuttgart. 153. Mitchison, J. M. (1952) Cell membranes and cell division: The structure of the cell membrane. Symp. Soc. Exp. Bioi. 6:105-127. 154. Mitchison, J. M. (1953) A polarized light analysis of the human red cell ghost. J. Exp. Bioi. 30:397- 432. 155. Mitchison, J. M., and M. M. Swann (1952) Optical changes in the membranes of the sea-urchin egg at fertilization, mitosis and cleavage. J. Exp. Bioi. 29:357-362. 156. Monne, L. (1939) Polarizationoptische Untersuchungen tiber den Golgi-Apparat und die Mitochondrien miinnlichen Geschlechtszellen eineger Pulmonaten Arten. Protoplasma 32:184. 157. Monne, L. (1944) Cytoplasmic structure and cleavage pattern of the sea urchin egg. Ark. Zool. 35A:l- 27. 158. Monne, L. (1945) Investigations into the structure of the cytoplasm. Ark. Zool. 36A:l-29. 159. Nakajima, H. (1964) The mechanochemical system behind streaming in Physarum. In: Primitive Motile Systems in Cell Biology (R. D. Allen and N. Kamiya, eds.). Academic Press, New York, pp. 111-123. 160. Nicklas, R. B. (1970) Mitosis. In: Advances in Cell Biology, Volume// (D. M. Prescott, L. Goldstein, INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 507 and E. H. McConkey, eds.). Appleton-Century-Crofts, New York, pp. 225-297. 160a. Nicklas, R. B. (1983) Measurements of the force produced by the mitotic spindle in anaphase. J. Cell Bioi. 97:542-548. 161. Noll, D., and H. H. Weber (1935) Polarisationsoptik und molekularer Feinbau der Z-Abschnitte des Froschmuskels. Pfluegers Arch. 235:234-246. 162. Okazaki, K., and S. Inoue (1976) Crystal property of the larval sea urchin spicule. Dev. Growth Differ. 18:413-434. 163. Okazaki, K., K. McDonald, and S. Inoue (1980) Sea urchin larval spicule observed with the scanning electron microscope. In: The Mechanisms ofBiomineralization in Animals and Plants (M. Omori and N. Watanabe, eds.). Tokai University Press, Tokyo, pp. 159-168. 164. Oncley, J. L. (1959) Biophysical sciences-A study program. Rev. Mod. Phys. 31:1-568. 165. Oosawa, F., and S. Asakura (1975) Thermodynamics· of the Polymerization of Protein. Academic Press, New York. 166. Oster, G. (1955) Birefringence and dichroism. In: Physical Techniques in Biological Research, Volume I (G. Oster and A. Pollister, eds.) Academic Press, New York, pp. 439-460. 167. Pattri, H. 0. E. (1932) Uber die Doppelbrechung der Spermien. Z. Zellforsch. mikrosk. Anat. 16:723- 744. 168. Perutz, M. F., M. Jobe, and R. Barer (1950) Observations of proteins in polarized ultra-violet light. Discuss. Faraday Soc. 9:423-427. 169. Perutz, M. F., and J. M. Mitchison (1950) State of haemoglobin in sickle cell anaemia. Nature 166:677- 682. 170. Pfeiffer, H. H. (1938) Double refraction measurements and structural changes in mitotic spindles dis• turbed by centrifugal force. Biodynamica 2:1-8. 171. Pfeiffer, H. H. (1941) Experimentalle Beitrage zur subrnikroskopischen Feinbaukunde (Leptonik) un• differenzierten Cytoplasmas. Ber. Dtsch. Bot. Ges. 59:288. 172. Pfeiffer, H. H. (1941) Mikrurgisch-polarisationsoptische Beitrage zur submikroskopischen Morphologie larvaler Speicheldrusenchromosomen von Chironomus. Chromosoma 2:77-85. 173. Pfeiffer, H. H. (1942) Polarisationsmikroskopische Dehnungs und Kontraktionsversuche an Chro• matinabschnitten von Chara-Spermatozoiden. Chromosoma 2:334-344. 174. Pfeiffer, H. H. (1951) Polarisationsoptische Untersuchungen am spindellapparat mitotischen Zellen. Cytologia 16:194-200. 175. Pfeiffer, H. H. (1949) Das Polarisationsmikroskop als Messinstrument in Biologie und Medizin. Vieweg, Braunschwieg. 176. Picken, L. (1960) The Organization ofCells and Other Organisms. Oxford University Press (Clarendon), London. 176a. Pickett-Heaps, J. D., and D. H. Tippit (1978) The diatom spindle in perspective. Cell14:455-467. 177. Platt, J. R., ed. (1964) Systematics of the Electronic Spectra of Conjugated Molecules. Wiley, New York. 178. Ramachandran, G. N., and S. Ramaseshan (1961) Crystal optics. In: Handbuch der Physik, Volume XXV/I (S. Flugge, ed.). Springer-Verlag, Berlin, pp. 1-217. 179. Rebhun, L. I., and G. Sander (1967) Ultrastructure and birefringence of the isolated mitotic apparatus of marine eggs. J. Cell Bioi. 34:859-883. 180. Rebhun, L. 1., and N. Sawada (1969) Augmentation and dispersion of the in vivo mitotic apparatus of living marine eggs. Protoplasma 68:1-22. 180a. Rebhun, L. 1., J. Rosenbaum, P. Lefebvre, and G. Smith (1974) Reversible restoration of the birefrin• gence of cold-treated, isolated mitotic apparatus of surf clam eggs with chick brain tubulin. Nature 249:113-115. 181. Rees, A. L. G. (1951) Directed aggregation in colloidal systems and the formation of protein fibers. J. Phys. Chern. 55:1340-1344. 182. Richartz, M., and Y. H. Hsu (1948) Analysis of elliptical polarization. J. Opt. Soc. Am. 39:136- 157. 183. Rinne, F. W. B., and M. Berek (1953) Anleitung zu optischen Untersuchungen mit dem Polarization• mikroskop. Stuttgart, Schweizerbart. 184. Ritter, H., S. Inoue, and D. Kubai (1978) Mitosis in Barbulanympha. I. Spindle structure, formation, and kinetochore engagement. J. Cell Bioi. 77:638-654. 184a. Roberts, K., and J. S. Hyams, eds. (1979) Microtubules. Academic Press, New York. 185. Ruch, F. (1956) Birefringence and dichroism of cells and tissues. In: Physical Techniques in Biolog• ical Research, Volume III (G. Oster and A. Pollister, eds.). Academic Press, New York, pp. 149-176. 186. Rimnstrom, J. (1928) Die Veranderungen der Plasmakolloide bei der Entwicklungs-Erregung des Seeigeleies. Protoplasma 4:338-514. 508 APPENDIX Ill 187. Salmon, E. D. (l975a) Pressure-induced depolymerization of spindle microtubules. I. Spindle birefringence and length changes. J. Cell Bioi. 65:603-614. 188. Salmon, E. D. (l97b) Pressure-induced depolymerization of brain microtubules in vitro. Science 189:884-886. 189. Salmon, E. D. (l975c) Spindle microtubules: Thermodynamics of in vivo assembly and role in chromo• some movement. Ann. N.Y. Acad. Sci. 253:383-406. 190. Salmon, E. D. (1984) Tubulin dynamics in microtubules of the mitotic spindle. In: Molecular Biology of the Cytoskeleton (G. Borisy, D. Cleveland, and D. Murphy, eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 99-109. 191. Salmon, E. D., and D. A. Begg (1980) Functional implications of cold-stable microtubules in ki• netochore fibers of insect spermatocytes during anaphase. J. Cell Bioi. 85:853-865. 192. Salmon, E. D., and G. W. Ellis (1975) A new miniature hydrostatic pressure chamber for microscopy: Strain-free optical glass windows facilitate phase contrast and polarized light microscopy of living cells. Optional fixture permits simultaneous control of pressure and temperature. J. Cell Bioi. 65:587-602. 193. Salmon, E. D., and G. W. Ellis (1976) Compensator transducer increases ease, accuracy and rapidity of measuriQg changes in specimen birefringence with polarization microscopy. J. Microsc. 106:63-69. 194. Salmon, E. D., M. McKeel, and T. Hays (1984) Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine. J. Cell Bioi. 99:1066- 1075. 195. Sato, H. (1969) Analysis of form birefringence in the mitotic spindle. Am. Zoo/. 9:592. 196. Sato, H. (1975) The mitotic spindle. In: Aging Gametes (R. Blandau, ed.). Karger Basel, pp. 19-49. 197. Sato, H., and J. Bryan (1968) Kinetic analysis of association-dissociation reaction in the mitotic spindle. J. Cell Bioi. 39:ll8a. 198. Sato, H., and S. Inoue (1964) Condensation of the sperm nucleus and alignment of DNA molecules during spermiogenesis in Loligo pealei. Bioi. Bull. 127:357. 199. Sato, H., G. W. Ellis, and S. Inoue (1975) Microtubular origin of mitotic spindle form birefringence. J. Cell Bioi. 67:501-517. 199a. Sato, H., T. Kato, T. C. Takahashi, and T. Ito (1982) Analysis of D20 effect on in vivo and in vitro tubulin polymerization and depolymerization. In: Biological Functions of Microtubules and Related Structures (H. Sakai, H. Mohri, and G. G. Borisy, eds.). Academic Press, New York. pp. 2ll-226. 200. Sawada, N., and L. I. Rebhun (1969) The effect of dinitrophenol and other phosphorylation uncouplers on the birefringence of the mitotic apparatus of marine eggs. Exp. Cell Res. 55:33-38. 201. Schmidt, W. J. (1924) Die Bausteine des Tierkorpers in po/arisiertem Lichte. Cohen, Bonn. 202. Schmidt, W. J. (1937) Die Doppelbrechung von Karyoplasma, Zytoplasma und Metaplasma. Pro• toplasma-Monographien Volume ll. Borntraeger, Berlin. 203. Schmidt, W. J. (1939) Doppelbrechung der Kernspindel und Zugfasertheorie der Chromosomen• bewegung. Chromosoma 1:253-264. 204. Schmidt, W. J. (1941) Einiges uber Optische Anisotropie und Feinbau von Chromatin and Chromo• somen. Chromosoma 2:86-100. 205. Schmidt, W. J. (1944) Die Doppelbrechung des Protoplasmas und ihrer Bedeutung fur die Erforschung seines submikroskopischen Baues. Ergeb. Physiol. 44:27. 206. Schmidt, W. J. (1951) Polarisationsoptische analyse der Verkniipfung von Protein-und Lipoidmolekeln, erlautert am Aussenglied der Sehzellen der Wirbeltiere. Pub/. Staz. Zoo/. Napoli 23:158-183. 207. Schmidt, W. J. (1952) Polarisationmikroskopische Beobachtungen an Zellmembrane, Kern, Cytoplasma Tentakel der Noctiluca miliaris Suriray. Zoo/. Anz. 148:115-131. 208. Schmidt, W. J. (1957) Instrumente und Methoden zur mikroskopischen Untersuchung optisch anisotroper Materielen mit Ausschluss der Kristalle. In: Handbuch der Mikroskopie in der Technik, Volume I (H. Freund, ed.). Umschau Verlag, Frankfurt am Main, pp. 147-314. 209. Schmidt, W. J. (1964) Wissenschaftliche Veroffentlichungen. Justus Leibig-Universitat, Giessen/Lahn. 210. Schmitt, F. 0. (1955) Cell constitution. In: Analysis ofDevelopment (B. Willier, P. Weiss, and V. Hambur• ger, eds.). Saunders, Philadelphia, pp. 39-69. 211. Schmitt, F. 0., and N. Geschwind (1957) The axon surface. Prog. Biophys. 8:165-215. 212. Schrader, F. (1953) Mitosis-The Movements of Chromosomes in Cell Division. Columbia University Press, New York. 213. Sebera, D. (1964) Electronic Structure and Chemical Bonding. Gina (Blaisdell), Boston. 214. Seeds, W. E. (1953) Polarized ultraviolet microspectrography and molecular structure. Prog. Biophys. 3:27-46. 215. Seeds, W. E., and M. H. F. Wilkins (1959) Ultra-violet microspectrographic studies of nucleoproteins and crystals of biological interest. Biochim. Biophys. Acta 11:417-427. 216. Shillaber, C. P. (1944) Photomicrography in Theory and Practice. Wiley, New York. INTRODUCTION TO BIOLOGICAL POLARIZATION MICROSCOPY 509 216a. Shurcliff, W. A. (1962) Polarized Light: Production and Use. Harvard University Press, Cambridge, Mass. 217. Skinner, C. A. (1925) A universal polarimeter. J. Opt. Soc. Am. 10:491-520. 217a. Sluder, G. (1976) Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs. J. Cell Bioi. 70:75-85. 217b. Sluder, G. (1979) Role of spindle microtubules in the control of cell cycle timing. J. Cell Bioi. 80:674- 691. 218. Smith, L. W. (1960) Diffraction images of disk-shaped particles computed for full Kohler illumination. J. Opt. Soc. Am. 50:369-374. 218a. Soranno, T., and E. Bell (1982) Cytostructural dynamics of spreading and translocating cells. J. Cell Bioi. 95:127-136. 219. Stephens, R. E. (1965) Analysis of muscle contraction by ultraviolet microbeam disruption of sarcomere structure. J. Cell Bioi. 25:129-139. 220. Stephens, R. E. (1969) Factors influencing the polymerization of outer fiber microtubule protein. Q. Rev. Biophys. 1:377-390. 221. Stephens, R. E. (1972) Studies on the development of the sea urchin Strongylocentrotus droebachiensis. II. Regulation of mitotic spindle equilibrium by environmental temperature. Bioi. Bull. 142:145-159. 222. Stephens, R. E. (1973) A thermodynamic analysis of mitotic spindle equilibrium at active metaphase. J. Cell Bioi. 57:133-147. 223. Strong, J. (1958) Concepts of Classical Optics. Freeman, San Francisco. 224. Sutherland, G. B. B. M., and M. Tsuboi (1957) The infra-red spectrum and molecular configuration of sodium deoxyribonucleate. Proc. R. Soc. London Ser. A. 239:446-463. 225. Swann, M. M. (1951) Protoplasmic structure and mitosis. I. The birefringence of the metaphase spindle and asters of the living sea-urchin egg. J. Exp. Bioi. 28:417-433. 226. Swann, M. M. (1951) Protoplasmic structure and mitosis. II. The nature and causes of birefringence changes in the sea-urchin egg at anaphase. J. Exp. Bioi. 28:434-444. 227. Swann, M. M., and J. M. Mitchison (1950) Refinements in polarized light microscopy. J. Exp. Bioi. 27:226-237. 228. Swann, M. M., and J. M. Mitchison (1958) The mechanism of cleavage in animal cells. Bioi. Rev. 33:103-135. 229. Takahashi, W. N., and T. E. Rawlins (1933) Rod-shaped particles in tobacco mosaic virus demonstrated by stream double refraction. Science 77:26-27. 229a. Takashima, S. (1968) Optical anisotropy of synthetic polynucleotides. I. Flow birefringence and 1T• electron polarizability of bases. Biopolymers 6:1437-1452. 230. Tasaki, I., A. Watanabe, R. Sandlin, and L. Carnay (1968) Changes in fluorescence, turbidity and birefringence associated with nerve excitation. Proc. Natl. Acad. Sci. USA 61:883-888. 231. Taylor, D. L. (1975) Birefringence changes in vertebrate striated muscle. J. Supramol. Struct. 3:181-191. 232. Taylor, D. L. (1976) Quantitative studies on the polarization optical properties of striated muscle. I. Birefringence changes of rabbit psoas muscle in the transition from rigor to relaxed state. J. Cell Bioi. 68:497-511. 233. Taylor, D. L. (1976) Motile models of amoeboid movement. In: Cell Motility, Volume 3. (R. Goldman, T. Pollard, and J. Rosenbaum, eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 797-821. 234. Taylor, D. L. (1976) The contractile basis of amoeboid movement. IV. The viscoelasticity and contrac• tility of amoeba cytoplasm in vivo. Exp. Cell Res. 105:413-426. 235. Taylor, D. L., and R. M. Zeh (1976) Methods for the measurement of polarization optical properties. I. Birefringence. J. Microsc. 108:251-259. 236. Taylor, D. L., R. D. Allen, and E. P. Benditt (1975) Determination of the polarization optical properties of the amyloid congo red complex by phase modulation microspectrophotometry. J. Histochem. 22:1105-1112. 237. Taylor, D. L., J. S. Condeelis, P. L. Moore, and R. D. Allen (1973) The contractile basis of amoeboid movement. I. The chemical control of motility in isolated cytoplasm. J. Cell Bioi. 59:378-394. 238. Taylor, D. L., J. A. Rhodes, and S. A. Hammond (1976) The contractile basis of amoeboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts. J. Cell Bioi. 70:123-143. 239. Taylor, E. W. (1965) The mechanism of colchicine inhibition of mitosis. J. Cell Bioi. 25:145-160. 240. Taylor, E. W., and W. Cramer (1963) Birefringence of protein solutions and biological systems. I. Biophys. J. 3:127-141. 241. Thornburg, W. ( 1957) The form birefringence of lamellar systems containing three or more components. J. Biophys. Biochem. Cytol. 3:413-419. 510 APPENDIX Ill 242. Thornburg, W., and E. DeRobertis (1956) Polarization and electron microscope study of frog nerve axoplasm. J. Biophys. Biochem. Cytol. 2:475-482. 243. Tilney, L. G., and K. R. Porter (1965) Studies on microtubules in Heliozoa. I. Protoplasma 60:317- 344. 243a. Timasheff, S. N., and G. D. Fassman, eds. (1971) Subunits in Biological Systems. Marcel Decker, New York. 244. Tippit, D. H., D. Schultz, and J. D. Pickett-Heaps (1978) Analysis of the distribution of spindle microtubules in the diatom Fragilaria. J. Cell Bioi. 76:737-763. 245. Tobolsky, A. V. (1967) Properties and Structures of Polymers. Wiley, New York. 246. Tuckerman, L. B. (1909) Doubly refracting plates and elliptic analysers. Univ. Nebraska Stud. pp. 173- 219. 247. Uretz, R. B., and R. P. Perry (1957) Improved ultraviolet microbeam apparatus. Rev. Sci. Instrum. 28:861-866. 247a. Vidal, B. D. C., M. L. S. Mello and E. R. Pimentel (1982) Polarization microscopy and micro• spectrophotometry of Sirius Red, Picrosirius and Chlorantine Fast Red aggregates and of their complexes with collagen. Histochem. J. 14:857-878. 248. Von Ebner, U. (1882) Untersuchungen iiber die Ursache der Anisotropie organischer Substanzen. Leipzig. 249. Von Hippe!, A. R., ed. (1965) The Molecular Designing of Materials and Devices. MIT Press, Cambridge, Mass. 250. Von Muralt, A. L., and J. T. Edsall (1930) Flow birefringence of myosin. Trans. Faraday Soc. 26:837- 852. 251. Wahlstrom, E. E. (1960) Optical Crystallography, 3rd ed. Wiley, New York. 252. Weber, H. H. (1934) Der Feinbau und die mechanischen Eigenschaften der Myosin Fadens. Pfluegers Arch. 235:205-233. 253. Weidner, R. T., and R. L. Sells (1965) Elementary Modern Physics, Volume 3. Allyn, Bacon & Cottrell, Boston. 254. Weisenberg, R. C., and A. C. Rosenfeld (1975) In vitro polymerization of microtubules into asters and spindles in homogenates of surf clam eggs. J. Cell Bioi. 64:146-158. 255. Weis-Fogh, T. (1961) Molecular interpretation of the elasticity of resilin, a rubber-like protein. J. Mol. Bioi. 3:648-667. 256. West, C. D., and R. C. Jones (1951) On the properties of polarization elements as used in optical instruments. I. Fundamental considerations. J. Opt. Soc. Am. 41:976-986. 257. West, S. S. (1970) Optical rotation and the light microscope. In: Introduction to Quantitative Cytochem• istry, Volume 2 (G. L. Wied and F. G. Bahr, eds.). Academic Press, New York, p. 451. 258. Wiener, 0. (1912) Die Theorie Des Mischkorpers fiir das Feld der stationaren Stromung. I. Die Mittel• wertzatze fiir Kraft, Polarisation und Energie. Abh. Math. Phys. Kl. Koenig!. Ges. Wiss. 32:507-604. 259. Wiener, 0. (1926) Formdoppelbrechung bei Absorption. I. Ubersicht. Kolloidchem. Beih. 23:189. 260. Wilkins, M. H. F., and B. Battaglia (1953) Note on the preparation of specimens of oriented sperm heads for X-ray diffraction and infra-red absorption studies and on some pseudo-molecular behaviour of sperm. Biochim. Biophys. Acta 11:412-415. 261. Wilson, E. B. (1925) The Cell in Development and Heredity, 3rd ed. Macmillan Co., New York. 26la. Wolman, M. (1970) On the use of polarized light in pathology. In: Pathology Annual 1970 (S. C. Sommers, ed.). Appleton-Century-Crofts, New York, pp. 381-416. 262. Wolstenholme, G. E. W., and M. O'Connor, eds. (1966) Principles of Biomolecular Organization. Churchill, London. 263. Wood, E. A. (1964) Crystals and Light: An Introduction to Optical Crystallography. Van Nostrand, Princeton, N.J. 264. Wood, R. E. (1934) Physical Optics, 3rd ed. Macmillan Co., New York. 265. Wright, F. E. (1911) The Methods of Petrographic-Microscopic Research: Their Relative Accuracy and Range of Application. Carnegie Institution of Washington, Washington, D.C. 266. Zemike, F. (1958) The wave theory of microscopic image formation. In: Concepts of Classical Optics (J. Strong, ed.). Freeman, San Francisco, pp. 525-536. 266a. Zirkle, R. E., R. B. Uretz, and R. H. Haynes (1960) Disappearance of spindles and phargmoplasts after microbeam irradiation of cytoplasm. Ann. N.Y. Acad. Sci. 90:435-439. 267. Zirwer, D., E. Buder, W. Schalike, and R. Wetzel (1970) Lineardichroitische Untersuchungen zur DNS• Organisation in Spermienkopfen von Locusta migratoriaL. J. Cell Bioi. 45:431-434. 268. Zocher, H., and F. Jacoby (1972) Flow dichroism. Kolloid Beih. 24:365. IV Addendum on Digital Image Processors In the two years since Walter and Berns completed Chapter 10, several companies have introduced new digital image processors and programs that are applicable to microscopy. Many of the new processors are Jess expensive, easier to use, and often perform quite complex functions faster than the more elaborate systems. In addition, they can perform many functions in real or near real time on-line, tend to be self-contained, and do not require hookup to, or backup from, centralized computers. In this appendix, I will survey the types of systems available that complement those in Table 10-1, and briefly discuss how one might get started with digital image processing and analysis for microscopy. In addition to the type of equipment developed and used at major installations at the National Institutes of Health, University of Chicago, the Lawrence Livermore Laboratory, etc. for large• scale image analysis and cell sorting (Chapter 10, Section 11.4), three types of commercially available digital image-processing and analysis systems are directly applicable to microscopy. A. Turnkey, Limited-Function Processors The first group of systems, costing in the $10,000 to $20,000 range, are generally self• contained turnkey units with limited, specific functions. Most of the equipment in this group is made to freeze and store a video frame, either unprocessed, or with digital summing or running• averaging to reduce random noise. Incoming images often can be subtracted from the stored image to produce difference images or to perform background subtraction. They are well suited for eliminating random as well as optical noise and the influence of shading. Some degree of control of gray scale and contrast manipulation is usually available, but most of the processing is limited to point operations. The processing is usually not programmable or expandable by the addition of an external computer. Not only are these processors considerably less expensive than the other types, but they are quite compact and very simple to use. Where the need for image processing is limited to noise averaging, background subtraction, and limited contrast enhancement, such a processor can be very effective. Examples of processors in this group are: Arlunya TF4000 series Temporal Filter TV Store System (The Dindima Group Pty., Ltd.); Hughes Model 794 Television Image Processor (Hughes Aircraft Co., now distributed by Dage/MTI); Crystal digital image-processing system (MCI!Link); VIP-300 (Pie Data Medical). B. Dedicated Processors with Extended Functions This group includes many of the new stand-alone processors that take advantage of the large• scale computation that can be carried out rapidly on the specialized programmable hardware that is now available. The built-in, or attached, computer usually needs only to instruct the dedicated 511 512 APPENDIX IV hardware to perform the processing tasks, but generally are not themselves used to. carry out the large-scale number-crunching operations required for image processing. In addition to the functions mentioned for the first group of processors, point operations allowing arbitrary modification of gray scale and contrast, or display in pseudocolor, are carried out in real time by look-up tables. Logical operations (addition, subtraction, ORing, exclusive ORing, etc.) can be carried out by the ALU in a single frame time between images stored in the (three or so) frame buffers. Convolutions involving comparison of brightness of adjacent pixels, and multiplications by weighting factors that filter, sharpen, edge• enhance, edge-detect, etc., are performed in fractions of a second. Histograms of pixel brightness distribution can be displayed, or used to stretch or equalize gray scale and enhance contrast of regions of interest. These images can be made to zoom, shrink, roam, etc., and a variety of quantitative tasks can be performed. Many of these functions can be performed speedily and, depending on the system, interactively. These image processors and analyzers are considerably more flexible than those in Group A, but their cost tends to run in the $10,000 to $65,000 range. Most of the units in this group are turnkey or menu-oriented systems and generally do not require knowledge of computer program• ming or operation. Naturally, some learning is required to master the power of these highly flexible systems. Processors and analyzers in this group include: Hamamatsu Photonic System; lmage-I from Universal Imaging Corporation; PSICOM 327 from Perceptive Systems; QX-9000 system from Quantex; and systems by G. W. Hannaway and Associates. The Hamamatsu Photonic System was specifically designed for microscopy with input from R. D. Allen of Dartmouth College. Several pushbutton functions and adjustments are built into the control unit. The program used in the Image-I system was developed for the author's laboratory, primarily for real-time and near-real-time image processing and quantitation in microscopy. It features ease of use, versatility, speed, and power. No programming is required since single key strokes trigger most functions, but a C-library package is also available for those wishing to assemble additional software for custom applications. The Perceptive Systems PSI COM 327 and related processors and analyzers were developed by K. Castleman. They are capable of performing pattern recognition functions such as chromosome karyotyping, quantitative cell image analysis, sandgrain classification, etc. The Quantex QX-9000 series processor is controlled by a built-in 68000 central processing unit, is versatile, and uses a good mix of hard-wired and software-controlled functions for rapid image processing. It is a natural extension of the earlier (primarily hard-wired) processors produced by Quantex. The processor and analyzer produced by G. W. Hanna way and Associates has available several innovative programs, but which require some degree of familiarity with computer programming and operation. The system can provide considerable flexibility, depending on the degree of program availability. Again, some of the programs were designed to meet the needs of microscopists. C. Major Processing and Analyzing Systems Produced by DeAnza, Grinnell, Kontron, and other companies that, for many years, have specialized in producing general image-processing and analyzing equipment, these general-purpose, modular image processors tend to use powerful main-frame computers to perform a large volume of high-speed image-processing computations. These are the most flexible and powerful systems, but also require greater familiarity with computer operation or programming to exploit their power. The cost of these systems varies, depending on the configuration, but generally falls in the $60,000 to $150,000 range. The basic capabilities of several of these systems are listed in Chapter 10. REMARKS In place of acquiring a complete image processing system, it is also possible to purchase advanced components made for OEMs (original equipment manufacturers) that can be assembled ADDENDUM ON DIGITAL IMAGE PROCESSORS 513 into, or coupled with, computers for image processing or analysis. This may appear to be an economical alternative at first, but one needs to keep in mind that the apparent savings in cost may be illusory. One needs to add the costs of ( 1) purchasing and assembling the full set of hardware and peripherals needed; (2) software acquisition or development; (3) the lack of the follow-up service that is available for the integrated system. While it is common to underestimate somewhat the cost of the first and third items, the cost of software development is the one most likely to be grossly underestimated. In a discussion at the Castleman and Winkler course on digital image processing in 1984, the consensus was that the ratio of cost for hardware acquisition and software development was generally greater than 50 : 50, probably around 40 : 60. In other words, $30,000 worth of hardware would probably require another $45,000 of software development to make the system functional (although finished software that functions with specific hardware configuration may be available for the order of one to a few thousand dollars, depending on the type). With very few exceptions, I would advocate the use of an assembled, complete system, which tends to be more economical in the long run, can be used right away, and whose whole system would receive backup service by experienced personnel. Another general point to consider: The systems in Group A are generally easier to use than those in Group B, and those in Group B easier than in Group C. The speed for the limited tasks in Group A may be just as fast as those in Group B or C; and those in Group B, using dedicated hardware to carry out some computationally intensive tasks, can be much faster than those achieved by the VAX and other powerful computers used in Group C. The final point to consider is that the price of image-processing and analyzing systems can be expected to drop significantly in the next few years relative to the power and sophistication of function. Powerful new memory and processing chips are being developed, not only for TV applications, as described elsewhere, but also for image processing generally. Already, one-mega• byte memory chips and ultra-large-scale integrated circuits (more powerful than the very-large-scale integrated circuits), and compact, high capacity magnetic and optical disks have been announced early in 1985. * Digital processing provides opportunities not available by analog processing. Video images are grabbed without deterioration in a single frame time; noise is averaged and background subtracted effectively. Various tasks of selective image filtering and enhancement, as well as quantitation, are achieved efficiently and more effectively. (See Baxes, 1984, for a comprehensive, nonmathematical description, illustrated with many photographs, of the type of image processing that can be carried out with modem digital processors.) However, some tasks of contrast enhancement, image im• provement, and simple quantitation can be carried out quite well by analog means (Chapter 9). Depending on the nature of the specific image-processing and analysis task, it is well to consider where in the whole system improvements are needed: the microscope optics; the selection of the specimen; video camera, recorder, monitor; analog enhancing devices; or digital processing and analysis. *At the same time, personal computers are using ever more powerful CPUs. My expectation is that by 1986 or 1987 several modular plug-in image processing boards and powerful software applicable to image processing and analysis in microscopy will become available for the IBM PC XT!AT and comparable microcomputers. References* Acker, D. E. (1983) Understanding digital TBC specifications. EITV April:33-40. [8.5) Agar, A. W. (1983) The use of a digitizing frame store in microscopy. J. Microsc. 130:339-344. [11.5] Agard, D. A. (1984) Optical sectioning microscopy: Cellular architecture in three dimensions. Annu. Rev. Biophys. Bioeng. 13:191-219. [11.5) Agard, D., and J. W. Sedat (1983) Three dimensional architecture of a polytene nucleus. Nature 302:676-681. [10.12; Fig. 10-51) Agard, D. A., and R. M. Stroud (1982) Linking regions between helices in bacteriorhodopsin revealed. Biophys. J. 37:589-602. [11.5] Aldersey-Williams, H. (1984) Liquid crystals in flat panel displays. Electronic Imaging 3(11):54-57 [7.5) Allen, R. D. (1985) New observations on cell architecture and dynamics by video-enhanced contrast optical microscopy. Annu. Rev. Biophys. Biophysical Chern. 14:265-290. [Fig. 1-7) Allen, R. D., and N. S. Allen (1983) Video-enhanced microscopy with a computer frame memory. J. Microsc. 129:3-17. [3.10, 11.3) Allen, R. D., G. B. David, and G. Nomarski (1969) The Zeiss-Nomarski differential interference equipment for transmitted-light microscopy. Z. wiss. Mikrosk. 69:193-221. [5.6) Allen, R. D., J. L. Travis, N. S. Allen, and H. Yilmaz (198la) Video-enhanced contrast polarization (AVEC• POL) microscopy: A new method applied to the detection of birefringence in the motile reticulopodial network of Allogromia laticollaris. Cell Motil. 1:275-289. [3.1, 11.3) Allen, R. D., N. S. Allen, and J. L. Travis (1981 b) Video-enhanced contrast, differential interference contrast (A VEC-DIC) microscopy: A new method capable of analyzing microtubule-related motility in the re• ticulopodial network of Allogromia laticollaris. Cell Motil. 1:291-302. [3.1, 11.3) Allen, R. D., J. Metuzals, I. Tasaki, S. T. Brady, and S. P. Gilbert (1982) Fast axonal transport in squid giant axon. Science 218:1127-1129. [11.3) Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson (1985) Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: Evidence for an active role of microtubules in cytoplasmic transport. J. Cell Bioi. 100: 1736-1752. [11.3) Ali-in-One Camcorders (1983) EITV July:35-45. [8.3) Altar, C. A., R J. Walter, K. A. Neve, and J. F. Marshall (1984) Computer-assisted video analysis of 3H• spiroperidol binding autoradiographs. J. Neurosci. Methods 10:173-188. [10.10) Andrews, D. W., and F. P. Ottensmeyer (1982) Electron microscopy of the poly-L-lysine alpha-helix. Ultra• microscopy 9:337-348. [11.5) Ashkar, G. P., and J. W. Modestino (1978) The contour extraction problem with biomedical applications. Computer Graphics and Image Processing 7:331-355. [10.13) Ashley, C. C. (1970) An estimate of calcium concentration changes during the contraction of single muscle fibers. J. Physiol. (London) 210:133P-134P. [Fig. 11-l] Bacus, J. W., and E. E. Gose (1972) Leukocyte pattern recognition. IEEE Trans. Syst. Man Cybern. 2:513- 526. [1.2) Baker, T. S., D. L. D. Caspar, and W. T. Murakami (1983) Polyoma virus 'hexamer' tubes consist of paired pentamers. Nature 303:446-448. [11.5] *See Appendix III (pp. 500-510) for additional references (Bibliography). 515 516 REFERENCES Ballard, D. H., and J. Sklansky (1976) A ladder structured decision tree for recognizing tumors in chest radiographs. IEEE Trans. Comput. 25:503-513. [10.13] Bally, G. von, and P. Greguss, eds. (1982) Optics in Biomedical Sciences. Springer-Verlag, New York. [11.5] Barer, R. (1956) Phase contrast and interference microscopy in cytology. In: Physical Techniques in Biological Research, Volume III (G. Oster and A. W. Pollister, eds.). Academic Press, New York, pp. 29-90.[11.3] Barlow, H. B., and J.D. Mollon, eds. (1982) The Senses. Cambridge University Press, London. [4.1] Barrows, G. H., J. E. Sisken, J. C. Allegra, and S.D. Grasch (1984) Measurement of fluorescence using digital integration of video images. J. Histochem. Cytochem. 32:741-746. [11.2] Bartels, P. H., and G. L. Wied (1973) Extraction and evaluation of information from cell images. Proceedings of the First Annual Life Sciences Symposium: Mammalian Cells, Probes, and Problems, LASL, University of California, Los Alamos, N. M., pp. 15-28. [1.2] Bartels, P. H., G. B. Olson, H. G. Bartels, D. E. Brooks, and G. V. Seaman (1981) The automated analytic electrophoresis microscope. Cell Biophys. 4:371-388. [10.13] Bartels, P. H., J. Layton, and R. L. Shoemaker (1984) Digital Microscopy. In: Computer-Assisted Image Analysis Cytology (S. D. Greenberg, ed.). Karger, Basel, pp. 28-61. [11.4] Baxes, G. A. (1984) Digital Image Processing: A Practical Primer. Prentice-Hall, Englewood Cliffs, N.J. [1.3, 6.5, Postscript, Appendix IV] Beck, C. (1938) The Microscope: Theory and Practice. Beck, London. [5.1] Beckerle, M. C. (1984) Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells. J. Cell Bioi. 98:2126-2132. [11.2] Bedford, A. V. (1950) Mixed highs in color television. Proc. IRE 38:1003-1009. [4.9, 7.4] Belling, J. (1930) The Use of the Microscope. McGraw-Hill, New York. [5.1] Bennet, A. H. B., H. Jupnik, H. Osterberg, and 0. W. Richards (1951) Phase Microscopy. Wiley, New York. [5.6] Bennett, H. S. (1950) The microscopical investigation of biological materials with polarized light. In: Handbook of Microscopical Technique (C. E. McClung, ed.). Harper & Row (Hoeber), New York, pp. 591-677. [5.6, 11.3] Benson, D. M., J. A. Knopp, and I. S. Langmuir (1980) Intracellular oxygen measurements of mouse liver cells using quantitative fluorescence video microscopy. Biochim. Biophys. Acta 591:187-197. [10.13, 11.2] Benson, D. M., J. Bryan, A. L. Plant, A.M. Gotto, Jr., and L. C. Smith (1985) Digital imaging fluorescence microscopy: Spatial heterogeneity of photobleaching rate constants in individual cells. J. Cell Bioi., 100:1309-1323. [11.2, 11.4] Berns, G. S., and M. W. Berns (1982) Computer-based tracking of living cells. Exp. Cell Res. 142:103-109. [10.13, 11.3, 11.5] Berns, M. W. (1974) Biological Microirradiation: Classical and Laser Sources. Prentice-Hall, Englewood Cliffs, N.J. [11.5] Berns, M. W., J. Aist, J. Edwards, K. Strahs, J. Girton, M. Kitzes, J. Hammer-Wilson, L.-H. Liaw, A. Siemens, M. Koonce, R. Walter, D. van Dyk, J. Coulombe, T. Cahill, and G. S. Berns (1981) Laser microsurgery in cell and developmental biology. Science 213:505-513. [ 10. 7] Berns, M. W., A. E. Siemens, and R. J. Walter (1984) Mitochondrial fluorescence patterns in Rhodamine 6G• stained myocardial cells in vitro. Analysis by real-time computer video microscopy and laser microspot excitation. Cell Biophys. 6:263-277. [10.13] Bertram, J. F., and A. W. Rogers (1981) The development of squamous cell metaplasia in human bronchial epithelium by light microscopic morphometry. J. Microsc. 123:61-73 [10.13] Bestor, T. H., and G. Schatten (1981) Anti-tubulin immunofluorescence microscopy of microtubules present during the pronuclear movements of sea urchin fertilization. Dev. Bioi. 88:80-91. [11.2] Biedert, S., W. H. Barry, and T. W. Smith ( 1979) Inotropic effects and changes in sodium and calcium contents associated with inhibition of monovalent cation active transport by ouabain in cultured myocardial cells. J. Gen. Physiol. 74:479-494. [9.5] Blifford, I. H., Jr., and D. A. Gillette (1973) An automated particle analysis system. Microscope 21:121-130. [1.2] Blout, E. R., G. R. Bird, and D. S. Grey (1950) Infra-red microscopy. J. Opt. Soc. Am. 40:304-313. [11.2] Blum, H. (1964) Transformation for extracting new descriptions of shape. In: Symposium on Models for the Perception of Speech and Visual Form (W. Wathen-Dunn, ed.). MIT Press, Cambridge, Mass., pp. 362- 381. [10.13] Bok, D., and R. W. Young (1979) Phagocytic properties of the retinal pigment epithelium. In: The Retinal Pigment Epithelium (K. M. Zinn and M. F. Marmor, eds.). Harvard University Press, Cambridge, Mass., pp. 148-174. [4.2] Born, M., and E. Wolf (1980) Principles of Optics, 6th ed. Pergamon Press, Elmsford, N.Y. [5.5; Fig. 5-21] REFERENCES 517 Bowie, J. E., and I. T. Young (1977a) An analysis technique for biological shape. II. Acta Cytol. 21:455-464. [10.13] Bowie, J. E., and I. T. Young (1977b) An analysis technique for biological shape. III. Acta Cytol. 21:739-746. [10.13] Boyes, E. D., B. I. Muggridge, and M. I. Goringe (1982) On-line image processing in high resolution electron microscopy. J. Microsc. 127:321-335. [11.5] Bracewell, R.N. (1978) The Fourier Transform and Its Application, 2nd ed. McGraw-Hill, New York. [11.3] Bradbury, S. ( 1983) Commercial image analyzers and the characterization of microscopical images. J. Microsc. 131:203-210. [11.4] Brady, S. T., R. J. Lasek, and R. D. Allen (1982) Fast axonal transport in extruded axoplasm from squid giant axon. Science 218:ll29-113l. [11.3] Braithwaite, L. (1985) The official Video magazine blank tape tests. Video 8(ll):78-ll2. [8.8] Brigham, E. 0. (1974) The Fast Fourier Transform. Prentice-Hall, Englewood Cliffs, N.J. [IO.ll, 10.12] Brooks, M. I. (1978) Rationalizing edge detectors. Computer Graphics and Image Processing 8:277-285. [10.11] Butler, J. W., M. K. Butler, and A. Stroud (1966) Automatic classifications of chromosomes. II. In: Data Acquisition and Processing in Biology and Medicine Volume 4 (K. Enslein, ed.). Pergamori Press, Elmsford, N.Y., pp. 47-60. [1.2] Butterfield, J. F. (1978) Video microscopy. Microscope 26:171-182. [12.7] Cagnet, M., M. Fran~on, and J. C. Thrierr (1962) Atlas of Optical Phenomena. Springer-Verlag, Berlin. [5.5, 5.9] Carman, G., P. Lemkin, L. Lipkin, B. Shapiro, M. Schultz, and P. Kaiser (1974) A real time picture processor for use in biologic cell identification. II. Hardware implementation. J. Histochem. Cytochem. 22:732-740. [1.2] Caspersson, T. (1950) Cell Growth and Cell Function. Norton, New York. [11.2] Caspersson, T. (1973) Fluorometric recognition of chromosomes and chromosome regions. In: Fluorescence Techniques in Cell Biology (A. A. Thaer and M. Semetz, eds.). Springer-Verlag, Berlin, pp. 107-123. [1.2] Caspersson, T., J. Lindsten, G. Lomakka, H. Wallman, and L. Zech (1970) Rapid identification of human chromosomes by TV techniques. Exp. Cell Res. 63:477-479. [1.2] Castillo, X., D. Yorkgitis, and K. Preston (1982) A study of multidimensional multicolor images. IEEE Trans. Biomed. Eng. 29(2):111-121. [10.5] Castleman, K. R. (1979) Digital Image Processing. Prentice-Hall, Englewood Cliffs, N.J. [1.3, 5.7, 6.5, 10.3, 10.4, 10.11, 10.12, 11.5, 11.3; Figs. 10-12, 10-34] Castleman, K., and R. Wall (1973) Automatic systems for chromosome identification. In: Chromosome Identifi• cation (T. Caspersson and L. Zech, eds.). Academic Press, New York. pp. 77-84. [1.2, 10.13]. Cederberg, R. L. T. (1979) Chain link coding and segmentation for raster scan devices. Computer Graphics and Image Processing 10:224-234. [10.13] Chamot, E. M., and C. W. Mason (1958) Handbook of Chemical Microscopy, 2nd ed. Wiley, New York. [5.6, 11.3] Cheng, S.-Y., F. R. Maxfield, J. Robbins, M. C. Willingham, and I. H. Pastan (1980) Receptor-mediated uptake of 3,3', 5-triiodo-L-thyronine by cultured fibroblasts. Proc. Natl. Acad. Sci. USA 77:3425-3429. [11.2] Cline, H. E., A. S. Holik, and W. E. Lorensen (1982) Computer-aided surface reconstruction of interference contours. Appl. Opt. 21:4481-4488. [11.3] Comsweet, T. N. (1970) Visual Perception. Academic Press, New York. [4.1] Cox, I. J., and C. J. R. Sheppard (1983) Scanning optical microscope incorporating a digital framestore and microcomputer. Appl. Opt. 22:1474-1478. [11.5] Crewe, A. V., and M. Ohtsuki (1982) A "stand-alone" image processing system for STEM images. Ultra• microscopy 9:101-108. [11.5] Currier, R. L. (1983) Interactive video disc learning systems. High Technology 3(11):51-60. [8.9] Cutcheon, J. T., J. 0. Harris, Jr., and G. R. Laguna (1973) Electro-optic devices utilizing quadratic PLZT ceramic elements. Report No. SLA 73-0777, Sandia Laboratories, Albuquerque, N.M. [12.7] Davidson, M. L. (1968) Perturbation approach to spatial brightness interaction in human vision. J. Opt. Soc. Am. 58:1300-1309. [Fig. 4-13] Davis, L. S., and A. Rosenfeld (1975) A survey of edge detection techniques. Computer Graphics and Image Processing 4:248-270. [10.13] Davis, R., and D. Thomas (1984) Systolic array chip matches the pace of high-speed processing. Electronic Design Oct. 31, pp. 207-218. [11.5] 518 REFERENCES DeForest, L. (1907) Space Telegraphy. U.S. Patent No. 879,532. [l.l] de Montebello, R. L. (1969) The RLM Synthalyzer technique and instrumentation for optical reconstruction and dissection of structures in three dimensions. Ann. N.Y. Acad. Sci. 157:487-496. [11.5; Fig. ll-12] Dempster, W. T. (1944) Principles of microscope illumination and the problem of glare. J. Opt. Soc. Am. 34:694-710. [3.8, 5.3, 5.4] De Weer, P., and B. M. Salzberg, eds. (1986) Optical Methods in Cell Physiology. Wiley, New York, in press. [11.2, Postscript] DiBona, D. R. (1978) Direct visualization of epithelial morphology in the living amphibian urinary bladder. J. Membr. Bioi. 40:45-70. [11.3] Diller, K. R., and J. M. Knox (1983) Automated computer analysis of cell size changes during cryomicroscope freezing: A biased trident convolution mask technique. Cryo-Letters 4:77-92. [10.13] D'Ormer, P., K. Pachmann, R. Penning, G. J'Ager, and H. Rodt (1981) Discrimination in immunofluorescence between labeled and unlabeled cells by quantitative microfluorimetry and computer analysis. J. lmmunol. Methods 40(2):155-163 [10.13] Dvorak, J. A., L. H. Miller, W. C. Whitehouse, and T. Shiroishi (1975) Invasion of erythrocytes by malaria merozoites. Science 187:748-750. [11.3; Fig. 1-4] Dyson, J. (1950) An interferometer microscope. Proc. R. Soc. London Ser. A 204:170-187. [5.6] Eckert, R., and G. T. Reynolds ( 1967) The subcellular origin of bioluminescence in Noctiluca miliaris. J. Gen. Physiol. 50:1429-1458. [1.2, 5.6, 11.2] Edelhart, M. (1981) Optical disks: The omnibus medium. High Technology 1:42-57. [8.9] Egger, M.D., and M. Petran (1967) New reflected-light microscope for viewing unstained brain and ganglion cells. Science 157:305-307. [11.5] Einck, L., and M. Bustin (1984) Functional histone antibody fragments traverse the nuclear envelope. J. Cell Bioi. 98:205-213. [11.2] Eisen, A., D.P. Kiehart, S. J. Wieland, and G. T. Reynolds (1984) Temporal sequence and spatial distribution of early events of fertilization in single sea urchin eggs. J. Cell Bioi. 99:1647-1654. [11.2] Ekstrom, A. (1910) Swedish Patent No. 32,220. [1.1] Ellis, G. W. (1966) Holomicrography: Transformation of image during reconstruction a posteriori. Science 154:ll95-ll96. [5.6, 11.5] Ellis, G. W. (1978) Advances in visualization of mitosis in vivo. In: Cell Reproduction: In Honor of Daniel Mazia (E. Dirksen, D. Prescott, and C. F. Fox, eds.). Academic Press, New York, pp. 465-476. [5.6, 11.3] Ellis, G. W.(1979) A fiber-optic phase-randomizer for microscope illumination by laser. J. Cell Bioi. 83:303a. [Appendix I; Table 5-1] Ellis, G. W. (1985) Microscope illuminator with fiber optic source integrator, J. Cell Bioi. 101:83a. [5.8] Ellis, G. W., S. Inoue, and T. D. Inoue (1986) Computer aided light microscopy. In: Optical Methods in Cell Physiology (P. De Weer and B. M. Salzberg, eds.). Wiley, New York, pp. 15-30. [3.10, 11.2, 11.3; Fig. 1-8] Ennes, H. E. (1979) Television Broadcasting: Equipment, Systems, and Operating Fundamentals, 2nd ed. Sams, Indianapolis. [Preface, 6.1, 6.3, 6.4, 6.5, 6.6, 7.4, 7.5; Figs. 6-21, 7-27,7-28, 8-8, 8-9; Table 6-4] Erickson, R. 0. (1973) Tubular packing of spheres in biological fine structure. Science 181:705-716. [12.1] Faroudja, Y., and J. Roizen (1981) Color-under recorders: Limitations, manual correction, and automated enhancement. SMPTE J. Dec.:ll52-ll57. [8.3] Fay, F. S., K. E. Fogarty, and J. M. Coggins (1985) Analysis of molecular distribution in single cells using a digital imaging microscope. In: Optical Methods in Cell Physiology (P. De Weer and B. M. Salzberg, eds.). Wiley, New York, in press. [11.5] Fiorentini, A. (1964) Dynamic characteristics of visual processes. Prog. Opt. 1:253-288. [4.4] Flory, L. E. (1951) The television microscope. Cold Spring Harbor Symp. Quant. Bioi. 16:505-509. [1.2] Flory, L. E. (1959) Scanning microscopy in medicine and biology. Proc. IRE Nov.:1889-1894. [1.2] Flory, L. E., and W. S. Pike (1953) Particle counting by television techniques. RCA Rev. 14:546-556. [1.2] Forman, D. S., and D. E. Turriff (1981) Video intensification microscopy (VIM) as an aid in routine fluores- cence microscopy. Histochemistry 71:203-208. [11.2] Forman, D. S., K. J. Brown, and D. R. Livengood (1983) Fast axonal transport in permeabilized lobster giant axons is inhibited by vanadate. J. Neurosci. 3:1279-1288. [11.3] Foster, J. (1984) High resolution acoustic microscopy in superfluid helium. 17th International Conference on Low Temperature Physics, Karlsruhe, West Germany. G. L. Report No. 3760. W. W. Hansen Laboratory of Physics, Stanford University. [11.5] Francron, M. (1961) Progress in Microscopy. Row, Peterson, Evanston, Ill. [5.1, 5.5, 5.6; Figs. 5-18 to 5-20] Franklin, G., and T. Hatley (1973) Don't eyeball noise. Electron. Des. 24:184-187. [6.5] REFERENCES 519 Freed, J. J., and J. L. Engle (1962) Development of the vibrating-mirror flying spot microscope for ultraviolet spectrophotometry. Ann. N.Y. Acad. Sci. 97:412-448. [1.2, 11.5] Freeman, H. (1970) Boundary encoding and processing. In: Picture Processing and Psychopictorics (B. Lipkin and A. Rosenfeld, eds.). Academic Press, New York, pp. 241-266. [10.13] Fuchs, H., S. M. Pizer, E. R. Heinz, S. H. Bloomberg, L.-C. Tsai, and D. C. Strickland (l982a) Design and image editing with a space-filling 3-D display based on a standard raster graphics system. Proc. Soc. Photo. Opt. lnstrum. Eng. 367:117-127. [11.5] Fuchs, H., S.M. Pizer, L.-C. Tsai, S. H. Bloomberg, and E. R. Heinz (1982b) Adding a true 3-D display to a raster graphics system. IEEE Comput. Graphics Appl. 2:73-78. [11.5; Fig. 11-13] Fuseler, J. W. (1975) Mitosis in Tilia americana endosperm. J. Cell Bioi. 64:159-171. [5.10] Gabor, D. (1948) A new microscope principle. Nature 161:777-778. [5.6] Gabor, D. (1949) Microscopy by reconstructed wave-fronts. Proc. R. Soc. London Ser. A 197:454-486. [5.6] Gabor, D. (1951) Microscopy by reconstructed wave-fronts. II. Proc. Phys. Soc. London Ser. B 64:449-470. [5.6] Gage, S. H. (1941) The Microscope. Cornell University Press (Comstock), Ithaca, N.Y. [5.1] Galbraith, W. (1982) The image of a point of light in differential interference contrast microscopy: Computer simulation. Microsc. Acta 85:233-254. [5.5] Galbraith, W., and G. B. David (1976) An aid to understanding differential interference contrast microscopy: Computer simulation. J. Microsc. 108:147-176. [5.6] Galbraith, W., and R. J. Sanderson (1980) The intensity distribution about the image of a point. Microsc. Acta 83:395-402. [5.5] Gallistel, C. R., C. T. Piner, T. 0. Allen, N. T. Adler, E. Yaden, and M. Negin (1982) Computer assisted analysis of 2-DG autoradiograms. Neurosci. Biobehav. Rev. 6:409-420. [10.7] Garbay, C., G. Brugal, and C. Choquet (1981) Application of colored image analysis to bone marrow cell recognition. Anal. Quant. Cytol. 3:272-280. [10.5] Gaunt, W. A., and P. N. Gaunt (1978) Three-Dimensional Reconstruction in Biology. University Park Press, Baltimore. [11.5, 12.7] Ghatak, A. K., and K. Thyagarajan (1978) Contemporary Optics. Plenum Press, New York. [Fig. 5-21] Gilev, V. P. (1979) A simple method of optical filtration. Ultramicroscopy 4:323-336. [12.1] Gilkey, J. C., L. F. Jaffe, E. B. Ridgway, and G. T.Reynolds (1978) A free calcium wave traverses the activating egg of medaka, Oryzias latipes. J. Cell Bioi. 76:448-466. [11.2; Fig. 11-1] Gingell, D., and I. Todd (1979) Interference reflection microscopy: A quantitative theory for image interpreta• tion and its application to cell-substratum separation measurement. Biophys. J. 26:507-526. [11.3] Glaser, E. M., M. Tagamets, N. T. McMullen, and H. Vander Loos (1983) The image-combining computer microscope-An interactive instrument for morphometry of the nervous system. J. Neurosci. Methods 8:17-32. [11.4] Glass, B. (1961) Light and Life. Johns Hopkins Press, Baltimore. [11.2] Gonzalez, R. C., and P. Wintz (1977) Digital/mage Processing. Addison-Wesley, Reading, Mass. [6.5] Goochee, C., W. Rasband, and L. Sokoloff (1980) Computer densitometry and color coding of [1 4C] deox- yglucose autoradiographs. Ann. Neurol. 7:359-370. [10.7] Goodman, J. W. (1968) Introduction to Fourier Optics. McGraw-Hill, New York. [6.5, 11.3] Gordon, G., and D. J. Palatini (1982) Enhanced visualization of enzymatic reaction product by means of image analysis. Histochem. J. 14:842-845. [11.5] Gould, R. G., M. J. Lipton, P. Mengers, and R. Dahlberg (1981) Investigation of video frame averaging digital subtraction fluoroscopic system. Proc. SPIE Int. Soc. Opt. Eng. 314:184. [10.4] Graff, G. (1984) Liquid crystals. High Technology 4(5):54-68. [7.5] Gregory, R. L. (1970) The Intelligent Eye. McGraw-Hill, New York. [4.8] Grinvald, A., L. B. Cohen, S. Lesher, and M. B. Boyle (1981) Simultaneous optical monitoring of activity of many neurons in invertebrate ganglia using a 124-element photodiode array. J. Neurophysiol. 45:829-840. [6.2, 11.4] Gruenbaum, Y., M. Hochstrasser, D. Mathog, H. Saumweber, D. A. Agard, and J. W. Sedat (1984) Spatial organization of the Drosophila nucleus: A three-dimensional cytogenetic study. J. Cell Sci. Suppl. 1: 223-234. [10.12] Gruner, S.M., J. R. Milch, and G. T. Reynolds (1978) Evaluation of area photon detectors by a method based on detective quantum efficiency (DQE). IEEE Trans. Nucl. Sci. 25:562-565. [11.4] Gruner, S.M., J. R. Milch, and G. T. Reynolds (1982) Slow-scan silicon-intensified target-TV X-ray detector for quantitative recording of weak X-ray images. Rev. Sci. lnstrum. 53:1770-1778. [9.4] Gundersen, G. G., C. A. Gabel, and B. M. Shapiro (1982) An intermediate state of fertilization involved in internalization of sperm components. Dev. Bioi. 93:59-72. [11.2] 520 REFERENCES Hall, J. A. (1980) Arrays and charge coupled devices. In: Applied Optics and Optical Engineering, Volume VII (R. R. Shannon and J. C. Wyant, eds.). Academic Press, New York, pp. 349-400. [7.1] Hannaway, W., G. Shea, and W. R. Bishop (1984) Handling real-time images comes naturally to systolic array chip. Electronic Design Nov. 15: 289-300. [11.5] Hansen, G. L. (1969) Introduction to Solid-State Television Systems: Color and Black and White. Prentice• Hall, Englewood Cliffs, N.J. [Preface] Harlow, C. A., and R. W. Connors (1979) Image analysis segmentation methods. In: Biomedical Pattern Recognition and Image Processing (K. S. Fu and T. Pavlidis, eds.). Verlag Chemie, Weinheim, pp. 111- 130. [10.13] Harosi, F. (1982) Recent results from single-cell microspectrophotometry: Cone pigments in frog, fish, and monkey. Color Res. Appl. 7:135-141. [11.4] Harris, J. L. (1964) Diffraction and resolving power. J. Opt. Soc. Am. 54:931-936. [10.12] Hartshorne, N. H., and A. Stuart (1960) Crystals and the Polarising Microscope: A Handbook for Chemists and Others, 3rd ed. Arnold, London. [5.6, 11.3] Harvey, E. N. (1957) A history of luminescence from the earliest times until 1900. Mem. Am. Philos. Soc. 44:1-692. [11.2] Harvey, E. N. (1965) Living Light. Princeton University Press, Princeton, N.J. [11.2] Hayden, J. H., and R. D. Allen (1984) Detection of single microtubules in living cells: Particle transport can occur in both directions along the same microtubule. J. Cell Bioi. 99:1785-1793. [11.3] Hecht, E., and A. Zajac (1974) Optics. Addison-Wesley, Reading, Mass. [5.7, 10.12] Hecht, S. (1984) Outlook brightens for semiconductor vector lasers. High Technology 4(1):43-50. [8.9] Hecht, S., and J. Mandelbaum (1939) The relation between vitamin A and dark adaptation. J. Am. Med. Assoc. 112:1910-1916. [Fig. 4-4] Hecht, S., and S. Shlaer ( 1936) Intermittent stimulation by light. V: The relation between intensity and critical frequency for different parts of the spectrum. J. Gen. Physiol. 19:965-977. [Fig. 4-15] Hecht, S., and E. L. Smith ( 1936) Intermittent stimulation by light. VI. Area and the relation between critical frequency and intensity. J. Gen. Physiol. 19:979-989. [Fig. 4-15] Hecht, S., and C. D. Verrijp (1933) The influence of intensity, color and retinal location on the fusion frequency of intermittent illumination. Proc. Natl. Acad. Sci. USA 19:522-535. [Fig. 4-15] Helmholtz, H. (1866) Handbuch der Physiologischen Optik. Voss, Leipzig. [4.7] Hiramoto, Y., andY. Shoji (1982). Location of the motive force for chromosome movement in sand-dollar eggs. In: Oji International Seminar on Biological Functions of Microtubules and Related Structures. (H. Sakai, H. Mohri, and G. G. Borisy eds.) Academic Press, Japan, pp. 247-259. Hiramoto, Y., Y. Hamaguchi, Y. Shoji, T. E. Schroeder, S. Shimoda, and S. Nakamura, (1981) Quantitative studies on the polarization optical properties of living cells. II. The role of microtubules in birefringence of the spindle of the sea urchin egg. J. Cell Bioi. 89:121-130. Hirschberg, I. (1985) Advances in high-resolution imagers. Electronic Imaging 4:56-62. [Table 7-3] Hirshon, R. (l984a) Flat panel display technologies. Electronic Imaging 3(1):40-48. [7.5] Hirshon, R. (1984b) Optical storage meets increased data demands. Electronic Imaging 3(3):30-35. [8.9] Hobbs, M. (1982) Servicing Home Video Cassette Recorders. Hayden, Rochelle Park, N.J. [8.2, 8.7] Hoffman, R., and L. Gross (1975) Modulation contrast microscopy. Appl. Opt. 14:1169-1176. [5.6] Hotani, H. (1982) Micro-video study of moving bacterial flagellar filaments. III. Cyclic transformation induced by mechanical force. J. Mol. Bioi. 156:791-806. [11.2] Huxley, A. F., V. Lombardi, and L. D. Peachey (1981) A system for recording sarcomere longitudinal displacements in a striated muscle fibre during contraction. Boll. Soc. /tal. Bioi. Sper. 57:57-59. [11.4] Inoue, S. (1944) Stereoscopic Apparatus. Japanese Patent No. 166,528. [12.7] Inoue, S. (1951) A method for measuring small retardations of structures in living cells. Exp. Cell Res. 2:513- 517. [9.5] Inoue, S. (1961) Polarizing microscope: Design for maximum sensitivity. In: The Encyclopedia of Microscopy (G. L. Clarke, ed.). Reinhold, New York, pp. 480-485. [11.3] Inoue, S. (1964) Organization and function of the mitotic spindle. In: Primitive Motile Systems in Cell Biology (R. D. Allen and N. Kamiya, eds.). Academic Press, New York, pp. 549-598. [11.3] Inoue, S. (1981a) Cell division and the mitotic spindle. J. Cell Bioi. 91(Part 2):131s-147s. [11.3] Inoue, S. (1981b) Video image processing greatly enhances contrast, quality, and speed in polarization-based microscopy. J. Cell Bioi. 89:346-356. [3.1, 8.4, 11.3, 12.1, Appendix I; Figs. 1-5, 1-6, 11-4, 11-5] Inoue, S., and K. Dan (1951) Birefringence of the dividing cell. J. Morpho/. 89:423-456. [Fig. 11-4] Inoue, S., and W. L. Hyde (1957) Studies on depolarization of light at microscope lens surfaces. II. The simultaneous realization of high resolution and high sensitivity with the polarizing microscope. J. Biophys. Biochem. Cytol. 3:831-838. [11.3] REFERENCES 521 Inoue, S., and H. Kubota (1958) Diffraction anomaly in polarizing microscopes. Nature 182:1725-1726. [5.5, 11.3] Inoue, S., and D. A. Lutz (1982) Simultaneous video display of fluorescence and polarized light, or DIC, microscope images in real time. J. Cell Bioi. 95:46la. [Fig. 5-35] Inoue, S., and H. Ritter, Jr. (1975) Dynamics of mitotic spindle organization and function. In: Molecules and Cell Movement (S. Inoue and R. E. Stephens, eds.). Raven Press, New York, pp. 3-30. [5.6] Inoue, S., and H. Sato (1966) Deoxyribonucleic acid arrangements in living sperm. In: Molecular Architecture in Cell Physiology (T. Hayashi and A. G. Szent-Gyorgyi, eds.). Prentice-Hall, Englewood Cliffs, N.J., pp. 209-248. [5.6, 9.5, 11.3] Inoue, S., and H. Sato (1967) Cell motility by labile association of molecules: The nature of mitotic spindle fibers and their .role in chromosome movement. J. Gen. Physiol. 50:259-292. [11.3] Inoue, S., and L. G. Tilney (1982) The acrosomal reaction of Thyone sperm. I. Changes in the sperm head visualized by high resolution video microscopy. J. Cell Bioi. 93:812-819. [5.6, 11.3] Inoue, S., D. Cohen, and G. W. Ellis (1981) High resolution, stereo video microscope. Bioi. Bull. 161:306. [12.7] Inoue, S., T. D. Inoue, and G. W. Ellis (1985) Rapid stereoscopic displayofmicrotubuledistribution by a video• processed optical sectioning system. J. Cell Bioi. 101:146a. [12.7] Intaglietta, M. (1970) Graphic display of television raster lines. Rev. Sci. Instrum. 41:1105-1106. [9.6] Intaglietta, M., N. R. Silverman, and W. R. Tompkins (1975) Capillary flow velocity measurements in vivo and in situ by television methods. Microvasc. Res. 10:165-179. [1.2, 9.5] Ishihara, A., and H. Hotani (1980) Micro-video study of discontinuous growth of bacterial flagellar filaments in vitro. J. Mol. Bioi. 139:265-276. [11.2] Izzard, C. S., and L. R. Lochner (1976) Cell-to-substrate contacts in Jiving fibroblasts: An interference reflexion study with an evaluation of the technique. J. Cell Sci. 21:129-159. [5.6, 11.3] Jacobson, K., E. Elson, D. Koppel, and W. Webb (1983) International workshop on the application of fluorescence photobleaching techniques to problems in cell biology. Fed. Proc. 42:72-79. [ll.4] Jenkins, F. A., and H. E. White (1957) Fundamentals of Optics, 3rd ed. McGraw-Hill, New York. [5.8] Johnson, C. H. S., S. Inoue, A. Flint, and J. W. Hastings (1985) Compartmentation of algal bioluminescence: Autofluorescence of bioluminescent particles in the dinoflagellate Gonyaulux as studied with image• intensified video microscopy and flow cytometry. J. Cell Bioi. 100:1435-1446. [11.2; Fig. ll-2] Johnson, L. D., M. L. Walsh, B. J. Bockus, and L. B. Chen (1981) Monitoring of relative mitochondrial membrane potential in living cells by fluorescent microscopy. J. Cell Bioi. 88:526-535. [10.13] Judge, A. W. (1926) Stereoscopic Photography. Chapman & Hall, London. [12.7] Julesz, B. (1971) Foundations of Cyclopean Perception. University of Chicago Press, Chicago. [4.8] Kachar, B. (1985) Asymmetric illumination contrast: A new method of image formation for video light microscopy. Science 227:766-768. [11.3; Fig. ll-9] Kachar, B., D. F. Evans, and B. W. Ninham (1984) Video enhanced differential interference contrast micros• copy: A new tool for the study of association colloids and prebiotic assemblies. J. Colloid Interface Sci. 100:287-301. [11.3] Karniya, R., H. Hotani, and S. Asakura (1982) Polymorphic transition in bacterial flagella. In: Prokaryotic and Eukaryotic Flagella (W. B. Amos and J. G. Duckett, eds.). Cambridge University Press, London, pp. 53- 76. [11.2] Kater, S. B., and R. D. Hadley (1982) Intracellular staining combined with video fluorescence microscopy for viewing living identified neurons. In: Cytochemical Methods in Neuroanatomy, Volume 1 (V. C. Palay and S. L. Palay, eds.). Liss, New York, pp. 441-459. [11.2] Kazan, B., and M. Knoll (1968) Electronic Image Storage. Academic Press, New York. [l.l] Keller, H. E., and W. C. McCrone (1977) Standardization in the design of microscopes. Microscope 25:169- 177. [5.9] Kelly, D. H. (1961) Visual response to time-dependent stimuli. J. Opt. Soc. Am. 51:422-429. [Fig. 4-16] Kirsch, R. A. (1971) Computer determination of the constituent structure of biological images. Comput. Biomed. Res. 4:315-328. [10.13] Koester, C. J. (1961) Interference microscopy: Theory and Techniques. In: The Encyclopedia ofMicroscopy (G. L. Clarke, ed.). Reinhold, New York, pp. 420-434. [5.6] Koester, C. J. (1980) Scanning mirror microscope with optical sectioning characteristics: Applications in ophthalmology. Appl. Opt. 19:1749-1757. [11.5; Fig. ll-ll] Koester, C. J., C. W. Roberts, A. Donn, and F. B. Hoefle (1980) Wide field specular microscopy: Clinical and research applications. Ophthalmology 87:849-860. [11.5] Kohen, E., C. Cohen, B. Thorell, and P. Bartick (1979) A topographic analysis of metabolic pathways in single living cells by multisite microfluorometry. Exp. Cell Res. 119:23-30. [10.3] 522 REFERENCES Kohler, A., (1893) Ein neues Beleuchtungsverfahren fiir mikrophotographische Zwecke. Z. wiss. Mikrosk. 10:433-440. [5.3] Koonce, M. P., and M. Schliwa (1985) Bidirectional organelle transport can occur in cell processes that contain single microtubules. J. Cell Bioi. 100:322-326. [11.3] Koonce, M. P., R. J. Cloney, and M. W. Berns (1984) Laser irradiation of centrosomes in newt eosinophils: evidence of centriole role in motility. J. Cell Bioi. 98:1999-2010. [10.13] Kopp, R. E., J. Lisa, J. Mendelsohn, B. Pernick, H. Stone, and R. Wohlers (1974) The use of coherent optical processing techniques for the automatic screening of cervical cytologic samples. J. Histochem. Cytochem. 22:598-604. [10.13] Koppel, D. E., J. M. Oliver, and R. D. Berlin (1982) Surface functions during mitosis. III. Quantitative analysis of ligand-receptor movement into the cleavage furrow: Diffusion vs. flow. J. Cell Bioi. 93:950-960. [11.2] Kowel, S. T., P. G. Kornreigh, and 0. Lewis (1976) Focus detection using direct electronic Fourier-transform detectors. Appl. Photogr. Eng. 2:113. [10.12] Kries, T. E., and W. Birchmeier (1980) Stress fiber sarcomeres of fibroblasts are contractile. Cell22:555-561. [11.2] Kristan, J., and M. Blouke (1982) Charge-coupled devices in astronomy. Sci. Am. 247(4):66-74. [7.1] Kubota, H., and S. Inoue (1959) Diffraction images in the polarizing microscope. J. Opt. Soc. Am. 49:191- 198. [5.5, 11.3] Lamy, J., P.-Y. Sizaret, J. Frank, A. Verschoor, R. Feldmann, and J. Bonaventura (1982) Architecture of Limulus polyphemus hemocyanin. Biochemistry 21:6825-6833. [11.5] Lancaster, D. (1976) TV Typewriter Cookbook. Sams, Indianapolis. [9.2] Land, E. H. (1977) The retinex theory of color vision. Sci. Am. 237(6):108-128. [4.7] Land, E. H. (1983) Recent advances in retinex theory and some implications for cortical computations: Color vision and the natural image. Proc. Nat/. Acad. Sci. USA 80:5163-5169. [4.7] Land, E. H., and J. J. McCann (1971) Lightness and retinex theory. J. Opt. Soc. Am. 61:1-11. [4.5, 4.7; Fig. 4-14] Land, E. H., E. R. Blout, D. S. Grey, M.S. Flower, H. Husek, R. C. Jones, C. H. Matz, and D.P. Merrill (1948) Color translating ultraviolet microscope. Science 109:371-374. [1.2] Lemkin, P., G. Carman, L. Lipkin, B. Shapiro, M. Schultz, and P. Kaiser (1974) A real time picture processor for use in biologic cell identification. I. Systems design. J. Histochem. Cytochem. 22:725-731. [1.2] Lemkin, P., B. Shapiro, L. Lipkin, J. Maize!, J. Sklansky, and M. Schultz (1979) Preprocessing of electron micrographs of nucleic acid molecules for automatic analysis by computer. II. Noise removal and gap filling. Comput. Biomed. Res. 12:615-630. [10.13] Leslie, R. J., and J.D. Pickett-Heaps (1983) Ultraviolet microbeam irradiations of mitotic diatoms: Investiga• tion of spindle elongation. J. Cell Bioi. 96:548-561. [11.5] Leslie, R. J., W. M. Saxton, T. J. Mitchison, B. Neighbors, E. D. Salmon, and J. R. Mcintosh (1984) Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching. J. Cell Bioi. 99:2146-2156. [11.4] Levi, L. (1980) Applied Optics: A Guide to Optical System Design, Volume 2. Wiley, New York. [4.1, 4.3, 11.4; Figs. 4-7, 4-10]. Levinthal, C., and R. Ware (1972) Three dimensional reconstruction from serial sections. Nature 236:207-210. [11.5] Levinthal, C., E. Macagno, and C. Tountas (1974) Computer-aided reconstruction from serial sections. Fed. Proc. 33:2336-2340. [11.5] Lewis, R. (1985) Solid-state cameras. Electronic Imaging 4(1):44-52. [This article includes a two-page-long vendor list; 7.1, Postscript; Table 7-3] Lipkin, B.S., and A. Rosenfeld, eds. (1970) Picture Processing and Psychopictorics. Academic Press, New York. [1.2] Lipkin, L., P. Lemkin, B. Shapiro, and J. Sklansky (1979) Preprocessing of electron micrographs of nucleic acid molecules for automatic analysis by computer. Comput. Biomed. Res. 12:279-289. [10.13] Lipton, L. (1982) Foundation of Stereoscopic Cinema. Van Nostrand Reinhold, New York. [12.7] Liu, C. L., and J. W. S. Liu (1975) Linear Systems Analysis. McGraw-Hill, New York. [10.12] Loveland, R. P. (1981) Photomicrography: A Comprehensive Treatise, 2nd ed. Two volumes. Wiley, New York. [5.1, 5.6, 5.9] Ludvigh, E., and E. F. McCarthy (1938) Absorption of visible light by the refractive media of the human eye. Arch. Ophtholmol. 20:37-51. [Fig. 4-6] Macfarlane, T. W. R., S. Petrowski, L. Rigutto, and M. R. Roach (1983) Computer-based video analysis of cerebral arterial geometry using the natural fluorescence of the arterial wall and contrast enhancement techniques. Blood Vessels 20:161-171. [11.5] REFERENCES 523 Marcil, G. (1975) Microdensitometric data acquisition. Proc. Soc. Photo-Opt. Instrum. Eng. 48:23-33. [10.3] Marino, T. A., D. Biberstein, P. N. Cook, L. Cook, and S. J. Dwyer, III (1981) A quantitative morphologic analysis of interatrial muscle cells in the ferret heart. Anat. Rec. 201:31-42. [10.13] Marr, D. (1982) Vision: A Computational Investigation into the Human Representation and Processing of Visual Information. Freeman, San Francisco. [4.8] Marriott, F. H. C. (1962) Colour vision: Other phenomena. In: The Eye, Volume 2 (H. Davson, ed.). Academic Press, New York, pp. 273-298. [4.7] Martelli, A. (1976) An application of heuristic search methods to edge-on contour detection. Commun. ACM 19:73-83. [10.13] Martin, L. C. (1966) The Theory of the Microscope. American Elsevier, New York. [5.1, 5.5] Mathog, D., M. Hochstrasser, Y. Gruenbaum, H. Saumweber, and J. Sedat (1984) Characteristic folding pattern of polytene chromosomes in Drosophila salivary gland nuclei. Nature 308:414-421. [11.5] Matsumoto, S., K. Morikawa, and M. Yanagida (1981) Light microscopic structure of DNA in solution studied by the 4', 6-diarnidino-2-phenylindole staining method. J. Mol. Bioi. 152:501-516. [11.2] Mayall, B. H., and B. L. Gledhill, eds. (1979) Sixth Engineering Foundation Conference on Automated Cytology. J. Histochem. Cytochem. 27:1-641. [ll.4] McCutchen, C. W. (1967) Superresolution in microscopy and the Abbe resolution limit. J. Opt. Soc. Am. 57:ll90-ll92. [11.5] McGregor, G. N., H.-G. Kapitza, and K. A. Jacobson (1984) Laser-based fluorescence microscopy of living cells. Laser Focus/Electro-Optics Nov.:84-93. [11.2, 11.4] McNeil, P. L., R. F. Murphy, F. Lanni, and D.L. Taylor (1984) A method for incorporating macromolecules into adherent cells. J. Cell Bioi. 98:1556-1564. [11.2] Mendelsohn, M. L., B. H. Mayall, J. M. S. Prewitt, R. C. Bostrom, and W. G. Holcomb (1968) Digital transformation and computer analysis of microscopic images. Adv. Opt. Electron Microsc. 2:77-150. [ 1.2] Michael, C. R. (1969) Retinal processing of visual images. Sci. Am. 220(May):104-ll4. [4.4] Michel, K. (1981) Die Grundzuge der Theorie des Mikroskops in elementarer Darstellung. Wissenschaftliche Verlagsgeselschaft M. B. H., Stuttgart. [5.5] Middleton, R. G. (1981) Effectively Using the Oscilloscope. Sams, Indianapolis. [7.5] Miles, C. P., and D. L. Jaggard (1981) The use of optical Fourier transforms to diagnose pleomorphism, size and chromatin clumping in nuclear models. Anal. Quant. Cytol. 3:149-156. [10.13] Miller, K. (1985) Call to the colors: Progress toward a three-dimensional standard. Photonics Spectra 19(2):75- 82. [Fig. 4-19] Mitchell, P. W. (1983) Digital signal processing comes to TV: The signal's unchanged, but new sets will enhance the picture. High Technology Dec.:16-20. [Postscript] Montgomery, P. 0., F. Roberts, and W. Bonner (1956) The flying-spot monochromatic ultra-violet television microscope. Nature 177:ll72. [1.2, 11.5] Moore, J. H., C. C. Davis, and M. A. Coplan (1983) Building Scientific Apparatus: A Practical Guide to Design and Construction. Addison-Wesley, Reading, Mass., pp. 242-271. [11.4] Mueller, C. G., and V. V. Lloyd (1948) Stereoscopic acuity for various levels of illumination. Proc. Natl. Acad. Sci. USA 34:223-227. [4-21] Muirhead, T. (1961) Variable focal length mirror. Rev. Sci. Instrum. 32:210-211. [11.5] Nagashima, H., and S. Asakura (1980) Dark-field light microscopic study of the flexibility ofF-actin complex- es. J. Mol. Bioi. 136:169-182. [11.2] Nipkow, P. (1884) German Patent No. 30,105. [l.l] Nomarski, G. (1955) Microinterferometre diffefentiel a ondes polarisees. J. Phys. Radium 16:S9-SI3. [5.6] Noronha, S. F. R. (1981) Time base correctors: A must for quality correction. Photomethods May:56-58. [8.5] O'Kane, D. J., R. E. Kobres, and B. A. Palevitz (1980) Analog subtraction of video images for determining differences in fluorescence distributions. J. Cell Bioi. 87:229a. [9.3] Okazaki, K., and S. Inoue (1976) Crystal property of the larval sea urchin spicule. Dev. Growth Differ. 18:413- 434. [11.3] Olins, D. E., A. L. Olins, H. A. Levy, R. C. Durfee, S. M. Margie, E. P. Tinnel, and S. D. Dover (1983) Electron microscope tomography: Transcription in three-dimensions. Scie~e 220:498-500. [11.5] Olson, A. C., N. M. Larson, and C. A. Heckman (1980) Classification of cultured mammalian cells by shape analysis and pattern recognition. Proc. Natl. Acad. Sci. USA 77:1516-1520 [10.13] Oppenheim, A. V., and J. S. Lim (1981) The importance of phase in signals. Proc. IEEE 69:529-541. [11.2] Oppenheim, A. V., and R. W. Schafer ( 1975) Digital/mage Processing. Prentice-Hall, Englewood Cliffs, N.J. [6.5] Ortner, E. H. (1984) Through the viewfinder. Popular Science Apri1:64. [12.1] 524 REFERENCES Osborn, M., T. Born, H.-I. Koitsch, and K. Weber (1978) Stereo immunofluorescence microscopy. I. Three• dimensional arrangement of microfilaments, microtubules and tonofilaments. Cell 14:447-488. [12.7] Osterberg, H. (1955) Phase and interference microscopy. In: Physical Techniques in Biological Research, Volume I (G. Oster and A. W. Pollister, eds.). Academic Press, New York, pp. 377-437. [5.6, 11.3] Padawer, J. (1967) The Nomarski intereference-contrast microscope. An experimental basis for image in• terpretation. J. Roy. Microscop. Soc. 88:305-349. [5.6] Palevitz, B. A., D. I. O'Kane, R. E. Kobres, and N. V. Raikhel (1981) The vacuole system in stomatal cells of Allium. Vacuole movements and changes in morphology in differentiating cells as revealed by epifluores• cence, video and electron microscopy. Protoplasma 109:23-55. [11.2] Panitz, J. A., and D. C. Ghiglia (1982) Point projection imaging of unstained ferritin molecules on tungsten. J. Microsc. 127:259-264. [11.5] Parpart, A. K. (1951) Televised microscopy in biological research. Science 113:483-484. [1.2] Pask, C. (1976) Simple optical theory of super-resolution. J. Opt. Soc. Am. 66:68-70. [10.12] Pastan, I. H., and M. C. Willingham (1985) Endocytosis. In: Morphological Methods in Endocytosis. Plenum Press, New York, in press. [11.2] Paton, K. (1975) Picture description using Legendre polynomials. Computer Graphics and Image Processing 4:40-54. [10.13] Pavlidis, T. (1978) A review of algorithms for shape analysis. Computer Graphics and Image Processing 7:243-258. [10.13] Pavlidis, T. ( 1979) Methodologies for shape analysis. In: Biomedical Pattern Recognition and Image Processing (K. S. Fu and T. Pavlidis, eds.). Verlag Chemie, Weinheim, pp. 131-152. [10.13]. Pavlidis, T., and S. L. Horowitz (1974) Segmentation of plane curves./EEE Trans. Comput. 23:860-870. [10.13] Persoon, E., and K. S. Fu (1977) Shape discrimination using Fourier descriptors. IEEE Trans. Syst. Man Cybern. 7:170-179. [10.13] Persson, B.-E., and K. R. Spring (1982) Gallbladder epithelial cell hydraulic water permeability and volume regulation. J. Gen. Physiol. 79:481-505. [11.3] Petniii, M., M. Hadravsky, M. D. Egger, and R. Galambos (l%8) Tandem-scanning reflected-light micro- scope. J. Opt. Soc. Am. 58:661-664. [1.2, 11.5] Pike, W. S. (1962) Some television image enhancement techniques. Ann. N.Y. Acad. Sci. 97:395-407. [1.2] Piller, H. (1977) Microscope Photometry. Springer-Verlag, Berlin. [5.1, 5.4, 5.5, 5.6, 11.4] Pirenne, M. H., and F. H. C. Marriott (1%2) Visual functions in man In: The Eye, Volume 2 (H. Davson, ed.). Academic Press, New York, pp. 3-217. [4.2, 4.3; Fig. 4-ll] Pittman, R. N., and B. R. Duling (1975) Measurement of percent oxyhemoglobin in the microvasculature. J. Appl. Physiol. 38:321-327. [9.5] Poenie, M., I. Alderton, R. Y. Tsien, and R. A. Steinhardt (1985) Changes of free calcium levels with stages of the cell division cycle. Nature 315:147-149. [11.2] Polyak, S. (1957) The Vertebrate Visual System. University of Chicago Press, Chicago. [Figs. 4-l, 4-2] Pratt, W. K. (1978) Digital/mage Processing. Wiley, New York. [4.5, 6.5, 10.4, 10.7, IO.ll; Figs. 4-12, 4-13, 10-15] Preston, K. (1978) Biomedical image processing. In: International Symposium on Advances in Digital/mage Processing: Theory, Application, Implementation (P. Stucki, ed.) Plenum Press, New York, pp. 125-146. [10.14] Preston, K. (1979) Computer hardware for biomedical pattern recognition. In: Biomedical Pattern Recognition and Image Processing (K. S. Fu and T. Pavlidis, eds.). Verlag Chemie, Weinheim, pp. 213-231. [10.14] Preston, K., and A. Dekker (1980) Differentiation of cells in abnormal human liver tissue by computer image processing: A preliminary investigation to diagnostic microscopy. Anal. Quant. Cytol. 2:203-220. [10.5, 10.13] Prewitt, J. M.S., and M. L. Mendelsohn (1966) The analysis of cell images. Ann. N.Y. Acad. Sci. 128:1035- 1053. [10.13] Pycock, D., and J. T. Christopher (1980) Use of the Magiscan image analyses in automated uterine cancer cytology. Anal. Quant. Cytol. 2:195-202. [10.13] Quate, C. F. (1980) Microwaves, acoustic and scanning microscopy. In: Scanned Image Microscopy (E. A. Ash, ed.). Academic Press, New York, pp. 23-55. [11.5] Ramachandran, G. N., and S. Ramaseshan (1%1) Crystal optics. In: Handbuch der Physik, Volume XXV/I (S. Flugge, ed.). Springer-Verlag, Berlin, pp. l-217. [5.6] Raster Graphics Handbook (1980) Conrac Corp., Covina, Calif. (Forthcoming editions will be published by Van Nostrand, Princeton, N.J.) [7.4, 7.5; Figs. 7-30 to 7-34, 7-41; Tables 6-l, 6-3] RCA Electro-Optics Handbook (1974) RCA Solid State Division, Electro-Optics and Devices, Lancaster, Pa. [4.1, 5.7, 7.2, 12.1; Figs. 4-l, 4-5,4-17,4-18,7-3 to 7-7,7-12,7-16, 7-18; Table 7-2] REFERENCES 525 Reedy, M. C., M. K. Reedy, and R. S. Goody (1983) Co-ordinated electron microscopy and X-ray studies of glycerinated insect flight muscle. II. Electron microscopy and image reconstruction of muscle fibers fixed in rigor, in ATP and in AMPPNP. J. Muscle Res. Cell Motil. 4:55-81. [11.5] Reid, M. H. (1982) Quantitative stereology and radiologic image analysis. Part 1: Computerized tomography and ultrasound. Med. Phys. 9:346-360. [11.5] Renwanz, K. (1983) Panasonic field report: Panasonic RECAM Camera Recording System. Broadcast En• gineering March:276-285. [8.3] Reynolds, G. T. (1964) Evaluation of an image intensifier system for microscopic observations. IEEE Trans. Nucl. Sci. 11:147-151. [1.2] Reynolds, G. T. (1966) Sensitivity of an image intensifier film system. Appl.·Opt. 5:577:-583. [11.4] Reynolds, G. T. (1972) Image intensification applied to biological problems. Quart. R-ev. Biophys. 5:295-347. Reynolds, G. T. (1972) Image intensification applied to biological problems. Q. Rev. Biophys. 5:295-347. [7.1, 11.2) Reynolds, G. T., and D. L. Taylor (1980) Image intensification applied to light' microscopy. BioScience 30:586-592. [11.2] Rich, E. S., Jr., and J. E. Wampler (1981) A flexible, computer-controlled video microscope capable of quantitative spatial, temporal and spectral measurements. Clin. Chern. (N.Y.)" 27:1558-1568. [10.13] Roos, K. P., and A. J. Brady (1982) Individual sarcomere length determination from i1!olated cardiac cells using high-resolution optical microscopy and digital image processing. Biophys.J. 40:233-;244. [11.4] Rose, A. (1947) The sensitivity performance of the human eye on an absolute scale.f Opt. Soc. Am. 38:196- 207. [4.1, 4.5] Rose, A. (1973) Vision: Human and Electronic. Plenum Press, New York. [4.1, ·4.9, 11.4] Rose, B., and W. Loewenstein (1975) Calcium ion distribution in cytoplasm visualized ~y aeqqorin: Diffusion in cytosol restricted by energized sequestering. Science 190:1204-1206. [11.2] Rose, B., and W. Loewenstein (1976) Permeability of a cell junction. and the' local cytoplasmic free ionized calcium concentration: A study with aequorin. J. Membr. Bioi. 28:87-119. [11."2] Salmon, E. D., M. McKeel, and T. Hays (1984a) Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine. J. Cell Bioi. 99:1066-1075. [9.5, 11.5] Salmon, E. D., W. M. Saxton, R. J. Leslie, M. L. Karow, and J. R. Mcintosh (l984b)Diffusion coefficient of fluorescein-labelled tubulin in the cytoplasm of embryonic cell of a sea urchin: Video image analysis of fluorescence redistribution after photobleaching. J. Cell Bioi. 99:2157-2164. [lL2, 11.4] Salzberg, B. M., A. Grinvald, L. B. Cohen, H. V. Davila, and W. N. Ross (i977) Optical recording of neuronal activity in an invertebrate central nervous system: Simultaneous monitoring of several neurons. J. Neurophysiol. 40:1281-1291. [6.2, 11.4] . Sanger, J. W., B. Mittal, and J. M. Sanger (1984) Interaction offluorescently labelled' contractile proteins with the cytoskeleton. J. Cell Bioi. 99:918-928. [11.2] Sato, H., G. W. Ellis, and S. Inoue (1975) Microtubular origin of mitotic spindle fohn birefringence: Demon• stration of the applicability of Wiener's equation. J. Cell Bioi. 67:501-517. [ll,.3]: Sauer, B. (1983) Semi-automatic analysis of microscopic images of the human cerebral cortex using the grey level index. J. Microsc. 129:75-87. [11.4] Saxton, W. M., D. L. Stemple, R. J. Leslie, E. D. Salmon, M. Zavortink, and J. R. Mcintosh (1984) Tubulin dynamics in cultured mammalian cells. J.·Cell Bioi. 99:2175-2186. [11.4} Schade, 0. H., Sr. (1975) Image Quality-A Comparison of Photographic ·and Television Systems. RCA Laboratories, Princeton, N.J. [11.2] Schatten, G. (1981) The movements and fusion of the pronuclei at fertilization ef the sea urchin Lytechinus variagatus: Time-lapse video microscopy. i· Morpho/. 167:231-247. [3.1] Schatten, G., H. Schatten, T. H. Bestor, and R. Balczon (1982) Taxol inhibits th~ nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs. J. Cell Bio/.'94:455..:.4&5. [11.3] Schmidt, W. J. (1924) Die Bausteine des Tierkiirpers in polarisiertem Lichte. COheil, ·Jiloim. [5.6, 11.3] Schmidt, W. J. (1937) Die Doppelbrechung von Karyoplasma, Zytoplasma und'MNaplasma. Protoplasma• Monographien, Volume II. Bomtraeger, Berlin. [5.6, 11.3] Schnapp, B. J., R. D. Vale, M. P. Sheetz, and T. S. Reese (1985) Single microtubules from squid axoplasm support bidirectional movement of organelles. Cell, 40:455-462. [11.3; Fig.fll~8] Schultz, M. L., L. E. Lipkin, M. J. Wade, P. F. Lemkin, and G. Carman (l9.74)difgh resolution shading correction. J. Histochem. Cytochem. 22:751-754. [10.10] Schwartz, A. A., and J. M. Soha (1977) Variable threshold zonal filtering. Appl. Opt;,16:1779-178l. [10.13] Shack, R. V. (1974) On the significance of the phase transfer function. Proc. SPIE'46:39- Shaw, G. B. (1979) Local and regional edge detectors: Some comparisons. Computer Graphics and Image Processing 9:135-149. [10.13] Sher, L. D., and C. D. Barry (1985) The use of an oscillating mirror for three-dimensional displays. In: New Methodologies in Studies of Protein Configuration (T. T. Wu, ed.). Van Nostrand-Reinhold, Princeton, N.J. in press. [11.5; Fig. 11-14] Shillaber, C. P. (1944) Photomicrography in Theory and Practice. Wiley, New York. [3.7, 5.1, 5.4, 5.5, 5.6, 5.9, 11.5] Shimomura, 0., and F. H. Johnson (1976) Calcium-triggered luminescence of the photoprotein aequorin. Symp. Soc. Exp. Bioi. 30:41-54. [Fig. 11-1] Shlaer, S., E. L. Smith, and A. M. Chase (1942) Visual acuity and illumination in different spectral regions. J. Gen. Physiol. 25:553-569. [4.4; Fig. 4-9] Shoemaker, R. L., P. H. Bartels, D. W. Hillman, J. Jones, D. Kessler, R. V. Shack, and P. Jukobratovich (1982) An ultrafast laser scanner microscope for digital image analysis./EEE Trans. Biomed. Eng. 29:82- 91. [10.3] Shore, S. H. (1975) An overview of sensitometry and densitometry for the macro and micro conditions. Proc. Soc. Photo-Opt. Instrum. Eng. 48:2-17. [10.3] Showalter, L. C. (1969) Closed Circuit 1V for Engineers and Technicians. Sams, Indianapolis. [Preface 7 .5, 9.2] Shulman, A. R. (1970) Optical Data Processing. Wiley, New York, pp. 324-357. [Appendix I] Siemens, A., R. J. Walter, L.-H. Liaw, and M. W. Berns (1982) Laser stimulated fluorescence of submicron regions within single mitochondria of rhodamine treated myocardial cells in culture. Proc. Natl. Acad. Sci. USA 79:466-470. [10.13] Sklansky, J. (1978a) On the Hough technique for curve detection. IEEE Trans. Comput. 27:923-926. [10.13] Sklansky, J. (1978b) Image segmentation and feature extraction. IEEE Trans. Syst. Man Cybern. 8:237-247. [10.7, 10.11, 10.13; Fig. 10-19] Sklansky, J., and V. Gonzalez (1979) Fast polygonal approximation of digitized curves. Pattern Recognition 11:604-609. [10.13] Smith, F. H. (1956) Microscopic interferometry. In: Modern Methods of Microscopy (A. E. J. Vickers, ed.). Butterworths, London, pp. 76-86. [5.6] Smith, K. T., S. L. Wagner, and M. J. Bottema (1984) Sharpening radiographs. Proc. Natl. Acad. Sci. USA 81:5237-5241. [11.5] Smith, W. J. (1966) Modern Optical Engineering. McGraw-Hill, New York. [II.2] Sokoloff, L., M. Reivich, C. Kennedy, M. Des Rosiers, C. Patlak, K. Pethigrew, 0. Sakurada, and M. Shinohara (1977) The [l"C]-deoxyglucose method for the measurement of local cerebral glucose oxidation: Theory, procedure and normal values in the conscious and anesthetized albino rat. J. Neurochem. 28:897- 916. [10.3] Spencer, M. (1982) Fundamentals of Light Microsopy. Cambridge University Press, London. [Preface, 5.1] Spotlight: The Super Slo-Mo System (1984) Video Systems Sept.:83-85. [8.6] Spring, K. (1979) Optical techniques for the evaluation of epithelial transport processes. Am. J. Physiol. 237(3):F167-F174. [9.5, 11.1, 11.3] Spring, K. R. (1983) Application of video to light microscopy. In: Membrane Biophysics, Volume II. Liss, New York, pp. 15-20. [11.1] Stein, P. G., L. E. Lipkin, and H. M. Shapiro (1969) Spectra II: General-purpose microscope input for a computer. Science 166:328-333. [1.2] Stroke, G. W. ( 1969) An Introduction to Coherent Optics and Holography, 2nd ed. Academic Press, New York. [5.6] Strong, J. (1958) Concepts of Classical Optics. Freeman, San Francisco. [5.6] Sullivan, J. A., and G. L. Mandell (1983) Motility of human polymorphonuclear neutrophils: Microscopic analysis of substrate adhesion and distribution ofF-actin. Cell Motil. 3:31-46. [11.3] Summers, K., and I. R. Gibbons (1971) Adenosine-triphosphate-induced sliding of tubules in trypsin-treated flagella of sea urchin sperm. Proc. Natl. Acad. Sci. USA 68:3092-3096. [11.2] Swann, M. M., and J. M. Mitchison (1950) Refinements in polarized light microscopy. J. Exp. Bioi. 27:226- 237. [11.3] Swinton, A. A. C. (1908) Distant electric vision. Nature 78:151. [1.1] Tanasugarn, L., P. McNeil, G. T. Reynolds, and D. L. Taylor (1984) Microspectrofluorometry by digital image processing: Measurement of cytoplasmic pH. J. Cell Bioi. 98:717-724. [11.2] Tannas, L. E. (1981) Flat-Panel Displays. Van Nostrand-Reinhold, Princeton, N.J. [Fig. 7-33] Taylor, D. L., and Y.-L. Wang (1980) Fluorescently labelled molecules as probes of the structure and function of living cells. Nature 284:405-410. [11.2] REFERENCES 527 Thaer, A. A., and M. Sernetz, eds. (1973) Fluorescence Techniques in Cell Biology. Springer-Verlag, Berlin. [5.6] Thaer, A., M. Hoppe, and W. J. Patzelt (1982) The ELSAM acoustic microscope. Leitz Mitt. Wiss. Tech. 8:61- 67. [11.5] Tilney, L. G., and S. Inoue (1982) Acrosomal reaction of Thyone sperm. II. The kinetics and possible mechanism of acrosomal process elongation. J. Cell Bioi. 93:820-827. [Figs. 9-ll, 9-12] Toda, T., M. Yamamoto, and M. Yanagida (1981) Sequential alterations in the nuclear chromatin region during mitosis of the fission yeast Schizosaccharomyces pombe: Video fluorescence microscopy of synchronously growing wild-type and cold-sensitive cdc mutants by using a DNA-binding fluorescent probe. J. Cell Sci. 52:271-287. [11.2] Tsien, R. Y. (1983) Intracellular measurements of ion activities. Annu. Rev. Biophys. Bioeng. 12:9l-ll6. [11.2] Tycko, B., C. H. Keith, and F. R. Maxfield (1983) Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line. J. Cell Bioi. 97:1762-1776. [11.2] van der Horst, C. I. C., C. M. de Weert, and M.A. Bouman (l%7) Transfer of spatial chromaticity-contrast at threshold in the human eye. J. Opt. Soc. Am. 57:1260-1266. [Fig. 4-13] van Wezel, R. (1981) Video Handbook (G. J. King, ed.). Butterworths, London. [Preface, 8.8, 9.2; Fig. 9-7] von Bally, G., and P. Greguss, eds. (1982) Optics in Biomedical Sciences. Springer-Verlag, Berlin. [11.5] Vossepoel, A.M., A. W. Smeulders, and K. van den Brock (1979) DIODA: Delineation and feature extraction of microscopical objects. Comput. Programs Biomed. 10:231-244. [10.13] Wahlstrom, E. E. (1960) Optical Crystallography, 3rd ed. Wiley, New York. [11.3] Walker, J. (1982) Simple optical experiments in which spatial filtering removes the "noise" from pictures. Sci. Am. 247(5):194-206. [10.12, Appendix I] Walker, P. M. B. (1956) Ultraviolet absorption techniques. In: Physical Techniques in Biological Research, Volume III (G. Oster and A. W. Pollister, eds.). Academic Press, New York, pp. 401-487. [11.2] Walter, R. J., and M. W. Berns (1981) Computer-enhanced video microscopy: Digitally processed microscope images can be produced in real time. Proc. Nat[. Acad. Sci. USA 78:6927-6931. [10.8, 11.3] Walton, A. J., and G. T. Reynolds (1984) Sonoluminescence. Adv. Phys., 33:595-660. [11.2] Wampler, J. E. (1985) Low-light video systems. In: Bioluminescence and Chemiluminescence: Instrumentation and Applications, Volume 2 (K. van Dyke, ed.). CRC Press, Boca Raton, Aa., pp. 123-146. [11.2] Wang, Y.-L. (1984) Reorganization of actin filament bundles in living fibroblasts. J. Cell Bioi. 99:1478-1485. [11.2] Wang, Y.-L., J. M. Heiple, and D. L. Taylor (1982) Auorescent analog cytochemistry of contractile proteins. Methads Cell Bioi. 258:1-ll. [11.2] Weber, D. M. (1984) Digital circuits point towards better TV sets. Electronics Week 13:49-53. [Postscript] Weiner, P. K., S. V. Forgue, and P.R. Goodrich (1950) The Vidicon photoconductive camera tube. Electronics 23:70-73. [1.2] Wenzelides, V. K., I. Hillmann, and K. Voss (1982) Zur Darstellung der Leberzellgrenzen mit Anilinblau und ihre vollautomatische Erfassung mit einem Bildanalysesystem. Acta Histochem. 70:193-199. [11.4] Weszka, J. S. (1978) A survey of threshold selection techniques. Computer Graphics and Image Processing 7:259-265. [10.5, 10.13] Weszka, J. S., and A. Rosenfeld (1979) Histogram modification for threshold selection./EEE Trans. Syst. Man Cybern. 9:38-52. [10.13] Wheeler, H. A. (1939) Wide band amplifiers for television. Proc. IRE 27:420. [1.1] Williams, M. A. (1977) Quantitative methods in biology. In: Practical Methads in Electron Microscopy, Volume 6 (A. M. Glauert, ed.) Elsevier-North Holland, New York, pp. l-234. [11.5] Willingham, M. C., and I. Pastan (1978) The visualization of fluorescent proteins in living cells by video intensification microscopy (VIM). Cell13:501-507. [11.2] Willingham, M. C., and I. H. Pastan (1983) Image intensification techniques for detection of proteins in cultured cells by fluorescence microscopy. Methads Enzymol. 98:266-283, 635 (addendum). [11.2] Wilson, A. (1984) Solid-state camera design and applications. Electronic Imaging 3(4):38-46. [Table 7-3] Wilson, T., and C. Sheppard (1984) Theory and Practice of Scanning Optical Microscopy. Academic Press, New York. [11.5] Wright, F. E. (l911)The Methods of Petrographic-Microscopic Research: Their Relative Accuracy and Range of Application. Carnegie Institution of Washington, pub!. no. 158, Washington, D.C. [11.3] Wright, W. D. (1940) Colour vision and chromaticity scales. Nature 146:155-158. [4.7] Wrigley, N. G., R. K. Chillingworth, E. Brown, and A. N. Barrett (1982) Multiple image integration: A new method in electron microscopy. J. Microsc. 127:201-208. [11.5] Yamazaki, S., T. Maeda, and T. Miki-Nomura (1982) Flexural rigidity of singlet microtubules estimated from 528 REFERENCES statistical analysis of fluctuating images. In: Biological Functions of Microtubules and Related Structures (H. Sakai, H. Mohri, and G. G. Borisy, eds.). Academic Press, New York, pp. 41-48. [11.2] Yanagida, T., M. Nakase, K. Nishiyama, and F. Oosawa (1984) Direct observation of motion of single F-actin filaments in the presence of myosin. Nature 307:58-60. [11.2] Young, I. T., J. E. Walker, and J. E. Bowie (1974) An analysis technique for biological shape. 1./nf. Control 25:357-370. [10.13] Young, I. T., R. L. Pevenini, P. W. Verbeek, and P. J. van Otterloo (1981) A new implementation for the binary and Minkowski operators. Computer Graphics and Image Processing 17:189-210. [10.13] Young, J. Z., and F. Roberts (1951) A flying-spot microscope. Nature 167:231. [1.2, 11.5] Young, T. (1802) On the theory of light and colours. Philos. Trans. 92:12-48. [4.7] Zahn, C. T., and R. Z. Roskies (1972) Fourier descriptors for plane closed curves. IEEE Trans. Comput. 21:269-281. [10.13] Zernike, F. (1955) How I discovered phase-contrast. Science 121:345-349. [5.6] Zernike, F. (1958) The wave theory of microscopic image formation. In: Concepts of Classical Optics (1. Strong, ed.). Freeman, San Francisco, Appendix K, pp. 525-536. [5.6] Zucker, S. (1976) Region growing: Childhood and adolescence. Computer Graphics and Image Processing 5:382-399. [10.13] Zworykin, V. K. (1931) Photocell theory and practice. J. Franklin lnst. 212:1-41. [1.1] Zworykin, V. K. (1933) Description of an experimental television system and the kinescope. Proc. IRE 21:1655-1673. [1.1; Figs. 1-2, 1-3] Zworykin, V. K. (1934) The iconoscope-A modern version of the electric eye. Proc. IRE 22:16-32. [1.1; Figs. 1-2, 1-3] Zworykin, V. K., and L. E. Flory (1951) Television as an educational and scientific tool. Science 113:483. [1.2] Zworykin, V. K., and G. A. Morton (1954) Television: The Electronics of Image Transmission in Color and Monochrome, 2nd ed. Wiley, New York. [1.1; Figs. 1-1, 12-15, 12-16] Zworykin, V. K., E. A. Ramberg, and L. E. Flory (1958) Television in Science and Industry. Wiley, New York. [1.1, 1.2] CITED PAMPHLETS Applications Guide for VII Test Instruments. Visual Information Institute, Inc., P.O. Box 33, Xenia, Ohio 45385. CCD: The Solid State Imaging Technology. Fairchild CCD Imaging, 3440 Hillview Avenue, Palo Alto, Califor• nia 94304. Display Devices. RCA Solid State Division, Electro-Optics and Devices, New Holland Avenue, Lancaster, Pennsylvania 17604. Electro-optic devices utilizing quadratic Pl..Z/' ceramic elements. Report No. SLA 73-0777. Sandia Laborato• ries, Albuquerque, New Mexico 87115. IEEE Standards on Television: Methods of measurement ofaspect ratio and geometric distortion. Std 202-1954. Reaffirmed 1972. Institute of Electrical and Electronic Engineers, Inc., 345 East 47th Street, New York, New York 10017. Imaging Device Performance in the C-1000 Camera. Hamamatsu Systems, Inc., 332 Second Avenue, Wal• tham, Massachusetts 02154. Imaging Devices. Publication IMD 101. RCA Solid State Division, Electro-Optics and Devices, New Holland Avenue Lancaster, Pennsylvania 17604. Kodak Films for Cathode-Ray Tube Recording. Publication P-37. Eastman Kodak Company, Rochester, New York 14650. Kodak Professional Black-and-White Films (1976) Publication F-5. Eastman Kodal Company, Rochester, New York 14650. Performance Test for Medica/Imaging Monochrome Display Devices. Visual Information Institute, Inc., P.O. Box 33, Xenia, Ohio 45385. Photographing Television Images. Publication No. AC-10. Eastman Kodak Company, Rochester, New York 14650. Sony Basic Video Recording Course. Sony Video Communications, 700 West Artesia Boulevard, Compton, California 90220. REFERENCES 529 Stereo-Color TV Projection System. 3D Technology Corporation, 4382 Lankershim Boulevard, North Holly• wood, California 91602. Technology in Retrospect and Critical Events in Science (1969). National Science Foundation, Washington, D.C. 20550. Test Procedure for Precision Raster Scan Displays. Visual Information Institute, Inc., P.O. Box 33, Xenia, Ohio 45385. Index Abbe, 133 Acoustic wave theory of microscope, 470-471 anisotropy of, 478', 478f Abbe limit, 13g; see also Airy disk; Confocal microscope, microscope using, 418n doubling resolution with; Resolution (microscope), velocity of, 478, 478f limit of Acrosomal process, growth kinetics, 319-320, 322f, 323f Aberration, 13g, 104, 133n Actin chromatic, 19g, 133-135, 133n, 138-139 form birefringence of, 492, 501 collector lens, 97f, 102f, 103f, 105, 105n, 138-139 growth kinetics of filaments, 322f, 323f coma, 20g, 133 mdi vidual threads of molecules condenser, 106, 127, 138, 139 darkfield microscopy of, 40 I correction collar, 13lt, 134, 135t fluorescence microscopy of, 400 effect on diffraction pattern, 114n, 115-118, 115n, 404, Action potential, montaged on monitor, 44lf 404n-405n, 497f, 498, 498n Active picture area, 14g, 156, 173f reference book on, 114 Active scan lines, 14g, 156 in eye, vs. illumination, 74 H-scan hne , 159f, 172, 173f vs. immersion, 127, 134 V field, 159f objective, 100, 133-136 Adaptation of human eye, 72-74, 76f ocular, 134-135 vs. threshold modulation contrast, 80f and phase errors, 469 see also Dark adaptation spherical, 48g, 133, 133n; see also Spherical aberration AID converter, 14g, 330, 330f vs. tube length, 134 special, high-speed, 295 see also Astigmatism; Distortion see also Digitizer Aberration-free lens Aequorin, 396, 398f-399f image fidelity in, 469 AGC: see Automatic gain control see also Perfect lens Aging Absorption vs. crystalline lens transmission, 77 contrast generation by, 120, 122 vs. UV output of mercury arc lamp, 132 diffraction pattern by, 119-120 AHFC, 14g, 187, 259, 291 3-dimensional, Ill, 117f Ahrens prism, 483; see also Prism, calcite by highly-absorbing materials, 413n Airy dtsk, 14g, 112-118, 112f, 113f, 114f, 470, 470f, internal calibration of, 323 497f by ocular medium, 74-77, 74n of absorbing objects, 119-120 Absorption curve 3-dimensional diffraction pattern, Ill, 116f isolated retinal rods and cones, 415 effect of nonuniform aperture, 127n of rhodopsin, 74n see also Diffraction pattern Accommodation of human eye, 13g, 72 Algebraic manipulation of two images, 356-358, 375, effect on microscope depth of field, 118 380f, 383-384; see also ALU Achromatic aplanatic condenser, 14g, 139 Aliasing, 14g, 17ln, 471-472, 472f; see also Nyquist Achrorr.atic lens, 14g frequency doublet, 95f, %f Alignment of microscope optics, 66, 105-107, 106n; see objective, 13lt, 135t, 136 also Aperture functton UV-visible, 401 Alignment tape, 14g, 299 Achromatic mirror objective, 401 ALU, 14g; see also Arithmetic logic unit Acknowledgements, ix-x, 145n, 392, 477n Amoeboid motility, tracking of, 384 as video title, 433 Amoeboid streaming, 410 Suffixes g,f,n, and t denote Glossary, figure, footnote, and table, respectively. For entries starting with a numeral, look under the letters immediately following the numeral. Thus "3-D video" appears be· tween the entries "Dust cap" and "Dynamic changes." 531 532 INDEX Amplifier Annulus, phase-contrast, 122, 139t characteristics of, 173- 176, 174f, 175f, 178- 179, 179f, Anode, 15g 181,229-231,246 in iconoscope, 3, 3 f clipping by, 179, 180f, 312 m intensifier tube, 195f gain of, 246, 311, 312f, 315f, 316f; see also Automatic in picture tube, 242, 243f gain control Anomalous diffraction, 404n-405n, 497f gamma of, 247f, 311, 313f, 314f, 315f Antenna (lens mount as), 67f gray level manipulation with, 311- 314, 312f, 313f, Antireflection baffle, 429; see also Stops 314f, 315f, 316f Antireflection coating nonlinear, 247f, 311-314, 313f, 314f, 315f, 316f appearance when wet, 145 noise in, 315-319 cleaning lenses having, 140 in vidicon cameras, 181 , 215, 217 flare in the absence of, 107, 132 preamplifier, 181 , 274 hot spots from lenses without, 67 processing, 42g, 185, 243 improved transmittance by, 13 2 see also Circuits multilayer, 132n Amplifying projection lenses, 135 for polarization microscopy, 145n Amplitude modulation, 14g, 272 reflection loss without, 107n, 132 Amplitude response, 14g Antitubulin fluorescence, 402f definition and measurement of, 203, 206f Aperture (microscope), lSg loss of, at high frequency, 257f role of regulating, 64, 108-110, 108f, 109f of vidicons, at 400 TV lines, 194t unevenly illuminated, 105 see also Amplitude response curve see also Condenser iris diaphragm; Condenser NA; Amplitude response curve, 14g Scrambler of Chalnicon, 204f, 205f Aperture (video), !Sg of Newvicon, 205f scanning, vs. camera resolution, 229- 231 of Saticon, 204f in vidicon camera tube, 192 of SIT, 207f see also Aperture correction of sulfide vidicon, 204f Aperture correction, lSg see also Amplitude response; Contrast transfer function; in high-resolution camera, 229-231 Limiting resolution; MTF, in video; Spatial in high-resolution monitor, 246, 258n frequency horizontal and vertical, 246n Anaglyph, red-green, 15g Aperture function, I Sg effect of color blindness on, 451 n aberrations introduced by non-standard, 114 for stereoscopic video, 451, 452f asymmetric shading in image from , 90 for stereoscopy, 450 corrected by polarization rectifier, 497f Analog enhancement effect on diffraction pattern, 114n, l!Sn, 118 of color contrast, 413-415 effect on high-spatial-frequency components, 374 in DIC microscopy, 322f, 408f, 410 influence on Fourier transform, 374f in polarized-light microscopy, 7f, 8f, 405-406, 405n, modified by nonuniform filter, 373-374 406f, 407f selectively modifying, 419-420 preceding digital image processing, 345-346, 405 Aperture mask, 260, 26lf see also Amplifier; Digitally enhanced image; Image Aperture plane, !5g processing, analog; Living cell (video microscopy); eyepoint as, 102-103, 102f Signal processing, analog in Koehler illumination, 97f, 101-103, 102f Analog signal processing: see Signal processing, analog masking to induce stereoscopy, 447-452, 449f, 450f, Analog-to-digital converter: see AID converter 452f, 453f Anaxial illumination, 412 Aperture-scanning microscopy, 419-420 video-enhanced thin optical sections, 412f Aphakia, 15g, 74n Animation recording Apochromatic objective lens, 15g, 133 customizing VTR for, 289n Applicator stick, 69 with OMDR, 289, 304 swab stick for lens cleaning, 143-145, 144f use in V-to-F transfer, 445 Archimedes spiral VTR with capability for, 187n, 278, 279t, 285n, 289 focusing screen moving as, 420-421, 420f Anisotropy, 15g, 478 in Nipkow disk, 2-3, 2f, 416-417 of acoustic velocity, 478, 478f Arc lamp, l28t-129t of dielectric polarizabilities, 480-481 , 480f, 485 carbon, 3 vs. isotropy, 478 caution on ignition, 61, 296 mechanical, 478, 488- 491, 490f nonuniform luminous density, 127n optical, 404, 477, 480- 492 see also Mercury arc lamp; Xenon arc lamp of absorbtion spectra, 483f, 484- 485, 485f, 486f Area, 377, 378f of refractive index, 480- 483, 480f, 488-492, 489f, average density of, 340-341 490f measured with gray level histogram, 339-340 in PVA, 485n, 489-490, 489f, 490f selective manipulation of imago in, 383-384, 387 reference books on, 404, 500-SO I see also Boundary; Edge map in wood, 478, 478f Arithmetic logic unit, !Sg see also Birefringence; Dichroism interactive, 387-391, 390f INDEX 533 ArithmetiC logic unit (cont.) Azimuth orientation video-rate of birefringent specimen, 487f, 488f, 489f, 492 logic operations with, 380f, 383, 387, 388t-390t, 512 of DNA in sperm, 498f, 499, 499f overlay masking with , 380f, 383-384, 387, 388t-389t Azimuth recording, 16g Array, photodiode: see Photodiode array in Beta and VHS VCRs, 266-267, 276 Array processor, 15gr 330-331 , 330f in Betamax format, 266f systolic, 422n introduction of, 5 Artifact optical, 413 in video microscopy, 11 Back aperture of objective lens, 16g, 98f Aspect ratio, 15g inspection of, 106, 109f, 110 and active scan line, 172, 173f, 473 see also Bertrand lens effect on distortion, 251, 358 Back focal plane of objective lens in high-resolution video, 165t as Fourier plane, 373 standard, 156, 156n, 243 see also Back aperture; Rear focal plane Assemble editing, 16g: see also Editing, assemble Background gray level Astigmatism, 16g effect on gray level thresholding, 356, 358f from calcite prism, 483n, 495f effect of ratioing on, 364 in video camera, 172 Background subtraction, 17g in monitor, 259 by analog processing, 7f, 311-312, 345-346, 405 Astronomy: see Extremely-low-light-level imaging by adjusting pedestal, 405 , 406f Attenuation, 16g in monitor, 246, 247f in optical scrambler, 126n, 127n, 139n, 241 by digital image processing, 342-345, 343f, 511-512 Audio by edge sharpening, 371 OMDR with capability for, 303n, 304t effect on fluorescence , 356, 358f vs. video and frame averaging, 183f, 184f, 409f bandwidth, 181 motion detection by , 356, 357f frequency response curve, 179f for mottle subtraction, 9f, 70, 410 magnetic recording, 263-264 for shading compensation, 9f, !Of, 69-70, 183f VU meter, 53g, 436 see also Analog enhancement; Contrast enhancement see also Audio dubbing; Audio track (analog); Contrast enhancement/stretching (digital) Audio/CfL stack, 268, 269f, 277f Background wave Audio cue, 298, 433, 437, 438f blocking out of, 119f Audio dubbing, 16g, 267 illuminating the specimen, 119-122, 119f, 120f after editing, 438 interference with diffracted wave, 120, 120f, 121 on 112-inch VCR, 437n Back porch, 17g; see also H back porch on VTR channel I, 287, 298 Ball chart, 256, 258f; see also Test pattern from VTR to VTR, 433f, 434f, 436 Band-pass filter: see Filter, band-pass Audio head, 16g Bandwidth, 17g location of in VCR, 268, 269f, 277f compression of AUDIO IN/ OUT jack, !6g, 298, 436 automatic, at low light levels, 16g, 13ln, 233n Audio track, 16g in color video encoding, 238-242 recording data on, 298, 423 and noise reduction, 13ln, 317, 318f SMPTE time code on, 275 by slow-scan video, 317 on VCR tape, 265f, 266f, 267 , 275-276 in VTR recording, 272-273 Audio transfer, 436 in high-resolution monitor, 258 Auto-black, 16g, 65f, 66, 108, 232 in high-resolution TV, 165t, 247 Autofluorescence and bioluminescence, 396, 400f vs. horizontal resolution, 175-176, 175n, 176t, 233 Auto-iris, 16g, 240-241 limitation of Automatic bandwidth compression, 16g, 233n for broadcast TV, 154n Automatic gain control, 16g in NTSC video, 260 in camera, 217, 233 in TV receivers, 242 in VTR, 273 vs. noise, 181, 182, 233 , 315-317, 318f Automatic horizontal frequency control, 16g, 187, 259, vs. rise time, 173-176, 175f, 176t 291 of video amplifiers, 231 Automatic pedestal control, 16g, 232 Beam-splitting mirror, 451-452, 455f Automatic target control, 16g, 216-217, 233 Benzene, 140, 14lf Auto-off, on VTR, 287 Bertrand lens, 17 g Autoradiographic grains, quantitation of, 364, 365f inspection with Auto-rewind, 287 for conoscopic image, II 0, 493f Average picture level, 16g, 259 of microscope optical train, 69 Averaging: see Image averaging; Noise averaging (spatial); of objective back aperture, 106 Noise averaging (temporal) Beta format, 17g Averted vision, 78 azimuth of magnetic fields in, 266-267, 266f Axial magnification, 35g, 97, 100 blank tapes for, tests on, 30ln Axial resolution, 101, 110, 118; see also Depth of field recording pattern, 266, 266f 534 INDEX Beta format (com.) Birefringence (cont.) specifications of, 266f, 276 weak, 8f, 9f, 325f, 407f, 409f, 492-493, 494f, 498f, tape path in, 277f 499f VCR for, 281, 282t detection of, 404-405, 493-498 Betamax: see Beta format see also Polarizing microscope Bias light, 17g Bit, 17g for reducing lag, 219-220, 222f, 223f 1-8 bit image, 332, 335, 337f, 384 Binary image see also Binary image produced by discontinuous ITF, 355, 355f Black and white, 17g; see also entries under Monochrome use in image segmentation, 383, 378-386 Black level, 17g, 160 see also Keying; Thresholded image adjustment of, 311-312 Binary search, 386 automatic control of, 65f, 66, 108, 232 to locate centroid, 384-386 manual control, 232 Binocular parallax reference , 159f, 163f in dissecting microscope, 446, 447f setup of, 160 generation of in compound microscope, 446-450, 448f, Black negative , 18g, 159, 162f 449f, 450f Black reset , 58, 62 with video, 451-455, 452f, 453f, 454f, 455f Blanking, 18g, 160 and stereoscopic acuity, 90-91 , 90f and flyback, 152, 153 and stereoscopy, 90-91, 446 H blank, 154f, 158f, 169f Biological fine structure, 404, 491-492, 500 V blank, 154f, 159f, 160, 161 , 169f Biological specimens: see Cell; Digitally processed images; see also Blanking interval; Blanking level Living cell; Living cell (video microscopy) Blanking interval Bioluminescence, 393, 396, 400 comparison of, 161, 164f Birefringence, 17g, 480-484 specifications of, 158t, 160f, 162f, 163f of actin, 492 see also Blanking axes of, 481-483, 485-488, 485f, 487f-490f, 492, Blanking level, 18g, 159-160, 159f 498f, 499 specifications of, 160f, 162f, 163f of bacterial flagella, 407f Blanking pulse, 164f of calcite, 481-483, 482f, 483f, 484f in monitor, 243 in cell membrane, 491, 501 Bleaching of excised rods and cones, 403, 415 in chromosomal spindle fibers, 494f Blemishes and chromosome movement, 500 in camera tube, 181, 193 in chromosomes, 50 I allowed in specifications, 228 of cilia, beating, 8f, 405 in microscope optics, 66-70, 139, 299, 419 of crystals, 404 , 481-483, 486, 487, 487f, 488f, 489f, in solid-state sensor, 203 500 sources in video microscopy, 228, 299 and dichroism, 484-485 Blood cells of DNA, 404, 491 , 49ln, 498f, 499, 501 automatic sorting and counting of, 5, 416 edge, 498n human red form, 491-492, 491n flow velocity of, in capillaries, 320 interference colors from , 405 invaded by malarial parasite, 6f, 410 of lipids, 491, 500 Blood flow, measurement with video, 5-6, 320 in living cells, 404, 500-501 Blood smear, 339 measured by internal calibration, 323, 404 histogram flattening of, 352-353, 353f measurement by compensation, 486-488, 489 Blooming, 18g, 59 microdensitometer trace of in ceo, 221 . 226f in cave cricket sperm, 498f in monitor, 251 in Chaetopterus spindle, 323, 325f in vidicons, 221 , 224f, 225f in DNA microdomains, 499 Blurring of microtubules, 492 by smoothing convolution mask, 368 and molecular structure, 488-492 trade-off with noise reduction in muscle, 409f, 50 I analog, 317, 318f in nerve, 501 digital, 368, 369f of myelinated nerve Schwann sheath, 491 of vertical edges in video picture, 174, 175f in nitrocellulose, 491 n BNC connector, 18g, 55- 56, 57f, 58f and optical anisotropy, 480-484 Bonds, conjugated, 413n, 491n and optical rotation, 488 Boundary of polymer, effect of side groups on, 49ln detectwn by human eye, 82-83, 84f of PV A, 488- 491 in digital image processing reference books on, 404, 500- 501 blurring of, 368 of rootlets in Stentor cilia, Sf of derivative image, 358 slow and fast waves, 485-487, 487f determination of centroid from , 384-386, 385f in spindle, 323, 325f, 404, 494f, 500 enhancement of, 378, 379 strain, and extinction factor, 492 measuring area within, 339-340 video enhancement of, 7f, 8f, 9f, 405-406, 406f, 407f, segmentation by, 378-384, 380f, 381f 409f see also Edge map; Image segmentation INDEX 535 Brewster angle, 482 Camera (video) (cont.) BR quality (video tape), 18g, 293, 301 low-light-level (cont.) Brightfield microscopy photon counting, 197 Airy disk of absorbing obJect, 120 see also Image intensifier choice of lenses for video microscopy, 136 monochrome, selected U.S. sources for, 235-236, 236t; contrast generation, in- and out-of-focus, 413, 413n, see also Monochrome camera 414f random interlace, 256, 290, 293 of Siemens Test Star, 115, 116f, 117f response curves of, 332f, 366f luminance level and typical video cameras for, 230t, solid-state, 198/-203, 20lf 395t specifications of, 202t MTF curve for, 123, 125f surveillance, 55-56; see also Random interlace camera at various NAs, 124f see also Camera tube; Camera tube specifications; Color video-enhanced camera; Video camera of lightly-stained histological slide, 414f Camera circuit, optimizing, 176; see also Circuits potential of, 413-415 Camera controls, automatic, effect on contrast, 65f, 66 of stained blood smear, 339, 352, 353f Camera lens, 61 f, 66 see also Microscopy for copying with video, 429, 430f, 431 Broadcast-quality tape: see BR in video microscopy, 56, 60, 6lf, 66, 98f, 137f, 240- Broadcast TV, 71 241 bandwidth limitations for, 154n, 238, 242 Camera noise, 315-319 vs. ccrv standards, 149, 153, 185 from preamplifier, 181, 215, 217 degree of lag objectionable for, 219 in slow scan, 317 EIA standards, 159, 159f, 161, 162f, 163f smoothing filter, 317 history of, 4 target integration, 317-319, 317n, 318f, 319n narrower bandwidth by encoding, 238 see also Camera tube, noise in see also NTSC; PAL; SECAM; TV Camera resolution Broadcast video format, 159-160, 162f, 163f, 164f vs. monitor resolution, 424 Buffer: see Frame buffer vs. VTR resolution, 424 Build-up lag, 18g; see also Lag Camera ringing vs. tube orientation, 235, 235n Bum, 18g Camera signal in ceo, 197, 224, 294n monitoring of, 432 in intensifier camera tubes, 221-224 synchronizing of, 185n, 186, 186f, 187f in vidicon tubes, 193, 224 Camera tube diameter of, nominal, 231, 232t general description of, 191-193, 192f Calcite, 481 noise in , 181, 217 birefringence of, 481-483, 482f, 483f, 484f character of, 217-218, 230t lattice of, 482f from preamplifier, 195, 215, 217 o- and e-rays in, 481 , 482f, 483f proper orientation of, 235, 235n polars, 145n, 484f, 495f persistence of; in stereo video, 452; see also Lag precession of e-ray image, 481, 482f, 483 sensitivity of, vs. microscope magnification, 130 see also Calcite crystal; Prism, calcite sensor size vs. performance of, 1991, 203, 231, 2311 Calcite crystal target integration in, 3-4, 49g, 193, 319 double image through, 482f yoke for, 192f, 193 path of light rays in, 481, 482f see also Camera tube specifications; Chalnicon; New• as UV polarizer, 483, 484f vicon; Plumbicon (PbO vidicon); Saticon; SEC; Sil• UV transmission, 483 icon; Sulfide vidicon; Ultricon; Video imaging Calcite prism: see Prism, calcite device; Vidicon (sulfide); Vidicons; Vistacon Calcium ion, localization in cell, 400 Camera tube faceplate, 59f, 98, 192f wave, following fertilization, 396, 398f-399f cleaning of, 143-145, 144f Calibration (video) magnification of microscope image on, 100 of distance in image, 319 optical noise from, 68f of magnification, 100 positioning of, 59f, 60f, 6lf of frequency response, 256 Camera tube specifications see also Test pattern amplitude response Calibration curve (video densitometry) curve, 203, 204f, 205f linearizing, 363-365 percent at 400 TV lines, 194t for Newvicon, 332f, 363 blemishes, 228 linearized, rattoed, 366f blooming, 221, 224f, 225f, 226f Camera (video) bum, sticking, 221-224 as digttizer, 332 contrast transfer function, 203, 207f color: see Color camera dark current, 194t, 1991, 217 gamma correction in, 247f vs. target voltage, 216f instrumentation: see Instrumentation camera vs. temperature, 216f low-light-level distortion, 227 automatic shutoff circuit in, 234, 404n dynamic range, 199t, 214, 217 digitizing from taped image of, 381 exposure damage limit, 199t 536 INDEX Camera tube specifications (cont.) CCD (cont.) faceplate illuminance, typical, 199t description of, 19g, 197-203, 201f, 202t, 235-236 gamma, 194t, 209 dynamic range of, 197-198, 235 lag Fourier-transform generating, 375 vs. bias illumination, 219f, 220 high-resolution, xxiii, 198 build-up, 220, 223f IR -blocking filter for, 206n decay, 218f, 219-220, 220f, 22lf, 222f lag in, 197, 294n third-field, 194t, 199t, 2001 linear array, 416 light transfer characteristics, 209, 214f, 215f luminance levels for, 394t-395t limiting resolution, 194t, 199t, 2001, 203 in microscopy, 394t-395t, 416 vs. faceplate illuminance, 208f noise in, 197-198 low-light-level, 2001 in photometry, 235, 236, 416 photocathode material, 194t, 199t, 203 photon integration in, 198, 235 resolution: see Camera tube specifications; amplitude quantum efficiency of, 198, 212f response resolution of, xxiii, 198, 202t responsivity, 194t, 1991, 205-206 sensitivity of, 198, 202t sensitivity, 194t, 205 slow-scan, 198 shading, 194t, 224-227 SiN ratio in, 202t signal current level spectral response of, 212f vs. faceplate illuminance, 214, 215 see also CCD camera typical, 1991, 200t CCD camera SiN ratio, 2001 chilled, photon integration in, 197-198, 235 vs. faceplate illuminance, 217 commercial sources of, 203n, 236t spectral response, 194t, 1991 description of, 20lf, 235-236 curve, 206, 210f, 2llf, 212f, 213f dynamic range of, 197, 235 spectral sensitivity, 203-204 in polarized-light microscopy, 405 usable light range, 199t see also CCD; Solid-state cameras Candela CCTV, 20g per mm2 of bright sources, 129t and broadcast TV, 71, 149, 185 definition, 18g, 205 early applications of, 4-5 Capacitor and electric field, 478, 479f, 480 and human vision, 71, 92 Capstan drive, 18g, 268, 269f in microscopy, 5, 149, 185 Carbon arc lamp, 3 scan rates and sync pulses in, 185 Carrier wave, 18g, 272; see also Color subcarrier; FM standards for, 158-161, 159f, 160f, 165f Cascaded series of devices Cell: see Blood cells; Cone cells; Cornea; Endothelial cell, MTF, 176-178, 469 living corneal; Eosinophil, tracking movement of; resolution, 176, 176t, 178 Epithelial cell; Ganglion; Hemocyte, contrast vs. rise time, 176 field diaphragm; Living cell; Living cell (video Cassette: see VCR microscopy); Nerve/neurons; Platelet, form Catadioptric, 18g birefringence of; Pollen mother cell, living, lenses, for UV- and IR-microscopy, 401 birefringence in; Rod cells; Sperm, living video projector, 444-445, 444n Cell boundary Schmidt correction plate in, 444, 444f enhanced by convolution, 384 Cathode, 19g mask using, 380f, 381f, 383-384 in intensifier tube, 196f see also Cell outline; Edge map in picture tube, 242, 243f, 244f Cell classification, 512 in vidicon tube, 192f, 193 cell sorting, 5, 416, 511, 512 see also Cathode ray tube; Electron gun; Photocathode by image segmentation, 386 Cathode ray oscilloscope, 19g malignant vs. normal, 378, 386 to display video waveform, 59n, 164, 255f, 256, 256n Cell, cultured: see Living cell (video microscopy), tissue waveforms culture digital reduction of SIT noise, 183f Cell, digital feature extraction H and field scans, 167f area, 377, 378f, 383 multiburst, 257f boundary, 380f, 381-386, 38lf NTSC color signal, 241 centroid, 384-386 see also Cathode ray tube; Oscilloscope circularity, 377, 378f Cathode ray tube, 19g, 242-243, 243f image segmentation in, 378-384 resolution in, 244-246, 245f perimeter, 377, 378f, 380f, 38lf, 384 UV-emitting, 417 preprocessing for, 377-378 phosphor characteristics of, 248, 2491-2501 Cell division in video monitor, 242-251, 244f automatic tracking of, 407 CCD birefringence of spindle fibers, 404, 500 blooming in, 221 , 226f Cell fine structure, with polarizing microscope, 404, 492, burn in, 197 500 characteristics of, 202t, 235-236 Cell fluorescence compared with Newvicon, 203 chromatin, optical sections, 375, 376f INDEX 537 Cell fluorescence (cont. ) Chromaticity, CIE FRAP, 416 diagrams, xxv(l), 88-90, 88f, 89f, 239f images, 6f standard color mixture curves, 87-90, 87f isolation and masking of, 380f, 381-384 Chromatin, OP.tical microtubules in PtK-2 cells, 402f 3-D fluorescence image, 375, 376f, 418 in pseudocolor, xxvi(l), 382f lightly stained, brightfield microscopy of, 414f Cell, living: see Living cell; Living cell (video Chrominance, 19g, 90 microscopy) reference wave, 240, 24If Cell, malignant: see Cell classification Chrominance signal, 19g, 240, 241f, 260 Cell motility: see Cell tracking; Living cell; Living cell Chromophores, orientation of, 485 (video microscopy) Chromosomes Cellophane, 485 birefringence, dichroism in, 50 I Cell outline birefringence in sperm, 499f video-enhanced optical sectioning, 418-419 3-D image reconstruction, 418 in complex tissues, 410 in flying-spot UV microsocpe, 417 see also Cell boundary Fourier-filtered optical sections, 375, 376f Cell sorting, 5, 416, 511, 512 identification of automatic, 386, 416 automatic karyotyping, 386, 512 Cell-substrate adhesion, 410 using fluorescence microscopy, 5 Cell tracking in living pollen mother cells, 494f by centroid location, 384-386, 385f movement, vs. spindle birefringence, 500 migrating cells, 407-408, 417n polytene using motorized stage, 386 3-D image of nucleosomes in, 422 Centration of illuminator and lenses, I 04-107, I 06n straightened image, 362f, 363 Centrifuge, for drying coverslips, 143, 143f see also Chromatin;. optical section Centroid vs. motion tracking, 384- 386, 385f CID, 19g, 198 CF (chrome-free) lens, lOOn camera, 201f CFF: see Critical flicker frequency CIE, 19g C format recording chromaticity diagrams, 87-90, 88f, 89f, 239f description, 19g, 275-276, 278, 278f color plate, xxvi VTR specifications for , 279t standard color-mixture curves, 87-90, 87f Chaetoptorus spindle; fiber birefringence, 494f Cigarette smoke photometry of, 323, 325f effect on floppy disks, 302 Chaga's disease, Trypanosoma cruzi in, 6f keeping optical components from, 145 Chalnicon, 19g Cilia, beating amplitude response curve, 204f, 205f birefringence of, Sf build-up lag, vs. bias illumination, 220, 223f in video still field , 405 , 424 burn tendency, 224 bending and action potential, 441f decay lag, vs. bias illumination, 220, 222f Circle, distortion of use in DIC microscopy, 410 in camera, 227 , 253f and dynamic range in microscopy, 234 in monitor, 248, 252f lag, 219, 222f, 223f Circle of confusion, 118 light transfer characteristics use in stereo microscopy, 447, 449f curve, 214f Circuit diagrams, v vs. target voltage, 209 books on video with, v, 311n noise character, microscopy application, 230t Circuits specifications: see Camera tube specifications aperture correction spectral response curve, 210f, 213f in high-resolution camera, 231 target material, 194t in high-resolution monitor, 246 in video microscopy, 55 , 394t automatic pedestal control, 16g, 232 see also Microscopy automatic shutoff, for high-gain cameras, 234, 404n Charge-coupled device, 19g; see also CCD clamping, 20g Charge coupling, 198 eliminating AC-Iine disturbance of, 181 Charged priming device, 19g; see also CPD for H-line DC restoration, 178n Charge injection device, 19g; see also C!D contrast inversion, 430 Chilling equalizing, 273f of CCD camera, 197-198 filtering commercial source for, 235 for edge crispening, 246 of SIT camera, 319 for noise reduction in camera, 234 Chip, 19g gamma compensation, 28g, 209, 247f high-resolution ceo. 198 high impedance, 29g, 63, 63f !-megabyte memory, 513 high resolution, 173-178 Chroma: see Chrominance one-line delay Chroma detector, 19g for dropout compensation, 24g, 181 , 274 in monitor and VTR , 242 for vertical aperture correction, 246n Chromatic aberration, 19g, 133, 133n, 138 peaking, 176 538 INDEX Circuits (cont.) Color burst (cont. ) signal processing omissiOn for monochrome scenes, 240n in camera, 231-235, 243 oscilloscope display of, 241 f in monitor, 244f, 246 standard in RS-170A, 163f in VTR, 273-274 Color camera, 20g, 236-242 75-ohm termination, 45g, 56, 63, 63f alignment of, 240 video delay, for synchronizing cameras, 185n DC operation of, 228n see also Amplifier; Auto-black; Automatic bandwidth RGB , vs. NTSC, 237 , 237f, 242 compression; Automatic gain control; Automatic single-tube type, 240 horizontal frequency control; Automatic target con• Trinicon, 50g, 242 trol; Circuit diagrams; Color killer; Comb filter; three-tube type, 49g, 237f Dropout compensation; Integrated circuit; Phase• in video microscopy, 229 locked loop; Preamplifier; Processing amplifier; in brightfield, 413 75-ohm Termination; White balance dynamic range of, 240-241 Clamping, 20g; see also Circuits, clamping vs. monochrome camera, 229 Classification recording interference colors with, 405 by digital image processing, 5, 416, 512 white balancing of, 240n by pattern recognition, 386 Color detection Cleaning method automatic, 242 optical surfaces, 139-145, 141f, 142f, 143f, 144f video enhancement of vital dyes, 413 VCR head, drum, 299-300, 300n Color encoder, 20g Clipping function of, 237 of gray level, 339, 339f, 383 Color encoding, 237-240 white, black, 179, l80f bandwidth compression in , 238-242 Clock, in CCDs, 198; see also Time-date generator loss of image resolution by, 238, 242 Closed-circuit TV, 20g; see also CCTV reference books on, 240n C mount schemes for, 164, 166t adapter for 35-mm camera lens, 429, 430f see also Color video format thread of, 59f, 60, 429 Color filters, complementary, 451n Coaxial cable, 20g, 55, 56f, 57f for stereoscopy, 450-451 checking conductivity of, 62 Color invariance, 83n, 90 impedance and termination of, 63-64, 63f, 64f, 185 Color killer, 20g, 242, 274 phase delay in, l85n Color matching equation, 87n as source of noise, 181, 297 Color mixing, 86-90, 87f, 238f; see also Chromaticity, see also Termination CIE, diagrams Coherence Color monitor, 20g in diffraction pattern, 113, l33n accepting PAL, SECAM, 164, 166, 260n of illumination, 115 adjustment of, 262 Coherence length of laser waves, 419 automatic balancing of hue in, 262 Colcemid, colchicine, 323, 500 need for critical termination in, 185n Collector lens/mirror, lamp high-resolution, 260-262 centering and alignment, 105-107 input from digital image processor, 348, 348f image planes of, 97f, 102f RGB, 237, 237f, 260, 424 in Koehler illumination, 101 use in stereo video display, 451 Color see also Monitor; Monitor picture CIE standard curves, 87-90, 87f Color perception: see Color vision distinguished by human eye, 356 Color picture tube, 260, 261f gray/white as, 89, 89n, 90, 238, 238f, 239, 239f, Color projector 241f three-tube type, 444 vs. hue, 86-90, 238 use in stereo video display, 451 vs . intensity discrimination, 81f Color recording, 430, 431 perceived, effect of luminance on , 90 NTSC, 277, 279t, 28\t, 282t, 290t and perception of image detail, 238 signal processing in, 273-274 sensation of, and hue, 238-240 Color reference subcarrier: see Color subcarrier spectral, on CIE chromaticity diagram, xxv, 88f, 239f Color reproductions video microscopy in, 242 CIE chromaticity diagram, xxv VTR, vs. monitor resolution, 424 flashing fluorescence in pseudocolor, xxvi see also Chromaticity, CIE; CIE; other entries under microsphere fluorescence in pseudocolor, xxv Color Color sensation, 238 Color bars, 20g loss in scotopic vision, 90 adjusting monitor with, 262 Color signal, 185 NTSC, 24lf encoded, vs. RGB, 237; see also Color encoding from test signal generator, 256 exact H-and V-scan rates in , 189n, 240n Color blindness, 90 limitation in video microscopy, 229 and complementary-color stereoscopy, 451, 451 n luminance scale in, 24lf Color burst, 20g nature of, 237-240 in NTSC video, 161, 167, 240 NTSC, 240n, 24lf INDEX 539 Color subcarrier, 20g, 240 Compensator (polarization optics) (cont.) absent m monochrome recording, 270 retardance of, 489f exact frequency of, 240n see also Compensation (polarizatiOn optics); Polarizmg Color temperature, 20g, 77 , 126 microscope (instrument) for vidicon calibration, 206, 209, 2llf Complementary-color stereoscopy, 451, 45ln, 452 Color, true, display with digital image processor, 348; see Composite video signal, 21g also False color; Pseudocolor display generation of, 157, 158f Color TV receivers voltage levels for vs. color monitors, 260 IEEE scale, 160n loss of resolution in, 260 P-to-P voltage of, 159- 160 Color-under recording, 276- 277, 277n separate sync signal from, 258-259 Color video, 149 as input to RGB monitor, 260-262 A and B frames in NTSC , 163f, 240n Compound microscope, binocular, generation of stereo im• dropout compensation in , 274n age in , 446-451 for conoscopic observation of crystals, 405 with video, 451-455 use of four fields for, 163f, 240n Compression (analog), 2lg gamma compensation for, 209, 247f effect on noise and resolution, 318f light valve projector for, 443f, 444 of video bandwidth monitor for, 260-262, 261f automatic: see Automatic bandwidth compression reference books on , 240n using smoothing filter, 317, 318f RGB vs. NTSC, 237, 237f, 242 white, 53g Color video camera: see Color camera Compression (digital), 2lg Color video encoding: see Color encoding; Color video of gray values, 345, 345f format nonlinear, of image histogram, 349f, 354 Color video formats, 161, 163f, 166f Compression (mechanical), 2lg NTSC, 38g, 237-242, 260-262 of video tape during storage, 30 I PAL, 39g, 166, 238, 260 Computer SECAM, 45g, 166, 238, 260 analysis of fluorescent images with, 383 Color vision home, video titles produced on, 435, 435f in complementary-color stereoscopy, 451 n host, 387 in humans, 86-90, 87f, 88f, 89f calculation of edge map by , 381, 383 mixing of primary colors, xxv, 87-90, 87f, 88f, 89f, centroid calculated by, 384-386 238, 238f, 239f in digital image processing, 330-331, 330f vs. MTF, 82n, 83f relief by ALU , 387 Retinex theory of, 90 image analysis with: see Digital image analysis Coma, 20g, 133 image processing with, v, 330f, 327-392, 511-513 Comb filter, 20g, 260, 260n interactive hardware and software, 33g; see also reference book on, 260n Interactive image processing, digital Comet tailing, due to tube lag, 293 microcomputer in Newvicon, in swimming sperm, 219, 221f in high-speed VTR, 295 in SIT, in swimming embryo, 219, 220f interlace pattern in, 161 n Commercial firms, addresses of plug-in image processing boards for, 513n additional, digital image processors, 458 use in microscopy, history of, 5 instrumentation video cameras, 236t tracking of cell motility with, 384-386, 385f main listing, xxi-xxiii Computer-controlled motorized stage, 384-386 related magazines, 458-459 Computer-enhanced images: see Contrast enhance• Compatibility, 2lg ment/stretching (digital) of color and monochrome equipment, 149 Computer-optimized lens design, 107, 133 and EIA standards, 158 Condenser of VCR, 286, 300-301 aberrations of, JOin, 106, 139 of video equipment and signal, 164, 184-189 achromatic aplanatic, 14g, lOin, 139, 139t Compensation (polarization optics), 489f centration of, I 06-107 additive and subtractive, 409f effect on diffraction pattern, 127n contrast reversal with , 407f focal plane at iris diaphragm, I 0 1-102, 102f vs. image contrast and brightness, 404, 493 focal planes of, 97f, 101-102, 102f for video contrast enhancement, 405 image planes of, 97f see also Compensator (polarization optics) long working distance, 139, 139t Compensation (video circuit) need for immersion, 127-128, 139 of non-ideal MTF, by spatial filtration , 375 role in microscopy, 101-110, 114-115, 119-122, 124f see also Dropout compensation; Gamma compensation; sources of optical noise on, 69 Shading compensation (analog) specified optical conditions for, 10 In, I 27 Compensator (polarization optics), 500 Condenser aperture determining fast and slow axes with, 486-488, 488f, vs. 3-D diffraction pattern, 115n 489f uniform illumination of, 127n location in microscope, 484f, 492, 495f Condenser iris diaphragm, 2lg mica, 499f vs. aberration, 106 measuring retardation with, 488n, 489f aperture planes, conjugate with, 101, 102f 540 INDEX Condenser iris diaphragm (com. ) Contrast enhancement (analog) (cont. ) vs . NA, IOS-110, IOSf, 109f with automatic pedestal control, 232 in Koehler illumination, 101-107, 102f, IOS-110, IOSf, of faint indicator dyes, 413 109f in human vision: see Contrast detection by human eye see also Condenser NA; Conoscopic observation by video camera, 231-232, 345-346, 40Sf Condenser NA, 109f, 110 of DIC image, 322f, 40Sf vs. depth of field, liS, 414f, 415 of fluorescent image, 5, 324f effect on flare, 110 of polarized-light image, 7f, Sf, 325f, 407f effect on MTF of microscope, 12~. 124f, 125f see also Contrast control, on monitor; Contrast enhance• effect on resolution, 115, 414f, 415 ment/stretching (digital) optimal for visual observation, 110 Contrast enhancement/stretching (digital), 21g raised by video microscopy, 110, 136, 414f analog preprocessing, 345-346 Condenser, rectified: see Rectified condenser by histogram equalization, 352-353, 352f-353f Conditioner, in microscope optical train, 93, 94f, 122 and histogram transformation, 342-356 Cone cells, 73f of brightfield image, 353f and color blindness, 90 of phase-contrast image, 352f, 355f distribution in retina, 77-SO, 7Sf with ITF isolated, dichroism of, 415 discrete, 344-347, 345f-347f in photopic vision, 74, 77 linear, 342-346, 343f-346f role in image resolution, 7S-79, 79f nonlinear, 346-356, 347f-355f see also Retina; Rod cells sigmoid, 353-354, 355f Confocal microscope, 21 g with mottle subtraction doubling resolution with, 418 by anaxial illumination, 412f Nipkow disk in, 416-417 of brightfield image , 414f slit-scanning type, 4IS , 4 I 9f of DIC image, 9f, 306f, 4llf Confocal optics, 2 Jg of fluorescence image , !Of, 402f with sun as a light source, 5 of polarized-light image, 9f, 306f, 409f Conjugated bonds vs. shading distortion, 333f light absorption by, 413n, 491n in VTR playback, 424 polarizability of, 49 In see also Contrast enhancement (analog) ; Digitally pro• Conjugate planes , points, 21g, 97f, 9Sf, 99f cessed image of perfect lens, 95, 96f Contrast generation in Koehler illumination in microscope, 93, 94f, 11S-119 aperture, 101-103, 102f modes in microscopy, 122 field, 103-104, 103f in phase contrast, 119-122 reciprocal, I 04 reference books on, 122 Connectors, BNC, UHF, !Sg, 50g, 55-56, 57f, 5Sf see also Microscopy Conoscopic image, 2lg, 110 Contrast range, 21g, 33S observation with color video, 405 Contrast reversal vs. orthoscopic image , liOn by anomalous diffraction, 404n-405n, 497f Conoscopic observation by defocusing, 115, 116f, 117f, 413 Bertrand lens for, 106n, 110, 493f Contrast transfer function, 21 g for centering microscope condenser, I06 vs . amplitude response curve, 203 with goniometer stage, 44Sf of CCDs, 202t for inspecting objective lens back aperture, 110 vs. focal levels, 115, 116f, 117f Contrast, 2Jg mathematical relationship to MTF, 46S effect of field diaphragm on, 65f measuring, 203, 206f, 256, 256n seen by logarithmic detector, 405 , 406f vs. MTF and OTF, 123 modes of generating: see Contrast generation in multicomponent system, 469 numerical definition of, 232 for SIT, 203, 207f of transparent specimen, 121 see also Modulation transfer function; MTF; Optical video enhancement of, early use in microscopy, 5 transfer function see also Amplitude response curve; CfF; Modulation Control head , 265f, 267, 26S , 269f, 277f transfer function; MTF; Phase-contrast image; Control pulse, 187, 265f, 276 Phase-contrast microscopy Control track, 21g, 265f, 266f Contrast control, on monitor, 2Jg, 56f in Beta and VHS formats, 266f, 276 adjustment for photographing monitor, 426 function of, IS7, 267, 270, 276 home settings for, 59 in U format , 265f, 276 use in video microscopy, 246, 247f Convergence, 2 I g Contrast detection by human eye in color monitor, 262 at edges, S2-S3, S4f of retinal elements, 79-80, SOn vs. limiting resolution in TV, 203 Convolution of sinusoidal object, S2f image, 3Jg threshold, SO-S3, SOf, Sin, Slf as an ALU function, 391 Contrast enhancement (analog), 2lg, 405-415 spatial, 47g by analog processing, 311-314, 312f, 314f, 315f, 346 see also Convolution operation INDEX 541 Convolution kernel/mask, 22g, 367, 367f Crystalline lens, 72f empirical and theoretical bases of, 371 accommodation of, 72 Laplacian, 34g, 369-370, 370f fluorescence in, 74n sharpening, 369-372, 369f Crystalline structure smoothing, 368-369, 368f, 369f cleavage plane vs: optical axes, 486 types of, 368-372 determined with polarization optics, 485-492 variable, 379 see also Calcite see also Convolution operation; Spatial frequency Crystal optics, reference books, 404, 500 Convolution operation, 367-368, 367f Crystal oscillator, quartz, 186 applications of, 368-372 Crystals, liquid, 500 digital filtering by, 367-372 CTF, 22g; see also Contrast transfer function digital processors performing rapid, 388t, 512 Cue, for VTR editing, 22g, 287 for enhancing cell boundaries, 384 audio, 298 vs. Fourier filtering, 375-377, 377f for depth perception, 90-91, 91n for image segmentation, 3 79 SMPTE time code as, 285, 287 with Laplacian filter, 34g, 369-371, 370f on VCR with pause function, 438f logarithmic, 372 Cursor box, for centroid following, 384--386, 385f noise introduction, 371 Cursor, drawing edge map with, 383 noise reduction and blurring by, 368-369, 368f, 369f Cuticular walls, JR microscopy of, 40 I nonlinear, 372 Cutoff frequency: see Frequency, cutoff see also Convolution kernel/mask; Spatial filtration Cytochalasin, effect on cleavage, 320 Copying and editing video tape, 432-438, 433f, 434f Cytology, automated, 416; see also Cell sorting mechanics of, 436-441, 438f Copying video optically, 430-432 DAC: see Dl A converter arrangement for, 432f DIA converter, 22g, 330f, 331 from color to monochrome, 431 Dark adaptation, 22g, 74, 76f time-lapsed scene to incompatible VTR, 290, 431 for low-light-level microscopy, 324f, 393, 492-493 of titles, 435-436 vs. response to various wavelengths, 74-77, 76f video monitor, need for, 430-431 Dark current, 22g, 209 Copying with video cameras, 429-432, 431f in CCDs, 202t of movies, 430-431 vs. light transfer characteristics, 214f of still scenes, titles, etc., 429-430, 431f noise pattern from, 217-218, 2301 Copy stand for photographing monitor, 426, 427f vs. operating temperature, 216f, 217 Cornea, 71, 72f vs. target voltage, 216f, 217 observation of endothelial cells, 418, 419f in various camera tubes, 199t refraction at surface, 71-72, 99f Darkfield microscopy Comer resolution, 170, 252f condenser immersion in, 127 Correction collar, 22g, 1311, 134, 135t image brightness in , 129 Cosine-squared law, 483f reference book on, 122 Costs: see Price video cameras for, 193 Counter, field/frame, 293 video-enhanced Counting F-actin molecule, rigidity, 401 blood cells, 5 flagellin, polymerization and conformation change, particles, 3 34 401 see also Classification single microtubule, rigidity, 401 Coupling tube see also Microscopy microscope to video camera, 59f, 60, 60f, 61f DC restoration, 58f, 62 reflections from , 67f, 68f, 68 3-D display Courses, short, related to video microscopy, v, 457, Synthalizer, 420-421, 420f 459 vibrating mirror, 421-422, 421f, 422f Coverslip 4-D display, 455n centrifuge for drying of, 143f Decibel cleaning of, 141-143 SIN ratio and noise statistics, 181-184 depolarization by, 495f appearance of line scan of, 182, 183f dirt on, 68-69, 141 influence on video picture, 182, 184f, 285 strain-free, 492 vs. voltage ratio, 182t thickness vs. aberration, 115n, 134, 134n Decoder, 22g reference book on , 134 in monitor, 237f, 238 CPD, 19g, 198 Definition, 22g camera, 198 picture, 285 color, 202t see also High-definition TV specifications, 202t Deflecting coils, 3f, 23g, 192f, 193 Critical flicker frequency, 22g, 83-86, 85f, 86f; see also and geometrical distortion, 227 Flicker Deflection, of scanning beam, 152, 153f Cropping, of picture, 423 , 426 Defocusing, contrast reversal by, 115, 116f, 117f; see also Cross talk, 22g, 266 Out-of-focus image 542 INDEX Delay line, 1-H, 274 DIC video microscopy comb filter as, 260n cameras appropriate for, 230t differential contrast image using, 314 concurrent display with low-level fluorescence, 147f, 148 Densitometry, using video digital subtraction, vs. division, 356, 358f from digital image, 363-365 edge maps in, 379 logarithmic converter for, 333 gray level histogram typical in, 339, 354, 354f use of image histogram for, 340-341 image of see also Microdensitometer acrosome elongation, 322f Depolarization, at optical interfaces, 495f; see also diatom, 306f, 316f, 3!8f, 408f, 412f Anomalous diffraction; Polarization rectifier; Rec• mitotic spindle, 292f tified condenser swimming sperm, showing lag, 219, 221f Depth of field, microscopy, 23g image taken with vs. condenser NA, 414f, 415 FV, 408 effect of accommodation on, 118 Newvicon, 221 f, 292f, 306f, 322f equation for, 118 SIT, 318f increased noise reduction by bandwidth compression, 318f in holography, 419 use of Plan Apo objectives in, 136, 306f, 408f, 412f in confocal system, 418 video-enhanced, 410-412 shallow see also Microscopy applications of, 418-420 Dielectric constant, 479-481 in confocal system, 418 of anisotropic medium, 480f in slit-scanning mirror system, 418 relation to refractive index, 479-480 see also Axial resolution Dielectric ellipsoid, 480-481, 480f Depth of focus, 23g Dielectric polarizability, anisotropy of, 480f, 485, 491 in light microscope, 68, 69f Differential interference contrast, 23g; see also DIC micro- Depth perception, visual cues for, 90 scope; DIC video microscopy Dew indicator light, on VCR, 287, 296 Diffracted orders Diaphragm: see Condenser iris diaphragm; Field diaphragm from Ronchi grating, 428-429 Diatom from video scan lines, 464 analog contrast enhancement, 7f Diffraction digitally enhanced anomalous, and rectifier, 404, 404n-405n, 497f, 498 anaxially illuminated, 412f causing phantom membranes and filaments, 413n high-resolution isointensity contour, Cover, 306f by microscope, vs. video ringing, 254 illustrating reverse polarity smear, 254f by pupil of eye, 74, 78 zoomed, thresholded image, 254f by Ronchi grating, 428-429, 428f FV image, 400f submicroscopic filament inflated by, 41\f scan line removal by spatial filtering, 464f, 465f, 466 see also Diffraction pattern SIT image, with noise reduction, 318f Diffraction image errors, in polarization microscopy, 404, vertical line scan of, 316f, 318f 404n-405n, 497f, 498, 498n DIC: see DIC microscope; DIC video microscopy Diffraction pattern Dichroic crystal vs. aberration, 133n chromophore orientation in, 485 Airy, 112-118, 112f, 113f, 114f, 470, 470f, 497f light transmission in, 484-485, 485f, 486f along axis of observation, Ill, 116f see also Dichroism and Bessell function, Illn Dichroism, 500 3-D, 49g, 111-113, 116f basis of, 485 and defocused image, 115-118, 116f, 117f of isolated retinal rods and cones, 415 effect of aperture function on, 115n, 118 in living cells, 500 effect of condenser on, 127n vs. molecular anisotropy, 477 and generation of image contrast, 122 negative, in 13-form DNA, 491 and image wave, 111-118, 112f polarized-light microscopy of, 404 and microscope resolution, 113-115, 113f, 114f of sheet Polaroid, 485 modified in tourmaline, 484-485, 485f, 486f anomalous, 404n-405n, 497f see also Dichroic crystal; Polarized light in DIC, 115n DIC microscope, 410-412 by oblique illumination, 66 condenser NA for visual observation, 110 photographs of, ll3n, 113f, 114n, 114f diffraction pattern, 114n, 115n VS. PSF, 470 immersion of condenser, 127 reference books on, Ill MTF curve for, 123, 125f relation to Fourier transform, 122 Nomarsld prism in, 412 see also Airy disk; Diffracted orders; Diffraction Nomarski-type, 410 Diffuser, ground-glass: see Ground-glass diffuser rectified plan apochromatic optics, 145n Diffusion of fluorescent dye, in sea urchin egg, 324f signal arising from, 129-130 Digital computer: see Computer Smith-type, 320, 322f, 410 Digital image analysis, 328, 377-384 transmittance of polarizers used in, 133 classification by, 5, 386, 416, 512 Wollaston prism in, 410, 412 densitometry by , 333, 340--341, 363-365 INDEX 543 Digital image analysis (cont.) Digital 1mage processing (cont.) early history, 5 volution operation; Digital image analysis; D•gital extracting features of cells by, 377, 381; see also Cell, image processing system; Digital image processors digital feature extraction; Edge map (equipment); Digitally processed image; Digitally using image processors, 381-386 processed images image segmentation by, 378- 381; see also Image Digital image processing system, 327-338 segmentation analog-to-digital converter in, 330, 424 motion tracking, 384-386, 385f and host computers, 330-33 I, 330f pattern recognition by, 386, 416, 511 , 512 cursor, interactive, 383 see also Digital image processing; Image analysis digital-to-analog converter in, 22g, 330f, 331 Digital image memory, 330-331, 330f frame store, 70; see also Digital image memory dynamic range of, 364, 368 freezing fields or frames with , 424 see also Digital image processors (equipment); Frame input from OMDR to, 304 buffer; Frame memory input to and from VCR, 293 Digital image processing interactive, 329 vs. analog, 345-346 output of, to color monitor, 348 averaging to improve SIN ratio, 335, 336f real time, 381, 384 background subtraction by, 9f, !Of, 70, 371, 409f tolerance, to signals from VTR, 189 boundary following in, 378-384 true-color display with, 348 camera calibration in, 332, 332f, 363-365, 366f see also Digital image processors (equipment) compensation for non-ideal MTF, 374-377, 374f, 375f, Digital image processors (equipment), 386-392, 457-459, 376f, 377f 511-513 conditional image manipulations by , 387-391, 390f ALUs in , 387-391, 388t, 390t cursor for locating box, 384-386, 385f background subtraction in, 511-512 dilation in, 23g commercial sources of, 388t-39lt, 458t, 511-513 display device, dynamic range of, 355-356 cursor in, 392 edge detection by, 24g, 370f for freeze frame, 511 by Sobel, Kirsch, Roberts operators, 372 gray scale manipulation, interactive, 511-512 edge enhancement by, 367 , 369-372, 370f, 378 interactive, 329, 347, 458t, 512 edge sharpening by, 371-372, 37lf interactive devices for, 389t, 39Jt, 392 equipment for, 328-329; see also Digital image pro- digitizing tablet, 23g, 328, 392 cessors (equipment) joystick, 383, 384, 392, 422 erosion in , 25g light pen, 389t, 391!, 392 feature extraction by, 377-386 look-up tables, 389t, 39lt, 512 filtering to suppress noise by, 368-369, 369f noise averaging with, 5 I I fluorescence quantitation with, 380f, 381-384 overlays in, 39 I fold-over frequency in, 27g price of, 329, 511-513 Fourier filtering vs. convolution, 375-377, 377f quantitation with, 512, 513 frame summing of very-low-light-level images by , 9f, real time, 458t, 511-512 !Of, 184f, 402f, 409f roaming in, 392, 512 · freeze frame of fluorescent flash by, 381 selective image filtration with, 513 geometrical decalibration, 358-363, 360f, 361f spatial filtration with, 5 I 2 gray values needed for, 335 text overlay with, 391 interactive, 34 7-348 turnkey systems, 511-512 interpolation, bilinear, 360-363, 360f Digitally processed image look-up table used in, 346-348, 347f background subtracted, 9f, !Of, 306f, 357f, 359f, 409f, linearizing, real-time, 364 414f mitochondrial fluorescence, detection of, 38 I binary image of, 355f, 380f, 385f new programs and hardware for, 457n binary masking of, 380f noise suppression by, 368-369, 369f binary search with, 385f, 386 optical noise subtraction by, 9f, !Of, 70, 371 , 409f boundary dilation, erosion in, 380f pseudocolor display, xxv, xxvi, !Of, 381, 382f color recording of, 283 real time, 331 contrast-enhanced, 9f, !Of, 184f, 306f, 333f, 352f, 353f, reference books on, 184, 368, 372, 373, 457n 366f, 370f, 409f, 414f shading reduction, 51 I; see also Shading correction (dig• displaying or recording of, 331 ital); Shading distortion distortion corrected in, 361 f stretching, of gray scale, 345, 346f, 349f, 352f, 353f, edge detection by, 359f 512 edge segmentation by, 380f summing to improve SIN ratio, 9f, !Of, 184f, 402f, Fourier filtering of, 376 409f; see also Noise averaging (spatial); Noise aver• frame-averaged, 9f, !Of, 306f, 336f aging; temporal frame-summed, 402f, 409f thresholding in, !Of, 32g, 254f, 355f frozen field in, 381 , 400f tracking of cell movement, 384-386, 385f geometrical decalibration of, 36Jf vertical resolution in, 17ln gray-value resolution of, 337f of very-low-light-level fluorescence , !Of, 402f with high condenser NA, 184f, 306f, 402f, 409f, 412f, see also Contrast enhancement/stretching (digital); Con- 414f 544 INDEX Digitally processed Image (cont.) Digitizing tablet, 23g, 328, 392 histogram clipping of, 339f Dilation, 23g histogram flattening of, 352f, 353f connecting separated edge segments, 379, 380f, 38!f image overlay of, 380f for producing bit plane mask, 383 image regions, deletion of, 380f, 383 3-D image reconstruction; see also Chromosomes; Muscle; image subtraction by, 357f Nucleosome, 3-D image of intensity contours in, I Of, 306f out-of-focus images, 418 Laplacian mask filtered, 370f 3-D intensity contour, xxv, IOf logical operation on, IOf, 381f Dinoflagellate, luminescence and autofluorescence in, 396-- motion detection in, 357f 397, 400f motion tracking of 385f Dirty lens, as source of noise component, determination of, 182-184, 183f, flare, 107 l84f, 335 optical noise, 68-69 from OMDR playback, 304, 306f, 307f, 402f, 409f, Dirty tape, danger of continued use, 2%, 297f 414f Discrete ITF, 344-348, 345f, 346f, 347f optical density, linearized to, 365f binary image produced by, 355f optical sectioning with, 376f, 412f, 414f program for converting histograms, 351-352, 35lf with Plan Apo objective, 184f, 306f, 402f, 409f, 412f, Disk, 24g 414f laser, 302-303 pseudocoloring in, xxvi, 382 magnetic, 301-302 ratioing of, 366f OMDR, 303-304, 304t, 305f-307f shading distortion in, 333f video eliminated by ratioing, 366f interactive system, 302 sharpening convolution for, 370f supplement to journal, 303 sigmoid-shaped ITF of, 355f Display, 3-D: see 3-D display smoothing convolution for, 369 Display, 4-D: see 4-D display straightening of, 362f Display, video subtraction of mottle in, 9f, 410 color TV receiver vs. RGB monitor, 260 thresholded, !Of, 254f, 380f reference book on, 242n from VTR playback, blurring of, 338 TV set vs. monitor, 242, 259 see also Contrast enhancement (analog); Contrast en• Dissecting microscope, 413, 446, 447f hancement/stretching (digital); Digitally processed binocular parallax under, 446 images; Living cell (video microscopy) Dissolve, 24g, 439 Digitally processed images Distance of blood cell measured on monitor picture, 251, 259-260, 319 eosinophil, 385f measurement calibration, 319-320 smear, 336f, 353f optimum, for viewing monitors, 442 of brain section, 36lf, 365f see also Projection distance, of ocular; Projection ocular of chromatin, testes, brightfield image, 414f Distortion, 24g, 133n of chromosomes, polytene effect of aspect ratio on, 251, 358 brightfield, 362f by curvature of monitor faceplate, 259-260, 259n fluorescence, 376f geometrical decalibration of, 358, 360, 360f of cortical granules, an axial illumination, 412f of highlight regions in VCR recording, 299 of critical test specimen, 184f, 409f of image proJected with ocular, 100, 134-135 of diatom frustule pores, 254 f, 306f, 412f measurement with Ball Chart, 255f, 256, 258f of dinoflagellate, 400f in monitor, 248-251, 252f of epithelial cell, buccal, anaxial illumination, 412f in overdriven monitor, 62 of fluorescent microspheres, I Of vs. proper cable termination, 63, 179, 185 of microtubules of recorded sound, 436 DIC image, vs. electron microscopy, 4llf by SIT camera, 253 DIC and polarization images, 9f test charts showing, 252f, 253f fluorescence images, 402f, 411f by video amplifier, 179, 180f of muscle section, SIT polarization image, 184f, 409f of video waveform, 178-180, 185 of nucleus, sea urchin egg, anaxial illumination, 412f in vidicons, vs. solid-state pickup devices, 227 of tissue cultured cell, 357f white clipping/punching to prevent, 234 myocardial, fluorescence, 380f, 382f see also Geometrical distortion; Shading distortion; PtK-2, phase-contrast image, 337f, 339f, 355f, 359f, Streaking 369f, 370f, 402f Dithering, 427 Digital signal, 23g, 310 DNA Digital signal processing birefringence of, 491n compared with analog, 310, 513 fluorescence and UV microscopy of, 5 reference books, 184, 352, 513 fluorescence video microscopy of see also Digital image processing; Digitally processed individual molecular threads of, 400 images in Trypanosoma cruzi, 6f Digital-to-analog converter, 23g, 330f, 331 microdomains in Digitizer, 8-bit, 335, 387, 388t, 390t analysis by polarized UV microbeam, 483, 498f INDEX 545 DNA (cont.) Dynamic range, mtrascene (cont.) microdomains in (cont.) of video sensor, 126 polarization microscopy, rectified, 498f, 499 vs. vidicon tube, vs. linearity, 193 SSEE microscopy, 412 Dynamic shading correction, 227 molecules of Dynamic tracking, 24g anangementof, 499 in helical-scan VCR, 285 orientation of DNA bases in 13-form, 491 Dynode, 24g, 195, 198f polarizability of, 491 sensitivity of polarization microscopy for detecting, Echo, 24g 404 from improper termination, 63 see also Chromatin, optical section; Chromosomes Edge birefringence, 498n Dove prism, 420f Edge detection, digital, 24g 3-D perception, 90, 446 convolution masks for, 369-372, 370f Drafting tape, 429 needs prior shading correction, 356, 358f Dropout, 181 , 300, 302 Edge enhancement, 24g Dropout compensation, 24g, 274 by analog processing, 314, 317f color video, 274n in monitors, 246 Drosophila salivary gland chromosomes convolution masks for, 369-372, 369f, 370f, 37If fluorescence, optical section, 375, 376f by offsetting two images straightened, 362f, 363 in analog, 314 Drum cylinder, 265f, 267, 277f digitally. 358, 359f cleaning, 300 Edge map, 378-386 damage of, 296 of cell fluorescence , 380f, 381-384 Dry mass, measured with interference microscope, 323n chain-coded, 379, 384 Dual video camera, combining SIT and Newvicon images, drawn with cursor, 383 147f, 148 generation with image processor, 381-386 Dubbing, 24g; see also Audio dubbing see also Edge segments Dust, 68-69, 69f, 140, 145 Edge segments Dust cap of video camera lens, 59 connection 3-D video, 421-422, 42lf, 422f; see also Stereoscopic by dilation/erosion, 379, 380f video by variable convolution masks, 379 Dynamic changes generation of, 378-379, 380f in acrosomal process of sperm, 319-320, 322f, 323f see also Edge map of birefringence, 404 Edge sharpening: see Edge enhancement distribution shown by line scan, 325 Edges, sharpness of in darkfield microscopy, 40 I vs. camera design, 233 of fluorescence, quantitation, 381, 382f, 401 faithful reproduction in video, 173-174, 175f, 178 diffusion of lucifer yellow in cytoplasm, 324f vs. frequency components, 469 in low-light-level video microscopy, 397, 398f-400f in human vision, 80, 83 see also Measurement; Quantitation (video) see also Blurring; Ringing Dynamic focusing, 24g Edited tape in camera tubes, 227 color video, splice point in, 437n in monitors, 246, 258n finished quality of, 435 Dynamic range, 24g picture resolution in , 283 of digital memory device, 364, 368 time base corrector to improve stability of, 433 of digitizer, vs. gray values utilized, 338-339, 339f Editing of display device, utilization of, 355-356 assemble, 16g, 285-286 factors limiting, 214-217 using VCR with pause function , 436-438, 438f noise limitations of, 214-215 followed by audio dubbing, 438 of photomultipliers, 415, 415n insert, 32g of scanning electron microscopes, 427n using editing deck, 285-286, 432, 437 of video camera, 232 with low-cost 1/2-inch VCR, 437n use in video microscopy, 233-234 selecting and preparing material for, 433-436, 434f of video imaging devices, 415 scene planning, 433-435, 434f video monitor vs. camera, 432 script for, 435 of vidicons in polarized-light microscopy, 406f SMPTE time codes used in, 285 see also Dynamic range, intrascene; Usable light range special effects in , 438-441 , 440f, 441 f Dynamic range, interscene: see Usable light range Editing cue Dynamic range , intrascene, 24g, 33g SMPTE time code as, 285 of camera tubes, 199t for VCR with pause function, 438f of ceo camera, 202t for VTR, 433 chilled, 197-198, 235 Editing deck, 24g, 277, 285-286 of human eye, 74 copying different formats with , 290 of image isocon, 197, 410 used for insert editing, 432, 437 of MOS camera, 202t Editing vtdeo tape of sulfide, vs. other vidicons, 215 equipment for, 432-433, 433f,.434f 546 INDEX Editing video tape (cont.) Elliptically-polarized light, 4S6 , 4S7f, 4S9f mechanics of, 436-441 Encoded signal, NTSC, 23S-242, 260; see also Color message conveyed, 433 encoding Editor: see Editing deck Encoder: see Color encoder EIA, 25g, 156n End of video field, 160f linearity chart, 256, 25Sf relation to sync pulses, 164f standards, 15S-161 , 169 Endothelial cell, living corneal, 41S see also entries under RS- ENG, 25g Eidophor, 25g; see also Light-valve projector portable VCRs for, 2S6 Eigengrau, 74n Recams used in, 274, 275f Eject switch, on VCR, 25g Eosinophil, tracking movement of, 3S4-3S6, 3S5f effect on tape threading, 26S Epifluorescence: see Fluorescence microscopy Electrical noise, 37g, 1S0-1S4, 1S3f, 310, 315-317, 369f Epithelial cell from amplifier, 315-319 buccal, anaxial illumination, 412, 412f from ceo sensor. 197- 19S corneal, observed in presence of light-scattering, 41S from electromagnetic interference, 65f, 67f, 233, 254 Equalizing pulses, 25g, 164f hum, 30g, lSI selected for even and odd fields, 161, 161n from intensifier, !Of, lSI, 196, 215, 21S, 317-319, Erase head, 25g, 26S, 269f, 277f, 437 31Sf, 396, 397, 402f, 409f flying, 279t, 2S6 measuring rms, ISl, !Sin, IS2t E-ray, in calcite, 4Sl, 4S2f from preamplifier, lSI, 215, 217 image precession of, 4S2f, 4S3 from vidicon camera, 9f, lSI, 217 , 335; see also Dark Erosion, 25g current; Noise (video camera) for sharpening dilated boundary, 379, 3SOf from VTR, lSI, 274 Error see also Glitch; Hash; Noise; Noise averaging (spatial); in diffraction image, 404n-405n, 497f, 49S Noise averaging (temporal), digital; SIN ratio parallax, in measuring distance on monitor, 319 Electric displacement vector, 25g, 4S0-4SI see also Distortion; Shading; Time-base error Electromagnetic focusing coil Ethanol, 143n, 144-145, 144f in picture tube, 244 E-to-E, 25g, 63, 63f, 64f, 432f, 440f in vidicon, 192, 192f Even and odd field Electromagnetic wave interlacing of, 154-156, 155f physical optics of, 500 selection of equalizing pulses for, 161 velocity of, 47S-4SO, 479f see also, Field (video); Interlace Electron beam Exact 2:1 interlace, 172, 307f, 426 in iconoscope, 3, 3f, 4f scan lines in, 256, 256n, 42Sn in image isocon, 197, 19Sf Exposure duration in picture tube, 242, 243f, 244 in high-speed recording, 293-295, 294t dynamic focusing of, 25Sn for photographing the monitor, 425, 425f, 426 sweep linearity and astigmatism control, 259 video camera vs. photography, 126 in SIT tube, 196, 197f see also Target integration in vidicon tube, 192-193, 192f Extended-play modes, for VCRs, 27S , 2S2t misalignment of, 66 thinner base tape for, 30 I Electron gun, 3, 25g SP,LP, SLP,47g, 276 in iconoscope, 3, 3f External sync, IS6-IS7 in picture tube, 242, 243f, 244 of camera, 1S6f, 1S7f, 235 in vidicon tube, 192-193, 192f locking VTRs to, during playback, 2S5 Electron-hole pair of monitor, 62, 259, 262 in CCDs, 19S see also Genlocking in photoconductive layer of vidicon, 193 Extinction factor, 403n, 406f, 492 in SIT, 196 Extremely-low-light-level imaging Electronic news gathering: see ENG detectors, used in astronomy, 197-19S, 319n Electronics-to-electronics: see E-to-E fluorescent, 324f, 400f Electron microscope lenses for, 130, 13lt closing gap with light microscope, 4 77 luminescence, 396-397, 39Sf-400f scanning, image quality in, 427, 427n video cameras for, 230t, 394t, 396-40 I tomography with, 419 Eye Electron multiplier accommodation of, 13g, 72 in Gen-II intensifier, 195, 196f effect on depth of field in microscope, liS in return-beam type camera tube, 197, 19Sf effect on focusing, 72 Electrons contrast discrimination by, SO-S3, Slf, S2f, 203 in intensifier tube, 195 convergence of signals in, 79-SO, SOn secondary, in microchannel plate, 195 dark-adaptation, 74, 76f, 324f, 393; see also Scotopic see also Electron beam; Electron gun; Electron-hole pair; vision Photoelectrons vs. gamma of video sensors, 229 Electrostatic lens image in , 97-99, 99f, 103f, 104 in image intensifier, 196f involuntary rapid movements, 79 in monitor picture tube, 244 iris, 72f INDEX 547 Eye (cont.) Field (video) (cont.) as logarithmic detector, 405-406, 406f; see also Weber even, odd, 154-155, !55f law first, second, 155, 155f, 160f, 163f MTF of, 123, 125 vs. frame, 27g, 154, 155f, 15Sf, 159f, 167f, 425 pupil of, 72-74, 72f, 99f, 102, 102f, 103f freezing with digital image processor, 424 refractile elements of, 71-74, 72f, 99f, 104 individual, limited image quality of, 425 resolution of, role of cones, 7S-SO, 79f skip, recording/playback, 46g, 2S9 spectral sensitivity of, 76 vertical frequency of, 51g vs. video camera tube, 415 see also Field rate; Freeze-field image; Freeze frame; structure of, 71-74, 72f, 73f Interlace unit magnification of, I 00 Field blanking, 26g see also Cornea; Crystalline lens; Ganglion; Human eye; Field-by-field Human visual system; Retina analysis Eyepoint with video motion analyzer, 293-295, 294t as aperture plane, 102, I 02f using VTR, Sf partial occlusion playback, Sf, 2S5 by pupil of eye, 449n see also Freeze-field image; Freeze frame for stereoscopic microscopy, 449-452, 452f, 453f Field diaphragm, 26g relation to pupil of eye, I 02, 102f adjustment of, 107-10S small, cf. projection distance, 6S, 69f conjugate planes in Koehler illumination, 101-104, 103f see also Ramsden disk vs. contrast in video microscopy, 65f, 66, 10S vs. hot spot, 67f, 6S Faceplate, 25g vs. illumination, 10Sf, 126 of camera tube, 59f-6lf, 9Sf image of, focused, JOSf, 109 cleaning of, 143-145. 144f see also Field planes defects affecting video microscopy, 6S r1eld-effect transistor, 26g, 61 magnification of microscope image on, I 00 Field/ frame counter, 293 size, vs. camera performance, 199t, 203 Field illuminance, 126-12S in vidicon tubes, 191-192, 192f vs . light scrambler, 414f of monitor picture tube, 242, 243f Field of view curvature causing distortion, 259-260 limited, vs. super-resolution, 41Sn reflections off, 425f, 426, 432 very large, by confocal scanning microscopy, 41S Faceplate illuminance in video microscopy, J36-13S, 475 vs. limiting resolution in intensifers, 203, 20Sf uneveness, 105; see also Shading low-light-level, camera tube performance at, 200! in wide-field specular microscope, 41S, 419f see also Camera tube specifications Field planes Fade, 26g, 439 in Koehler illumination, 26g, 103f, 104 Fall-off, 246; see also Shading optical noise arising near, 69, 139 False color, 26g, 149 Field rate, 154, 156 Far-red sensitivity exact value for NTSC, 161 , 240n of ceo, 212f for high-resolution video, 165t, 256-25S of human eye, vs. vidicon, 415 special monitors accepting multiple, 25S of intensifier camera, 400 Field-scanning of silicon vidicon, 221 microscope, 417-41S of vidicons, 206, 206n, 210f, 213f, 403 ophthalmoscope, 4JS, 419f Feature extraction, 26g, 37Sf Field-sequential.playback, 26g, 2S5 by digital image processing, 377-3S6 of high-speed recording, 294t, 295 see also Boundary; Classification; Edge map; Image Field stop, ocular, 9Sf, 99f, 103f, 104, 10S feature; Image segmentation; Motion tracking, digi• Filter (optics) tal; Pattern recognition barrier, 17g Fiber -optic for color contrast, 413 bundle, 26g, 195, 195f, 196f complementary color, for stereoscopy, 450, 450f light scrambler, 127n, 495f, 496f excitation, 25g plate, distracting patterns from, 22S interference, transmittance, 133 Fidelity: see Distortion; High-definition TV; Image fidelity; neutral-density, 126, 241 effect of phase on spatial: see Spatial filtration; Spatial frequency Fiducial line, with scan display see also Half-shade filter; IR-blocking filter; Polaroid slanted, 324f, 325f Filter (video) vertical, 316f, 3ISf band-pass, 176, 243 Fiducial marks, 319, 392 in camera, 233-234 added to VTR playback, 43S comb, 20g, 260n for measuring distances in video, 319-320, 320f high-pass, for blocking V-sync pulses, 243 see also Cursor, drawing edge map with notch, 3Sg, 260 Field (video), 26g, 154-156 in VTR , 273-274, 273f 4 per A, B color frame, 163f, 240n digital active, 159f, 173f for edge and boundary enhancement, 37S-379 end of, relative to sync pulse, 164f involving convolutions, 365-372 548 INDEX Filter (video) (cont.) Fluorescence microscopy (cont.) digital (cont.) and feature extraction, 381-384 involving Fourier transfonn, 372-377 of FITC-stained proteins, 6f selective, 513 and Fourier filtering, 375, 376f see also Circuits; Convolution kernel/mask; Convolu• image brightness in, 128-129 tion operation; Digital image processing; Fourier image of microtubules in, 402f, 4llf, 447 filtering of individual molecular threads white-balancing, 240, 240n of actin, 400 Filtering circuits: see Circuits of DAPI-stained DNA, 401 Filtering device, microscope optical train, 93, 94£, 122 intensity of, change with time, xxvi, 381-383, 382f, 401 FITC stain of proteins, 6f intensity contour of, xxv(f), !Of Flagella, bacterial low-light-level, 397-401 darkfield microscopy, 40 I of lucifer yellow-labeled swimming embryo, 219, 220f flagellin polymerization, in dark field, 401 of microspheres, xxv(f), !Of polarized-light microscopy, 405 , 407f of mitochondrial flashing, xxvi, 381, 382f Flagging, 26g objective lenses for, 13lt, 135t, 136, 136n relation to AHFC in monitor, 187, 259 pickup by SIT, on dual camera system, 147f, 148 of video picture, 26g, 188f, 291-293, 292f with Plan Apo objective, 402f Flare, 26g of propidium iodide-stained DNA, 6f effect of condenser NA on, 110 pseudocolor display in, xxv(f), xxvi(f), !Of, 381, 382f vs. image contrast, 107-108, 133 quantitation, vs. background subtraction, 356, 358f in modem objective lenses, 107, 133 of quinacrine mustard-stained DNA, 5 reduction of resolution, vs. NA, 397n with flying-spot microscope, 5, 418, 419f review in IR region , in video camera, 206n in biomedical science, 457 by Koehler illumination, 101 general, 122 by lens coating, I 07, 132 on video intensification, 397n, 399, 400 by oil immersion, 127 of rhodamine 6G-stained mitochondria, 381 , 382f sources of, in microscope, I 07, 132 vs. spectral response of vidicons, 206 Flashes super-resolution in, 418 bioluminescent, time course of, 396, 396n, 400f very-low-light-level images, 324f, 397-401 bright, vs . flicker, 83 , 85f, 86f lag in SIT, 219, 220f fluorescent, in mitochondria, 381 noise reduction in, !Of, 402f Flash exposure with video camera, 126n, 294, 294t video cameras for , 193, 229, 2301, 394t, 396-401 Flashing, of UV-emitting CRT, 417 video intensification for , 5, 397-401 Flat-screen picture tubes, 259-260 Fluorescence recovery after photobleaching, 27g, 416 Flicker, 27g, 83-86 Fluorite objective lens, 27g in alternate-field stereo video, 451-453, 452f, 453f, 454f aberrations corrected in , 27g, 133n frequency, vs. threshold modulation, 86f for epifluorescence microscopy, 136n in motion picture, vs . video, 84-86, 153-154 Flyback, 27g, 152-153, 153f photopic, vs. scotopic, 83 Flying erase head, 279t, 286 produced by bright flashes, 83 Flying-spot UV microscope, 417 split-field stereo video, absence in , 453-455, 455n quantitative analysis using, 5 vs. stimulus location and diameter, 84, 85f FM, 27g vs. video scanning rate, !54 demodulation, in VTR playback, 274 vs. wavelength and illumination, 83 , 85f recording, 272-273 see also Critical flicker frequency; Jitter sound, for data storage on video tape , 441 Flicker scope, for 3-D reconstruction, 420 of TV set, 242 Floppy disks, storage of video image on , 301 - 302, 331 video signal Fluorescence in laser disk playback, 302 of crystalline lens in human eye, 74n for OMDR recording, 303 dynamic changes, quantitation of, 381, 382f, 401 F number. 28g, 58 multispectral analysis of, 342 Focal distance, 95-99, 96f quantitation by video, 5, 323, 324f, 401 Focal length, 96£ Fluorescence microscopy vs. accommodation of eye, 72 of bioluminescent particles, 400f of condensers, 139t of chromatin, optical sections, 375, 376f Focal plane for chromosome identification, 5 of condenser, 97f, 101-102, 102f combined with DIC or polarized-light microscopy, 147f, diffraction pattern above and below, Ill , 116f 148 of video camera, 59 , 59f 3-D image of chromosomes, 375-377, 376f, 418 Focus, 27g of dinoflagellate, viewed with PV, 396, 400f of perfect lens, 93-95, 95f, 96f dye diffusion in cytoplasm of egg, 324f Focusing epifluorescence automatic, of microscope, 375 use of fluorite objectives in , 136n dynamic: see Dynamic focusing image brightness vs. objective NA, 128, 397n Focusing screen, moving as Archimedes spiral, 420, 420f video cameras for extremely-low-light-level, 2301 Fold-over frequency: see Aliasing INDEX 549 Footnotes, vi Freeze frame , 28g Fonnat, 27g with animat1on recorder, 279t, 289 special, for time-lapse VCRs, 296n with digital processor, 387, 424, 511 see also Camera tube, diameter of, nominal; Video with dynamic tracking VCR , 282t, 285 fonnats; VTR fonnats vs. freeze field Fonn birefringence, 491-492 choice of, 425 Four-dimensional display: see 4-D display scan lines in, 307f Fourier analysis, 372 jitter in, 92 , 293 Fourier descriptors of shape, 379 with modified editing deck, 289n Fourier filtering with OMDR, 304 vs. convolution operation, 375-377, 377f Frequency, 28g to correct degradation in optical system, 375f AC line, 181 optical sectioning of fluorescent chromatin, 375, 376f bond stretching, IR microscopy, 40 I to produce modified aperture function, 373-374 cutoff for removing hannonic noise, 377 for light microsocpe, 473 Fourier plane, 373 of MTF, 469-471 , 470f Fourier theory, 4 70 radio, 42g Fourier transfonn, 27g, 372f, 373f temporal, vs. spatial, and MTF, 124-125 influence of aperture function on, 374-375, 374f, in video tape recording, 263-264 375f see also Frequency response; Spatial filtration; Spatial vs. convolution, choice of, 377 frequency and digital filtering, 372-377 Frequency modulation: see FM of a Gaussian, 4 73 Frequency response, 28g hardware for perfonning, 391 curve for, 177n inverse, 122, 373, 375 audio, vs . video, 179f pattern recognition involving, 386 expressed in decibels, 182 relation to diffraction pattern, 122 vs. horizontal video resolution, 174-178, 176t relating OTF and point -spread function , 470-4 71 of monitor, 246 and super-resolution, 377 vs. SIN ratio, 124, 182 see also Spatial frequency of system MTF, 176-178, 177n Fovea, 27g, 77 for video, 178 central, visual acuity in, 77-79, 79f in VTR recording, 270-274, 273f distribution of rods and cones in, 77-79, 78f see also Bandwidth; Circuits, filtering see also Retina Fresnel coefficient of reflection, 132n, 482 Frame, 27g, 154-155, 155f Fresnel ellipsoid, 480-481 , 480f Frame averaging Fringes, moire and background subtraction, 183f, 184f, 409f in color video, 171, 171n effect on noisy image, 182-183, 184f, 336f following splicing point, 437n Frame buffer, 28g, 387, 388t-391t, 391, 512; see also noise pattern, 181,235, 375 Image memories in optically copying a monitor, 431 Frame-by-frame analysis: see Freeze frame see also Ringing Frame grabbing, 28g Frustrated total internal reflection, 122, 410 into digital image processor, 513 F-stop, 28g, 58 see also Frame memory F-to-V transfer, 430 Frame memory, 330-331, 330f, 364, 368, 474 !-megabyte, 513 pixel number vs. microscope magnification, 474 Gain, 28g Frame summation, 182-184 adjustment of, with gamma unchanged, 232, 312f of low-light-level fluorescence image, !Of, 396, 402f, amplifier, 311-312, 312f, 315f 409f control of vs. VCR resolution, 424 in camera, 232, 234 of very low contrast DIC and polarization images, 9f in monitor, 246, 247f of very low contrast polarization image, 184f, 409f vs. noise, 315 of very-low-light-level luminescence image, 397 see also Automatic gain control; Contrast control , on see also Digitally processed image monitor FRAP, in living cells, 27g, 416 Gamma, 28g Freeze-field image, Sf, 285 of amplifier, 311, 313f absence of jitter in, 92 of intensifier camera tubes, 397, 405n of cilia, Sf knee in curve, 214, 215f comet tailing in, 219, 220f, 221f, 293 of monitors, 246-248, 247f photographing, 425 for photometry, 209, 397 prominent scan lines in, 307f, 407f of solid-state cameras, 202t resolution and SIN of, 425 of sulfide vidicon, 209, 219f, 246 with and without Ronchi grating, 407f vs . video gain adjustment, 232 scan lines and staircasing in , 322f of video imaging devices, 209- 214 see also High-speed video; Motion analyzer, analog; vs. eye and photographic emulsion, 229, 405n Still-field display of vidicons, 194t, 209, 221 550 INDEX Gamma compensation, 28g Gray level histogram, 29g; see also Gray value, histogram for color video, 209, 246, 247f transformation of; Histogram; Histogram Ganglion transformation cells in retina, 73f Gray scale, 29g convergence of rod cells to, 79-80, SOn for color, vs. monochrome camera, 229 optical recording of neuron firing in, 416 in NTSC color signal, 241 f Gaussian steps, in camera specifications, 233 distribution of scan lines, 17lf, 244-246, 245f stretching: see Stretching, of gray scale Fourier transform of, 473 video test signal, 233 , 256 MTF curve approximated by , 473 see also Gray wedge, stepped Gen I intensifier: see Image intensifier Gray value, 334-335 GenII intensifier, xxiii, 28g , 195, 196f, 197, 230t at 0 and 255 in binary image, 355f Genlocking, 28g captured by video camera, 232-233 camera to special effects generator, 439, 440f of digitally manipulated pixels, 360-363 external syncing of cameras, 186, 235 expanding of range with pseudocolor, 356 synchronizing video cameras with, 186-187, 187f after histogram flattening, 353f for stereo video, 451, 452f histogram transformation of, 340-345, 343f-347f Geometrical distortion, 28g through discrete ITF, 345-347, 345f-347f circle appearing as ellipse, egg-shape, 156n, 248-251, through linear ITF, 342-345, 343f-346f, 355f 252f, 253f through nonlinear ITF, 348-356, 349f-355f mapping function to correct, 358-363, 360f, 36!f, 362f see also Histogram; Histogram transformation in monitors, 248-251, 252f human eye, steps distinguished by, 335 pincushion distortion, 40g, 248 linearizing for measuring optical density, 363-365, 365f, in intensifier camera, 227, 253f 366f in solid-state pickup devices, 227, 294n manipulating with look-up table, 387 test charts for measuring, 248-251 , 252f, 253f, 255f, measurement of average, 383 258f, 307f and nonlinear calibration, 363-365, 365f, 366f in vidicon tubes, 193, 227 steps needed for accurate image reproduction, 335 see also Distortion variation due to shading, 363 Geometrical manipulation of image, 362f, 363; see also vs. video camera input, 333 Geometrical distortion, mapping function to correct see also Gray level; Gray scale; ITF Ghost Gray wedge, stepped, 29g echo, 24g line trace of, 315f in optical copying, 429-430 modification of, 312f-315f and termination, 63, 185 test signal, 313f Glan-Thompson prism, 483, 483n, 495f; see also see also Gray scale Polarizer; Prism, calcite Ground-glass diffuser Glitch, 28g, 67f, 186, 29lf, 297f high-intensity arc lamp, 107n at junctions of scenes, 298, 437 low light transmittance of, 132, 138-139 see also Hash; Noise; Noise bar Grounding, vs . noise, 67f Glycerol: see Immersion medium Ground loop interference, 181, 209, 235 Goniometer stage, 448f Growth kinetics Graphics tablet, 328, 392 of actin filaments, 322f, 323f Graphite, 413n of tlagellin, 40 I paricle, diffraction pattern from, 119 Guard band, 29g Grating in Beta and VHS formats , 266-267, 266f colored, and MTF of eye, 82n, 83f in U format, 265f, 266, 276 modulated sine wave, visual response to, 81-82, 82f Guard-band noise, 29g video response to, 173-176, 174f in non-standard speed playback, 277 see also Amplitude response; Ronchi grating in playback of time-lapsed tape, 27lf Gray sources of, 270 in CIE chromaticity diagram, 239f see also Noise bar as a color, 86, 90, 238-240 Guide rabbet, 265f, 267, 301 Gray level, 29g Guide rollers, 265f, 267, 300 analog manipulation, 311-314, 312f-316f in digitized video image, 331-332 in images, 1- through 8-bit, 332, 335, 337f, 384 Half-shade filter resolution for stereo microscopy, 450, 450f of 8-, 10-, and 12-bits, look-up tables with, 387 in stereo video microscopy, 451 in digital video image, 334-335 Halogen lamp, characteristics of, 128t-129t effect on image reproduction, 337f Hard-copy device, 331 in human eye, 335 , 356 Hard disk, 302, 331 thresholding Hardware , 29g effect of nonuniform background, 356, 358f for digital image processing, 328-329, 386-391, 457n, for generating binary image, 340, 378-379, 380f 511-513 see also Gray value for performing Fourier transforms, 391 INDEX 551 Harmonic noise, removal of H1gh-resolution microscopy (cont.) Fourier filter vs. convolution, 377 brightfield, 353f, 414f with spatial filtering , 375 DIC, 9f, 322f, 408f, 4llf Harmonics fluorescence, 6f, 4llf higher frequency, in square wave, 175n polarization, 7f, 9f, 407f, 409f, 493-499, 498f, 499f spurious, 179 stereo, 451-455 Hash, 67f, 181, 296, 297f with confocal optics, 418 H back porch, 159f High-resolution video of color signal, 240, 24lf cameras, U.S. sources, 236t see also Color burst color monitor, 260 Head, 29g, 264, 264f, 265f, 267-268 digital image processing system, 279t, 388t, 390t, 458t Head gap, 272, 272f image isocon, 197 dimensions, 266 monitor, 246, 256-258, 427 Beta format, 266f commercial sources of, 258n VHS format, 276 OMDR, 303-304, 304t slanted, 266-267, 276 slow-scan camera, 417 Heart cells, cultured, velocity of movement of, 320 TV standards for, 165t Helical-scan recording, 29g VTRs, 279t, 284, 284t control tracks on, 265f, 266f, 267 time-lapse, 289, 289n freeze field vs. running image, 425 U format, 424, 424n track angle on, 265f, 267-268, 268f see also Ultra-high resolution video tracks in: see Video tracks High-speed video see also Helical-scan VTR camera, 294-295, 294n, 294t Helical-scan VTR, 265-268, 265f, 269f, 277f recorder, 293-295, 294t control pulse in, 187 Histogram, 344f see also Drum cylinder; Head; Head gap; VCR; VTR bimodal , 340, 340f Hemocyte, contrast vs. field diaphragm, 65f binary, 355, 355f Hemoglobin oxidation measured in live animals, 323 calculation Heterodyne recording, 277n of density and area from , 339-341 Heuristic processing routines, 379 hardware for video rate, 391 H-HOLD control, 29g, 56f, 62 , 255 to display dynamic range used, 338 High-definition TV, 295 flat, approximation of, 354 High-extinction microscopy, 29g gray value, after ratioing, 366f DIC, 9f, 306f, 322f, 4llf image, 3lg extinction factor, 403-406, 403n, 406f, 492-493 of pixel gray value, 340, 512 fluorescence, !Of, 324f, 400f, 402f sigmoid-shaped, 353-354, 355f luminance ranges in, 394t, 403 see also Gray value, histogram transformation of; Histo• optical noise in, 69 gram transformation polarization, 7f-9f, 184f, 325f, 403-406, 407f-409f, Histogram transformation 492-499, 494f-499f brightfield image, stained blood smear, 352, 352f video cameras for, 193, 2301, 394t clipping of highlight, 339f High-frequency response, VTR, 270-272, 272f, 273f and contrast enhancement, 342-349, 343f, 346f, 349f, High impedance: see Hi-Z 352f-355f Highlight, 29g, 234 contrast manipulation by , 512 analog expansion of, 314f flattening, 350, 350f clipping of Fortran program for, 35lf by amplifier, 179, 180f for mapping of ITF, 34 7 histogram showing, 339f multi-dimensional, in edge-mapping, 379 distortion of nonlinear, 354-356 in amplifier, 179, 180f phase-contrast 1mage in VCR recording, 299 vs. background, 353-354, 354f response of video camera to, 66 tissue culture cell, 352f see also Streaking ratioing, to correct shading, 364, 366f High light-gathering power see also Histogram effect of condenser immersion, 127-128 Hi-Z, 29g, 63-64, 63f, 64f, 434f, 440f m fluorescence microscopy, 128, 397n H-line scan , 167f, 173f, 183f; see also Line scan importance in video microscopy, 130-133 Holographic microscopy, 30g, 122 objective lenses with, 130, 131t image transformation during reconstruction, 419 of Plan Apo objectives, 136 Hookup High luminous density, 127 an array of video equipment, 184-189, 440f light sources having, 127n, 128t-129t camera and monitor, 63f High-NA lenses, 131t special effects generator, 440f extinction factor with , 492 stereo video, 452f-455f in video microscopy, 136, 306f two cameras, 186f, 187f High-resolution microscopy video analyzer, 319-320, 320f anaxial illumination, 412f video processor, 64f, 445f 552 INDEX Hookup (cont.) Human eye (cont.) VTR, 64f, 150f, 431f-434f crystalline lens see also Connectors, BNC, UHF; Termination accommodation of, 72 Horizontal blanking, 30g, IS4, 154f, 158f-164f, 158t, fluorescence in, 74n 167f-169f; see also Blanking dark adaptation, 76f Horizontal resolution for low-light-level microscopy, 393 of camera, monitor, vs . recorders, 424, 442 for polarization microscopy, 492-493 definition of, 170, 172, 173f response to various wavelengths, 74-77, 76f, 205 in monitors, 246 see also Scotopic vision non-standard scan rate, 185 diffraction by pupil, 74, 78 and optimal viewing distance, 442 distribution of rods and cones in, 73f, 78-79, 78f vs. phase of frequency components, 175n, 176 gray shades distinguished by, 335, 356 vs. rise time, video bandwidth, 172-176, 175n, 176t image parameters modeled by Rose, 81, Sin of VTRs, 263-264, 2811, 282t, 283-284, 284t, 424 intensity discrimination, vs. spectrum, 81f VHS color, 285 lens transmission, effect of aging on, 77 of video cameras, 229-231 light adaptation, vs . wavelengths, 74-77, 76f; see also of vidicon tubes, 193 Photopic vision see also Amplitude response curve; Limiting resolution; light transmission in, 74-77, 77f Resolution (video); Resolution (video camera); Res• vs. microscope depth of field, 118 olution (video monitor); Resolution (VTR) motion, perception of, 424-425 Horizontal scan, 30g, 151-153, 153f, 167f, 173f quantum efficiency of, 81 n across white bars, 172-176, 174f, 175f, 471-472, 472f radiance response of, 229 interval, 156 reference books on, 71 , 72f see also H-scan rate; Scan lines refraction in, 71-72, 99f Host computer: see Computer refractive index of parts of, 72f Hot spot, 30g, 66, 67f resolution limit of, 77-80, 79f eliminated by stops, 68, 68f self light in, 74n vs. field diaphragm, adjustment of, I 08 spectral sensitivity of, 76f, 205, 238 influence on video contrast, 66 vs . video imaging devices, 205, 403, 415 sources of, 67-68, 68f Stiles-Crawford effect, 74n in target of camera tube, 181 storage time and SiN ratio, Sin Hough transform, image segmentation by, 379 structure of, 71-74, 72f, 73f H-scan rate, 156, 156n threshold contrast of, 80-83, 80f, 81f, 203 exact, 189 threshold delectability of, 79, 79f for monochrome, vs. color, 189n threshold sensitivity of, 74 non-standard, 185 see also Cone cells; Cornea; Eye; Human visual system; see also Horizontal scan Retina; Rod cells H-sync pulse, 30g, 156-157, 157f-164f, 158t Human testis, brightfield, thin optical section, 414f detail in Human visual system, 80, 82, 91, 92 RS-170, 161, 162f boundary detection in, 82-83, 84f RS-170A, 161, 163f, 240n in designing TV, 92 RS-330, 160f, 161 and flicker, 83-86, 85f, 86f; see also Critical flicker displayed in pulse-cross, 169f frequency duration of, 158t MTF of, 81-83, 82f, 83f, 123, 125 line scan displaying, 167f noise averaging in , 80 selective blocking of, 243 response to gradients, 82-83, 84f, 90 see also RS-; Sync pulse signal processing in , 79-80, 82-83, 83n, 84f, 91, 91n Hue(s), 30g smooth motion, perception of, 424-425 vs. color, 86-90, 238-240 stereoscopic acuity in, 90f, 91, 9lf mixing three primary, xxv(f), 86-90, 89f stereoscopy, 90-91, 90f, 91 f, 446; see also Stereoscopy in NTSC signal, 238-240, 241f vs. video camera, 92 in video monitor see also Eye; Human eye automatic balancing, 262 Humidity, effect on video tape, 268, 287, 296 gamma compensation for, 246 Huygens wavelet, Ill, llln, ll2f see also Chromaticity, CJE, diagrams; Color Hum, 30g, 181 lconoscope, I, 3f, 4f Human eye IEEE scale, 30g aberration, vs. illumination, 73-74 voltage for video and sync signals, 160n, 160f, 163f accommodation, 13g, 72 , 118 Igniting arc lamps, caution, 61-62, 296 angular resolution of, 77-80, 78f. 79f Illuminance, 31g, 77 , 205 aphakia, 15g, 74n levels, in nature, 75f averred vision, 78 retinal, and standardized pupil area, 77; see also Troland brightness range sensed by, 72-74, 75f, 125 see also Faceplate illuminance; Image brightness colors distinguished by, 356 Illumination color vision in, 86-90, 87f. 88f, 89f; see also Color; Hue(s) brightness of, 126-133 contrast detection by, 203 in epifluorescence, 128 INDEX 553 Illumination (cont. ) Image averaging (cont.) coherence vs. resolution, 113n to reduce random noise cone angle of, I 08f, I 09-11 0, 109f spatial, 183, 368, 368f, 369 oblique temporal, 50, 182 and asymmetric diffraction, 66 see also Frame summation; Image integration; Noise appearance of 3-D relief, 90, 446 averaging (spatial); Noise averaging (temporal) stereoscopy with , 449 Image brightness video-enhanced optical sections by , 412 vs. compensator, 404, 493 with uniform aperture, 127n, 495f vs . condenser N A , 136 image with, 306f, 414f in epifluorescence, 128 , 397n see also Scrambler; Shading and illumination, 127-130 Illuminator, microscope vs . magnification, in video microscopy, 125-126 adjustment of, 105- 107 trading brightness for, 130-131 , 136-138, 4 7 5 ground-glass diffuser in, 105, 138-139 measurement, instantaneous, 363 with high luminous density, 127, 128t-129t, 404, in microscope, 126-133 495f and objective NA, 130, 131t Image, 31g time-lapse, vs . high-speed VTR, 288, 295 binary, 355, 355f, 378-386 total, in area, 340-341 1- through 8-bit, 332, 335, 337f, 384 Image contrast, microscopy calibrating distance in, 100, 319, 320f vs. compensation, 404, 493 computer-enhanced, 8; see also Contrast enhance- vs. flare , 107, 108 ment/stretching (digital); Digitally processed image; generated in optical train, 93 , 94f Image enhancement; Image processing of condenser generation of, 118-122 iris: see Condenser iris diaphragm; Conoscopic modes of generating, 122, 393-418 observation vs. spatial frequency, 118, 122-126, 469 conjugate: see Conjugate planes vs. objective and condenser NA , 123, 124f conoscopic, 2Jg, 110, liOn, 405 see also Contrast; Contrast transfer function; Modulation 3-D, 420-422, 42lf, 422f transfer function; MTF in eye, 71-74, 99-100, 99f Image converter tube, 5, 401 of field diaphragm, 65f, 97f, 101, 102f, 104, 106, 107, Image convolution, 31g; see also Convolution 108, 108f Image degradation formation of, in microscope, 97-100, 97f-99f compensated by digital processing, 329, 338 Koehler illumination, 101-104, 102f, 103f by gray level clipping, 339, 339f, 383 formed by perfect lens, 93-97, 95f, %f by smoothing filter, 317, 318f, 368, 369f orthoscopic: see Orthoscopic image in video, 172-176, 178-185 of point object, 470-471 , 470f; see also Diffraction vs . vignetting, 64-66, 65f, 68f pattern see also Aberration; Blemishes; Blooming; Blurring; Dis• pseudocolor, fluorescence tortion; Electrical noise; Glitch; Hash; Jitter; Lag; of cell, xxvi Noise; Noise bar; Optical noise; entries for of microspheres, xxv Resolution; Shading; Snow by SIT camera, frame averaging, 182, 183f, 184f, 402f, Image dissector, 3lg, 236, 333, 416; see also Image• 409f scanning camera; Photodiode array stereo, 446-455 Image distance video, 1-4, 149 adjustment of, with projection ocular, 135 on video camera tube, 57f, 59f, 60, 60f, 61f, 64-70, for standard ocular, 60, I 00 65f, 67f, 68f, 69f; see also Magnification, see also Distance microscope Image distortion: see Distortion video-enhanced: see Contrast enhancement (analog); Im• Image enhancement, 31g, 396-415 age enhancement; Living cell (video microscopy); by analog processor, 310-319 Low-light level in camera, 232, 234 see also other Image entries digital, 70, 182, 183f, 184f, 329, 338-363, 365-377, Image analysis, 31g 512 analog, 5-6 in monitor, 246, 247f providing line scan, 319-320, 320f, 323, 324f, 325- see also Contrast enhancement (analog); Contrast en• 326, 325f hancement/stretching (digital); Digital image pro• digital: see Digital image analysis cessing; Digitally processed image; Video image, image analyzer, 310 enhancing see also Feature extraction Image error Image-analyzing systems artifacts, II, 413 analog, 3 I 0, 325 in digitized image, 335-338 digital, 416, 458t, 511-513 aliasing, 14g, 17ln, 471-472, 472f Image averaging, 31g, 70, 182 in polarization microscopy, 404n-405n, 497f, 498, analog, 317, 318f 498n digital, 9f, IOf, 183f, 184f, 335, 336f, 338 see also Distortion; Optical noise with smoothing convolution, 368, 368f, 369f Image feature by photography, 182, 319 describing individual objects, 377, 378f 554 INDEX Image feature (com. ) Image planes, 97f-99f, 101-104, 373, 373f representation of, by pseudocolor, xxv , xxvi, 10, 149, in Koehler illumination, 101-104, 102f, 103f 381, 382f in microscope, 97f-99f, 112f, 374f statistics of, 378f, 379 see also Intermediate image plane see also Feature extraction Image processing, 32g Image fidelity, effect of phase on, 172-176, 469; see also analog, 69-70, 309- 319 Image degradation; Image error camera, 232, 234 Image formation: see Image monitor, 2461-248 Image histogram: see Histogram processor, 325, 433 , 434f Image integration VTR, 274 in camera tube target, 3-4, 49g, 193, 317-319 applications in cell classification, 386 in ceo camera, 198, 235 courses, seminars on, 457, 459 by frame summation, 9f, !Of, 184f, 402f, 409f interactive, 347-348, 387, 458 by photography, 70, 182, 424-425 plug-in boards for small computers, 513n see also Image averaging real-time, 381-384, 458, 512 Image intensifier, 5, 3lg, 195-197, 195f, 198f, 20lf in retina and brain, 79-86, 90-92 distortion in, 227 software for, 351, 455n, 512, 513, 513n four-stage type, 396-397, 398f-399f and video cameras, 234 lag, vs. faceplate illuminance, 219f see also Digital image processing; Digitally processed light-transfer characteristics, 215f image; Image analysis; Image enhancement; Signal in microscopy, low-light-level, 394t, 396-403 processing; Video enhanced image, analog aequorin luminescence image, 398f-399f Image-processing systems (equipment and software) noise character, microscopy applications, 230t analog, 64f, 325 noise vs. photoelectron statistics, 298f digital, 386-392, 388t-391t, 458t, 511 - 513; see also photon statistics as limiting, 196, 208f, 218, 415 Digital image processors (equipment) sensor size, 199t-200t, 231, 232t idealized, 330f, 334 SIN ratio, vs. faceplate illuminance, 217f Image resolution: see entries under Resolution specifications, 199t-200t, 2llf Image rotation see also Gen II intensifier; Image integration; Image digital, 360 isocon; Image orthicon; PN; Intensifier camera Dove prism for, 4 20f tubes; Intensifier isocon; I-SEC; !SIT; PVidicon; Image-scanning camera, 227, 236, 415-418, 458t Low-light level; SEC; SIT linear light-transfer characteristics in, 333 Image isocon, 31 g low geometrical distortion in, 236, 333 description of, 197, 198f, 410 wide-field specular, 418, 419f fluorescence image with, 6f Image segmentation, 32g, 377-378 gamma curve of, 214, 215f binary image used in, 378-381 lag, vs. faceplate illuminance, 219f Hough transform for, 379 with large photocathodes, 199t use of edge map in, 380f, 381-384 phase-contrast image with, 6f using heuristic processing routines, 379 potential for microscopy, 410 see also Boundary; Digital image processing; Edge map protection circuits for, 221 Image statistics, based on edge map, 379 resolution Image, stereo: see Stereo microscopy; high-resolution; Ster• and dynamic range of, 197, 410 eoscopic video; Stereoscopy vs. illuminance, 208f Image-storage device signal current, vs. illuminance, 215 iconoscope, 3-4, 3f SIN ratio, vs. faceplate illuminance, 200t, 217f intensifier camera tube as, 317-319 specifications of (35-mm), 199t-200t vidicon target as, 193, 319 see also Intensifier isocon; Microscopy Image subtraction, 511 Image manipulation conditioned by overlay, 387, 388t, 390t, 390f with interactive ALU, 387, 390f for fixed noise removal: see Background subtraction warping, 358-363, 362f for motion detection, 356, 357f see also Image processing Image summing: see Frame summation Image memories, 330-331, 387, 388t, 390t, 391-392; see Image thresholding also Frame buffer analog, 312, 314f, 316f Image orthicon, 32g, 197 pseudocoloring by, 314 gamma curve of, 214 with special effects generator, 313, 440 lag, vs. faceplate illuminance, 219f digital, !Of, 32g, 254f, 355f, 379, 380f, 384, 385f, 410; low-light-level DIC image with, 410 see also Digital image processing in microscopy, 6f, 410 enabling use of higher NA, 410 resolution, vs. illuminance, 208f see also Thresholded image; entries under Thresholding SIN ratio, vs. faceplate illuminance, 217f Image tube: see Image intensifier specifications of (45-mm), 199t Image wave, and diffraction pattern, 111-118, 112f, 113f, Image overlay, for area-selective manipulation, 380f, 383- 116f 384, 387f Imaging Image parameters, modeled for human eye, 81, 81 n with and without camera lens, 60, 60f, 6lf Image pickup tube: see Camera tube microscope components performing, 93, 9,4f INDEX 555 Imaging device, video: see Camera tube; CCD; Image Intensifier isocon, 33g intensifier; Solid-state sensors 35-mm photocathode, characteristics of, 199t-200t Imaging systems 40-mm, resolution vs. illuminance, 208f confocal, 5, 2lg, 416-418, 419f sensitivity, noise, microscopy applications, 230t 3-D, 420-422, 420f-422f see also Image isocon 4-D, 455n Intensifier vidicons, 33g, 196, 408 quantitative analysis of, by MTF, 467 non-linearity in, 397 see also Linear systems analysis see also Intensifier camera tubes Immersion medium, 32g, 131!, 139 Intensity contour cleaning oil from lens surfaces, 140-141, 141f of3-D diffraction pattern, 115, 116f for condenser, 127-128, 139 of diatom frustule pores, Cover, 306f effect on aberration, 134 of fluorescent microspheres, xxv, JOf glycerol, 1311, 134, 140 Intensity scan, 5, 324f; see also Line scan reduction of flare by, 127 Intensity transformation function, 33g: see also ITF as source of optical noise, 69 Intensity transformation table , 33g; see also Look-up water, 1311, 134, 135t, 140, 142f table Impedance, 32g Intensity, video measurement of, 323, 363-365 and termination, of coaxial cable, 63-64, 63f, 64f, 185 birefringence, 323, 325f see also Termination digital calibration, 383 Impulse response, 471 fluorescence, 324f FN, FV, PV, 32g, 396-397; see also PVidicon Interactive ALU, 387, 390f Incandescent illuminator: see Lainps Interactive devices, 389t, 391!, 392; see also Digital image 112-inch format: see Beta format; M format; VHS format processors (equipment); Digitizing tablet; Joystick; Incompatibility, 149, 164, 184-189, 235, 286, 433n Light pen Index ellipse, axes of, 480f, 481, 486-492, 487f, 488f, Interactive ima'ge processing, digital, 32g, 329, 347, 458t, 489f, 490f 512; see also Digital image processing Index ellipsoid, 480-481 , 480f, 482f Interactive video disk systems, 302 Index pin, 32g, 191, 192f Interference colors vs. ringing, 235, 235n seen between crossed polars, 491n Infra-red: see Far-red sensitivity; IR microscopy; IR video recording of, 405 response Interference filter, transmittance of, 133 F-Newvicon, 394-396 Interference microscopy sensitivity, noise character, microscopy applications, measurements with, 409 2301, 394t, 396-397 and potential of video, 408-4 10 Insert editing: see Editing, insert Interlace, 33g, 154, 154f Instrumentation camera, 32g, 55 1:1, 38g, 161n, 185, 256 high-resolution, 236t 2:1, 50g, 56n, 155-156, 155f, 185 , 256 horizontal resolution in , 231 exact, 156n, 172, 172f, 463 U.S. sources of, 235, 236t scan lines in, 256n, 307f, 426, 428n Integrated circuit, 33g microcomputer, 161 n IR microscopy of, 403 non-interlace, 37g large-scale, 34g, 197 random, 42g, 56n, 161 , 172, 185 very-large-scale, 51g random interlace camera, 256, 290, 293 Integrated optical density, 340-341 see also Freeze-field image; Scan lines Intensifier camera Intermediate image plane, 33g, 97, 98f, 99f. I 03f, I 04 threshold sensitivity, 397 faceplate of camera tube at, I 00 in video microscopy, 394t, 396-401 , 398f, 399f, 400f, in stereo microscopy, 447f, 450f 402f lnterscene dynamic range: see Usable light range see also Image intensifier; Low-light level lntrascene dynamic range: see Dynamic range, intrascene Intensifier camera tubes Intrinsic brightness: see Luminous density gamma of, 397, 405n Inverted microscope: see Microscope, inverted general description, 1%-197, 197, 198f Iodine crystals lag in, 218f, 219-220, 219f. 220f dichroism in, 485n light-transfer characteristics, 215f light absorbtion by, 413n limiting resolution, vs. faceplate illuminance, 208f Iontophoresis, 324f noise in, 181, 217-219 I and Q vectors, 30g, 239-240 character of, 230, 396-397 IR-blocking filter, 194t pincushion distortion in, 227 contrast loss without, 206n sensitivity class, microscopy applications, 230t, 394t, IRE scale, 33g 396-403, 400f, 402f, 408f, 409f NTSC color video, 163 f signal output, vs. faceplate illuminance, 215f RS-330, 160f SIN ratio, vs . faceplate illuminance, 217f Iris, automatic, m video camera, 125 specifications of. 199t-200t, 211f vs. video microscopy, 233n, 240-241 4-stage, 396, 398f-399f see also Condenser iris diaphragm vs. vidicons, noise limitation, 215, 217-219 IR microscopy, 40 I , 403 see also Image intensifier history of video for , 1.4 556 INDEX IR response Lag , 34g, 219 of CCDs, 212f build-up, 220, 223f of human eye to, 74, 403 in Chalnicon, Plumbicon, 219, 222f, 223f of vidicons, 206, 210f, 213f, 403 decay, 219-220, 222f spectral range of, 397n degree of, objectionable for broadcast TV , 219 I-SEC in intensifier tubes, 199t-200t, 219f 40-mm photocathode, specifications for, 199t-200t in Newvicon, image of swimming sperm, 219, 22lf resolution, vs. illuminance, 208f in SIT, image of swimming embryo, 218f, 219-220 ISIT, 33g, 196 producing comet tailing, 219, 220f, 293 in low-light-level microscopy, 394t, 396-397 third-field, 219-220 16-mm, light transfer characteristics of, 215f in various camera tubes, 194t, 199t 40-mm, specifications for, 199t-200t vs. faceplate illuminance, 219f resolution, vs. illuminance, 208f in vidicon tubes, 194f Isocon: see Image isocon see also Camera tube specifications, lag Isointensity contour: see Intensity contour Lamp aging, differential loss of UV output, 132 Isotropy, vs . anisotropy, 478; see also Anisotropy Lamp and mirror centering, 105, 107 ITF, 342 Lamp log, 298 discontinuous, produced by binary image, 355, 355f Lamps discrete carbon arc, 3 FORTRAN program for calculation of, 35lf, 352 with high luminous density, 127, 127n, 128t, 129! implemented by look-up table, 347f mercury arc to transform gray value, 345, 345f-347f aging vs. UV output, 132 for generating pseudocolor display, 348, 348f caution in igniting, 61-62 relation to gray level histogram, 342-344, 343f, 344f ground-glass diffuser in, 107n linear, 342-346, 343f-346f stand-by, in VCR, 48g, 296 linearizing digitizer input with, 363-365 tungsten nonlinear, 346, 348-356, 349f-355f color temperature of standard, 200n, 206, 209, 2llf, sigmoid-shaped, 354, 355f 217f see also Look -up table dimming of, vs. red sensitivity of Newvicon, 415 F-Vidicon: see P-Vidicon halogen, 128t to observe UV microbeam, 417 intensity variation with time, 384 I'Vidicon see also Light source; Xenon arc lamp for low-light-level microscopy, 20lf, 396, 397, 400f Landing beam, picture tube, 243-246 microscopy applications Laplacian filter, 34g, 369-371, 370f luminance level, 394 Large-scale integrated circuit, 34g, 197 sensitivity, noise, 230t Laser 40-mm, resolution vs. illuminance, 208f for aperture-scanning microscopy, 419 argon ion, with phase randomizer, 463 for holographic microscopy, 419 Jaggies, 33g, 322f, 360, 380f, 427 in V -to-F transfer, 445-446 Jitter, 34g, 62, 92, 293, 338, 425 Laser disk recording, 302-307; see also OMDR absence of in frozen field, 92 Laser disk video player, as confocal microscope, 418 Journals, relating to video microscopy Laser field-scanning microscopy, 417-418 frequently containing articles on, 457 Laser microbeam irradiation magazines and trade journals, 458-459 reference books on, 417 with video disk supplement, 303, 423 used in FRAP, 416 Joystick , 392 Lateral magnification, 34g, 97, 100; see also rotating 3-D display with, 422 Magnification; microscope used for outlining regions, 383, 384 Lateral resolution: see Axial resolution; entries under Resolution Karyotyping, 5, 386, 416, 458t, 512 Learning set, 386 Kell factor , 34g, 171-172, 231 Lens Kernel, convolution: see Convolution kernel/mask aberration-free, image formation by, 93-97 Keying , 34g achromatic used in montaging video images, 313, 440, 441 f doublet, 95f, 96f Keystone correction, 34g, 445 objective, 14g, 136 Kinescope , I, 4f, 34g catadioptric, 401, 444n in V-to-F transfer, 445-446, 445f cleaning of, 139-145, 14lf, 142f Kirsch operator, 3 72 computer-optimized, 107, 133 Knee , in light transfer characteristics curve enlarger, for video copying, 429 effect on SIN ratio, vs. illumination, 217f with high light-gathering power, 131t of image isocon, 214, 215f macro of image orthicon, 214 photographing video monitor with, 425f, 426n Koehler illumination, 34g, 102f, 103f video copying with, 429 methods for achieving, I 01-110 perfect, 93-97, 95f, 96f need for, 64-66, 101, 224, 492 diffraction pattern formed by, Ill, 113f, 116f reduction of flare by, 68, 10 I strain-free, 492, 493 INDEX 557 Lens (cont. ) Light scrambler: see Scrambler zoom: see Zoom lens, in dissecting microscope; Zoom Light source ocular argon ion laser as, 419, 463 see also Collector lens/mirror; lamp; Condenser; Objec• centration of, 105-107 tive lens; Objective lenses; Ocular; Oculars; Perfect color temperature of, 209 lens high luminous density, 127, 127n, 128t, 129t Lens aberration: see Aberration mercury arc lamp, 61-62, 463f, 484f, 495f Lens barrel xenon arc lamp, 61-62, 420f, 443f, 444 as source of flare, 107 luminous density of, 126--127 as source of hot spots, 67f, 68f with scrambler: see Scrambler Lens coating standard, for camera tube calibration, 205-206 hot spots in absence of, 67 see also Lamps improved transmission by, 132 Light transfer characteristics, 35g, 209 reduction of flare by, 132 of camera tube, 214-215 Lens mount and video photometry, 227 C-mount thread, 59f, 60, 429, 430f vs . target voltage, 209, 214f, 215f quality of machining in, 235 Light-valve projector, 35g, 242, 442-444, 443f as source of hot spots, 67f, 68f Light waves, 478-480 Lens paper, 140-144, 144f forming Airy disk, 111-118, 112f; see also Diffraction choice of, 140, 140n, 144 in Koehler illumination, 102f, 103f rice paper, 144, 144f through perfect lens, 94-95, 95f, 96f Lenticular screen, for stereo proJection, 450 plane and spherical, 94-95, 96f, 102, 102f, 103f, 104, Light 112f, 119f, 120, 120f absorption: see Absorption; Brightfield microscopy; polarized, 478-492; see also Polarized light Dichroism in Zemike's experiments, 119-122, 119f, 120f diffraction of: see Diffraction see also UV double refraction of: see Birefringence Limiting resolution, 35g, 175n emission of: see Electromagnetic wave; Luminescence of camera and intensifier camera tubes, 199t-200t fluorescence of: see Fluorescence and MTF, 177-178 interference of: see Interference colors; Interference of video imaging device, vs. eye, 203 microscopy of vidicon tubes, 194t phase retardation of: see Phase retardation of light of VTR, vs. camera and monitor, 177-178 polarized: see Polarization microscopy; Polarized light; see also Amplitude response curve; Horizontal resolution; entries under Polarizing Modulation transfer function; MTF reflection of: see Reflection Limit of resolution refraction of: see Refractive index of human eye, 77-80, 79f transmittance of: see Transmittance of light of light microscope, 101, 113-115, 113f, 114f, 116f, Light-absorbing particle, 119-120 122-125, 124f, 125f, 471 Light-adapted eye, 72-77, 76f; see also Human eye visualizing specimens below, cover, 9f, 254f, 306f, Light baffle/shield, 60, 60f, 136 322f, 400-401, 40lf, 402f, 404-405, 407f, 410, as source of hot spot, 67-68, 68f 41lf, 412, 418, 418n, 488-492, 499 vignetting from, 66 see also entries under Resolution; Super-resolution Light-gathering power Linear imaging device, 227, 333, 416 of condenser, 108f, 139 Linearity chart: see Ball chart of objective, 130, 1311, 136 Linearity control, monitor, 251, 259 Light microscopy Linear vs. loganthmic detector, 406f cutoff frequency for, 473 response to birefringence retardation, 404-405, 406f vs. electron microscopy, 4 77 Linear position sensors, 227, 416 photography vs. video in, 309-310 Linear response reference books on, vi, 93n, 121n, 122, 457, 500 of ceo. 198, 416 tomography in, 418-422 of photomultiplier, 415 , 415n see also Microscopy of vidicon, 209-214, 332, 397, 415 Light pen, 389t, 3911, 392 Linear systems analysis, 373-377, 374f, 375f, 472-475 Light rays Linear track VTR, 264, 264n, 294t, 295 displayed with uranium-glass blocks, 108f, 109n LINE-IN/OUT terminal, 35g, 433f, 434f, 436 through calcite crystal, 482f Line pair, vs. TV line resolution, 170, 424, 474; see also through microscope Amplitude response curve; Honzontal resolution imaging components, 97-100, 97f-99f Line scan, 35g Koehler illumination, 101-110, 102f, 103f, 109f of chromosome, 5 polarized UV microbeam, 484f of color video, 24lf polarizing, high-extinction, 495f of composite video, 158f, 167f producing flare, 67-68, 68f diagonal, 324f, 325f from Ramsden disk, 68, 69f of noise averaging, 183f stereo, 447-450, 447f-450f vertical, 315f, 316f, 318f through perfect lens, 94-97, 95f, 96f see also Horizontal scan see also Imaging Lint, 69, 145, 404, 492 Light scattering, 5, 120, 401, 417, 418 Lipids, birefringence of, 491 558 INDEX Liquid crystal display device, 35g, 242, 457 Livmg cell (video microscopy) (cont.) Living cell UV flymg-spot, 417 anaxial illumination of, 412, 412f UV microbeam, 417 birefringence of, 8f, 325f, 404, 407f, 491 - 492 , 494f, see also Living cell, polarization microscopy of 498f, 500 Logarithmic converter, for densitometry, 333 DIC microscopy of, 322f, 410 Logarithmic detector fluorescence of, 324f, 400f eye as , and photographic emulsion, 80n, 405, 406f flying-spot UV microscopy of, 5, 417 vs. linear detector, 406f FRAP of, 416 Logarithmic response, of human eye, 80n luminescence of, 396, 398f-399f, 400f Log, for video tape, 298 motion detection of, 356, 357f Logical operations between images: see Arithmetic logic phase contrast of, 337f, 339f, 352f, 355f, 359f, 369f, unit 370f, 385f Logical operations, in real time, 387, 512; see also polarization microscopy of Arithmetic logic unit sperm DNA, 412, 498f, 499, 499f Long-working distance spindle fibers , 494f condensers with, 139t reflected-light interference microscopy of, 410 objectives with, 134, 135t, 139t slit-scanning ophthalmoscopy of, 418 Longitudinal (axial) magnification, 35g, 97; see also tracking of, 384-386, 385t Magnification, microscope UV damage of, 5 Look-up table, 35g, 346-348, 387, 389t, 3911 video intensification microscopy of, 397-401 , 398f, for discrete ITF, 347f 399f, 400f as linearizing function, 364-365 see also Living cell (video microscopy) logarithmic, 348 Living cell (video microscopy) pseudocoloring with, 348, 348f, 387, 512 blood cell for real-time gray value manipulation, 387, 512 motion analysis in vivo, 320 sigmoid-shaped 354, 355f oxyhemoglobin content in vivo, 323 see also ITF cilia, Stentor , birefringence, 8f Loop through: see E-to-E cornea, endothelium, wide-field scanning, 418, 419f Low-distortion monitor, 251 , 426-427 dinoflagellate, luminescence, fluorescence, 396-397, Low-frequency response, video, 178, 246 400f causing shading in monitor, 246 egg, Chaetopterus, spindle birefringence scan, 323, 325f Low-light level egg, medaka, aequorin luminescence, 396, 398f, 399f automatic bandwidth suppression at, 233n egg, sea urchin flashes at, 38\ , 396, 400f anaxial illumination, 412f fluorescence video microscopy, reviews, 397-401, 397n fluorescence intensity scan, 324f noise integration of video image at, 402f; see also Image embryo, sea urchin, fluorescence, comet tailing, 220f integration; Noise averaging (spatial); Noise averag• eosinophil, phase-contrast, motion-tracking, 384-386, ing (temporal) 385f photon-limited, 40g, 196, 198, 218, 230t, 415 flagella video microscopy at, 394t, 396-403, 398f-399f, 400f, bacterial, birefringence, 405 , 407f 402f sperm, DIC, showing lag, 22lf Low-light-level microscopy, 393-403 fluorescence, quantitation photography vs. video in, 6f bleaching, 416 Lucifer yellow, in injected cell dynamic changes, 324f, 401 comet tailing from camera Jag, 220f FRAP, 416 diffusion in time course of, 324f hemocyte, sea urchin, contrast vs. auto-black, 65f Luminance levels, video microscopy, 394t organelle transport, DIC, 410 Luminance signal, 35g, 197n, 238-240, 24Jf parasite, malaria , DIC, invading blood cell, 410 in monochrome VTR recording, 270 retinal outer segments, preparation in IR, 403 Luminescence, 393-397, 394t, 396n sperm, acrosomal process, DIC, growth rate , 319-320, calcium-ion induced, 396, 398f-399f 322f in dinoflagellate, 396, 400f spermatocyte, grasshopper, birefringence, F-to-V, 271f sonoluminescence, 396n sperm, sea urchin, DIC, showing lag, 221f Luminosity, photopic vs. scotopic, 76f, 77n spindle, birefringence, 323, 325f Luminous density, 35g tissue culture of light source, 126-127, 128t-129t cell-substrate adhesion, reflected-light interference, 410 non-uniform, of arc lamp, 127n fluorescence, 397-399, 38lf Lux, 36g, 205 motion analysis, 320, 385f illuminance in natural scenes, 75f motion detection, 356, 357f myocardial cell, fluorescence, mitochondrial flash , 380f, 381-384 Mach band, 83, 83n pinocytosis, 397 Macro lens: see Lens, macro pseudocolor, fluorescence flash, xxvi(O, 381 , 382f Magazines PtK-2, phase-contrast, 337f, 339f, 355f, 359f, 369f, trade, listing digital image processors, 458t-459t 370f, 385f video microscopy-related, 457 INDEX 559 Magnetic disk recorder I recording Measurement (coni ) error rate, 302 of dtstortton (cont.) high capacity, 513 see also Distortton for motion analysis, 285 of fluorescence distribution, 323, 324f video image storage with, 301-302 of horizontal resolution, 172, 173, 203 , 206f, 256 , 256n , Magnetic field (VTR) 257f azimuth of, in Beta and VHS formats, 266-267, 266f internal optical calibration for, 323 induced, in pickup head of, 270-274, 272f, 273f microspectrophotometry, 40 I , 405 see also Magnetic recording; Magnetic tape muscle , minute movement in, 416 Magnetic focusing, 36g of optical, vs . electrical properties, 4 79 in intensifier tube, I%f of optical path difference, 323n, 409 in vidicon tube , I92, I 92f dry mass, 323n Magnetic permeability, vs. wave velocity, 479 of phase retardation Magnetic recording with interference microscopy, 409 audio vs . video, 263-264 in polarization microscopy, 488n, 489f frequency response for video, 270-274, 273f small amounts of, 407 in helical-scan VCR , 265-267 with video, 363-365 perpendicular, 39g of responsivity, units for , 205, 206 prices of, 331 of rms noise , 181n vertical field, 51g of stereoscopic acuity , 90f, 9 I wavelength of, 270, 270n, 272f see also Microdensitometer; Photometry , by video; see also VCR, recording with; VTR, recording Quantitation (video) Magnetic tape Mechanical tube length, 134, 134f; see also Tube length choice and care of, 300-301 Mercury arc lamp, high luminous density, 128t-129t, 495f print-through, vs. temperature, 301n inhomogeneity in, 127n Magnetic vector, of electromagnetic field , 479, 479f see also Lamps Magnets, adjustable, deflection, in yoke , 227 Message, in edited video tape, 433 Magnification, microscope, 36g, 99-I 00 Metal oxide semiconductor, 36g, I98, 202t axial, 35g, 97 , 100 M format , 36g, 274n , 275f, 276- 277 calibration, using stage micrometer, I 00 Micelles, and anisotropy, 488-491, 490f vs. camera tube sensitivity, 130-131, 472-475, 475f liquid crystals, 500 balanced, using zoom ocular, 136-138, 137f, 138f, Microbeam irradiation, 4 I6 , 4 I 7 146f, 147f polarized UV, 483, 484f, 448 MTF curves for, 475, 475f reference book on , 417 on camera tube target, 100, 474 Microchannel plate, 36g vs. image brightness, 130- 131 , 475 in Gen II intensifiers, I95 , I96f , 458t lateral, 34g, 97, I 00 for low-light-level microscopy, 394t, 400 by ocular or relay lens, 100, 474 vs. photomultiplier tube , I95 uneven H- and V-, in video image, 36If, 363 Microcrystalline domains of DNA, 498f, 499, 499f unit, definition of, 100 Microdensitometer Magnified copying with video, 407f, 43 I scanning recording, 415 Malaria parasite, 6f, 410 trace, of birefringence, 498f, 499 Malignant, vs. normal cell, image segmentation of, 378 see also Densitometry, using video Mapping function, 359, 360-363, 360f, 362 Microelectrode Mask record , montaged with video picture, 44If binary, 378-386, 380f, 381 n used for iontophoresis, 324f bit plane, 383 Micrometer scale convolution, 367-372, 367f-371f for calibration in video microscopy, I 00 as Fourier filter, 375-377, 377f on video tape record , 423 Laplacian, for edge sharpening, 369-371, 370f Microneedle, black glass, 413 smoothing, 368 Microscope variable, for image segmentation, 379 acoustic, 418n partial, for stereo microscopy, 449-452, 449f-453f aperture planes in , 15g, 101-104 shadow , in color picture tubes , 260, 26 If masking to induce stereoscopy, 449-452, 449f, 450f, MATCH, histogram-converting program, 351-352, 351f 452f, 453f Measurement aperture-scanning, 419-420 of area, 339-340 adjusting for Koehler illumination, 104-110 of birefringence distribution, 323, 325f, 404 cleaning, of optics, 139-145 of camera tube sensitivity, 206 cleanliness of, and video recording, 299 of CTF and MTF, 177, 177f, 203, 206f, 256, 256n, compound, stereo pair production with, 446-455, 448f- 257f 455f of distances on monitor picture, 251 , 319-320 conditioning device in optical train, 93, 94f, I22 of distortion contrast generation, modes of, 122 Ball chart for, 255f, 256, 258f and MTF, 123 , 125f and geometrical decalibration, 358, 360, 360f coupling video to, 56f, 57f, 59f, 60- 62 , 60f, 6If, 64- test signal generator for, 256 70; see also Microscope, inverted, with zoom ocular 560 INDEX Microscope (cont.) Microscopy (cont.) depth of field: see Depth of field , microscopy objective lenses diffraction pattern, 111-115, 113f, 114f with high light-gathering power, 13lt 3-D, 115-118, 116f, 117f with long working distance, 135t see also Airy disk reference books on, 93, 134 dissecting, 413 , 446, 447f video cameras for, luminance levels of, 395t eyepoint of, 102-103, 102f, 109 sensitivity class and noise character of, 230t occluding, for stereo, 449, 453f see also Anaxial illumination; Aperture-scanning micros• see also Ramsden disk copy; Brightfield microscopy; Confocal microscope; field planes in , 26g, 101-104 3-D display; 4-D display; Darkfield microscopy; field-scanning, 418, 419f DIC; Field-scanning; Fluorescence; Holographic mi• fine-focusing control of, 118 croscopy; Interference microscopy; IR microscopy; sources of flare in , 107-108; see also Flare Light; Low-light level; Microspectrophotometry; illumination of: see Koehler illumination Modulation contrast microscopy; Phase-contrast mi• illuminator: see Illuminator, microscope; Light source croscopy; Polarization microscopy; Reflected-light image, formation on retina, 71-72, 79, 97-99, 99f interference microscopy; Single-sideband edge-en• image formation in, 93-I 04 hancement microscopy; Stereo microscopy, high• Zernike's wave theory of, 119-122 resolution; UV microscopy; Video microscopy imaging system, model using Fourier transform, 372- Microspectrophotometry, 40 I 375, 374f, 375f, 470-471 polarizing, of rods and cones, 415 inverted, 134, 138f, 145-148, 146f, 495f, 496f UV,401 long-working-distance objectives for , 134, 135t Microspheres, fluorescent, xxv(f), !Of optical bench, 138 Microtubules stability of, for video microscopy, 145 alignment and assembly of, 404 with zoom ocular, 137f, 138, 146f, 496f flexural rigidity of single, 401 with zoom rifle scope, 138f fluorescence image of, 402f, 41lf, 447 lens aberrations, 133-136, 133n; see also Aberration form birefringence of, 492 references for, effects on diffraction pattern, 114n individual visualized, with video, 9f, 402f, 410, 41lf reference for, effects on image, 133n sliding doublets, 401 light transmission through, 132-133 stereo-pair photographs of, 447 matching resolution to video recording, 283-284, 475f; Migration of cells, tracking of, 407, 417 see also Magnification, microscopes, vs. camera Military video format, 161, 164f tube sensitivity Mirror MTF in, 122-125, 124f, 125f, 467-471 elliptical, parabolic, 129 Ramsden disk, vs. projection distance, 68, 69f first-surface , cleaning of,l45 relay optics, 474, 474f; see also Microscope, coupling oscillating, 418, 419f video to vibrating, 421-422, 42lf, 422f resolution of, 111-117 see also Catadioptric standard dimensions in, 134, 134f Mistracking, of video tape, 270, 27If, 288 tube length for: see Microscope tube Mitochondria, flashing fluorescence in, 380f, 381 , 38lf see also Bertrand lens; Condenser; Electron microscope; Mitosis, 500; see also Spindle Microscopy; Objective lens; Objective lenses; Mixer: see Video mixer Ocular; Oculars; Video microscopy Modulation, 36g, 177, 177f Microscope resolution: see Resolution (microscope) of amplitude response, 206f Microscope slide of single sideband, in microscopy, 412 keeping clean, 69 see also Amplitude modulation; FM; Modulation transfer cleaning, 141 - 143 function; MTF effect on condenser performance, 139 Modulation contrast microscopy, 122, 412 optical noise from , 68 video camera suitable for, 230t strain-free, for polarized-light microscopy, 492 Modulation transfer function, 36g, 123-125; see also MTF Microscope stage analysis of, in video microscopy, 467-475 used in locating optical noise, 68 compensation of, for non-ideal optics, 375 motorized, 384 curves Microscope tube , 68 deriving, 177, 177f tube length: see Tube length for microscope lens, 124f video coupling: see Light baffle/shield for various kinds of microscopy, 125f Microscope-video camera system; analysis of MTF, 472- of human visual system, 82, 83f 475, 474f vs. point-spread function, 338 vs. magnification of relay optics, 475f vs. resolution, in microscope, 118 see also Magnification, microscope, vs. camera tube Moire pattern, 171, 431 sensitivity Molecular organization, 404, 488-492, 500 Microscopy Molecular structure and physical properties, 500 automated, using computers, 5 polarizability, 489f, 490f, 491 coherence of imaging waves, 113 Molecules of contrast modes, 122, 230t, 394t actin, 322f, 323f, 400, 401 critical test specimen for, 405, 409f beta-form proteins, 491 INDEX 561 Molecules of (cont.) Mom tor (cont.) DNA, 400, 404, 491 stgnal-processing ctrcuits m, 244f, 246 flagellin, 401 synchronizing to video stgnal, 157, see also AHFC PYA, 488-491 , 489f termination of, 58, 58f, 63- 64 , 63f tubulin: see Microtubules underscanning, 167, 258 , 426 Monitor, 36g, 242-262 use with time-lapse VTR , 259, 291 AHFC of, 14g, 187, 259, 291 V-HOLD control, 58 , 61 , 255-256, 256n , 259 aperture correction in, 246, 246n, 258n see also Color monitor; Monitor picture; TV set astigmatism and focus controls, 259 Monitor, color: see Color monitor brightness control, 56f, 246, 247f Monitor picture home settings for, 58-59, 433, 433f, 434f blooming, 18g, 251 set too high, 251; see also Streaking break-up of, 29lf bringing out faint picture details in , 246, 247f flagging of, 26g, 167, 187, 188f, 255 , 259, 291-293, color: see Color monitor; RGB monitor 292f compatibility with signal, 149, 164, 184-189 fringes in, 254 connections and switches of, 58f hash in, 67f contrast control, 56f, 246, 247f, 251 from OMDR, 306f, 307f, 414f, 424n home setting for, 59; see Monitor, brightness control rolling of, 44g, 58, 164 adjustment for photography, 426 of scratched tape, 297f controls of, 62; see Monitor, brightness control; Monitor, shimmering of, 291, 29lf contrast control; Monitor, H-HOLD control; Monitor, streaking, 48g, 234, 251 , 254f, 435f linearity controls on , Monitor, v-HOLD control from VTR playback: see VTR , in playback distortion in, 248-251, 252f, 253f, 255-256, 255f see also Color monitor; Monitor by curvature of faceplate, 259 Monochrome camera dynamic focusing in, 246, 258n vs. color dynamic range, vs . photographic emulsion, 432 for polarized-light microscopy, 405 faceplate, reflections from , 425f, 426, 431 for video microscopy, 229 features of, 256-260 as part of color camera, 236 flagging, 26g, 292f; see also Monitor picture pseudocolor display with , 237 vs. AHFC, 187, 259 sources of instrumentation cameras, 236t in higher-priced, 259 Monochrome monitor: see Monitor frequency response in, 246 Monochrome signal, 36g gamma of, 28g, 246, 247f standards for, 159, 159f, 161 H-HOLD COntrol, 58, 61, 255, 259 Monochrome VCRs high resolution high-resolution, 284, 284t, 424 aperture correction in, 246, 246n recording, 270-274 shading correction in, 258n advantage over color, 283 , 424 high scan rate , 165, 256-258 Monochrome video, 149 horizontal resolution of, vs. VTR, 424 Monochrome video projector, 444, 444f H oscillator in , syncing of, 161 Montaging image quality, and noise in, 182, 184f an inserted image, 429, 431, 438-441, 44lf linearity controls on, 251, 259 data with microscope image, 440, 44lf low-distortion by keying , 313 flat-faced, 426 Morphometry, 416; see also Feature extraction high-quality, 251 MOS pickup device, 36g, 198 used in V-to-F transfer, 445, 445f camera specification, 202t vs . low-frequency response, 246 Motion analysis monochrome, vs. color, 185 magnetic disk recorder for, 285 use of multiple, 442 copied from tape, 4 24 NTSC, 149, 166, 237f, 260, 260n with OMDR, 304 vs. MTF of video train , 246 Motion analyzer, analog , 302, 319-323, 424 optically copying, with video, 430-431 Motion picture projector, use in F-to-V transfer, 430 optimum viewing distance for, 442 Motion picture, vs. video, flicker in, 84-86, 154; see also overdriving of, 251 Flicker paired, for stereoscopy, 451-452, 455f Motion tracking, digital, 320, 356, 384-386, 385f; see also phosphors in , 248, 249t-250t Tracking photographing of, 259-260, 424-429, 425f Mottle subtraction, 9f, 70, 410; see also Background picture size, 243-244, 245f subtraction power supply for, 259 Moving projection screen , for 3-D imaging, 420-421, 420f pulse-cross display with: see Pulse-cross display MTF, 37g, 124-125, 467-475 vs . receiver-monitor, 43g, 242 of human eye, 123, 125 reference books on, 242n of human visual system, 82-83, 82f, 83f resolution of, 244-246, 255-258, 257f and colored gratings, 82n, 83f scan rate of, and compatibility with signal, 185, 258 of imaging systems, 467 screen sizes of, 245f in microscope; see also MTF curve shading in, 227n, 251 vs. contrast, 118-119 562 INDEX MTF (com.) NA, 37g, 109f, 114 in mtcroscope (cont.) of condenser, wor~ing, 109f, 110, 115 and diffraction pattern, 469-472 vs. brightfield image of chromatin, 414f directional effect in DIC, 123 vs. depth of field, 118, 415 vs. Fourier transform, 470- 471 for DIC microscopy with rectifier, 410 modificallon by aperture scanning, 420 effect on flare, 110 vs. point-spread function, 470, 47lf effect on image resolution, 115, 410 vs. resolution, 470-471, 470f, 47lf vs. iris diaphragm, 108f, 109f, 110 and spatial frequency, 122-125 in Koehler illumination, 101 vs. optical and contrast transfer functions, 38g, 123, vs. lateral resolution and depth of field, 415 123n, 468 vs. MTF of microscope, 124f vs. phase transformation, 467-469 vs. oil immersion, 127 of photographic film, 123, 125 vs. optical sectioning, 118 reference book on, 125, 170 in video microscopy, 136, 360f reference books on, 123, 125, 170, 176, 470 for visual observation, II0 of system, 177 and image resolution, 114 of cascading series of devices, 125, 176-178 vs. MTF of mtcroscope, 4 70 vs. frequency response curve, 177, 177 n of objective and condenser microscope-video, 474-475, 474f vs. extinction factor, 492 optimizing, vs. noise, 123 vs. resolving power and contrast, 124f vs. percent modulation, 177f of objective lens, 114 reference books on, 176 vs. image brightness, for epifluorescence, 128 of video train, 231 with long working distance, 139t vs. temporal and spatial frequency, 124 objective lens with high, 130, 13lt in video effect of tube length and cc1Verslip thickness, 134 vs. camera resolution, 473 vs. light-gathering power of, 130, 13lt effect of VTR on, 475 optical sectioning in fluorescence with, 418 horizontal and vertical, 468 setting accuracies with, 118 of monitor, vs. v ideo train, 246 simultaneous stereo pairs through, 446-456 standard signals for measuring, 256 for video microscopy, 136, 306f of vidicons, 205f, 473, 473f vs. resolution, 1 13-115, 114f see also Contrast transfer function; Modulation transfer fluorescence microscopy, 397n function; MTF curve vs. stray light, polarization microscopy, 493 MTF curve Nerve/neurons for brightfield microscope, 124f, 12Sf action potentials, optical recording of, 4 16 contrast at 400 TV lines, 194t, 473; see also Amplitude particle movement in, 410 response Schwann cell, form birefringence of, 491 for DIC, 123, 125f stereoscopy of Golgi-stained, 449n Gaussian approximation, 473 Neutral-density filter, 3 7g of human eye, 81-83, 82f, 83f for calibration of digitized image, 363 microscOPil magnification vs. video, 474-475, 474f, for reducing illumination, 126 475f of color camera, 241 of microscopes, 122-125 Newvicon, 37g, 55, 145n, 234 vs. NA, 123, 124f amplitude response curve for, 205f for phase-contrast microscope, 123, 125f analog enhancement with, 405, 40Sn, 410 of simple imaging device, 82 DIC image, 6Sf, 22lf, 322f for SSEE, 123, 1 25f polarization image, 7f, Sf, 32Sf, 407f variation with DIC prism orientation, 123 calibration curves for, 363 of video camera, 473 digitally enhanced see also Amplitude response curve; Modulation transfer brightfield image, 414f function, MTF DIC image, 306f Multiburst test pattern, 256, 257f phase image, 337f, 339f, 352f, 355f, 359f, 369f, Multiplexing, 37g 370f, 38Sf of ALU output, 391 polarization image, cover, 306f monitor, in stereoscopic video, 455n and F-to-V transfer, 430 Multispectral analysis FN, 396-397 pseudocolor display in, 348 IR-blocking filter for, 206n of specimen fluorescence, 342 lag, in DIC image of sperm, 219, 221f Muscle light transfer characteristics of, 214f 3-D image reconstruction, 418 microscopy applications, typical, 230t, 394t-395t focus-dependent contrast in, 413n MTF of, 20Sf, 473, 473f ocular, 80 noise character of, 230t of pupil, 72-74, 72f response curve, effect of shading on, 332f section, as critical test specimen, 184f, 409f and scan line removal, 464f, 465f, 466 special optics and ceo images of, 416 scannong beam hold-off, 319 Myocardial cells, fluorescently stained m1tochondna in, sensitivity 380f, 381' 38lf classified, 230t INDEX 563 Newvicon (cont.) NOise bar (cont. ) sensitivity (cont.) on playback of VTR , 29g, 270, 27lf, 277 compared with CCD, 203 sources of, in time-lapse VTR, 270. 27lf, 2SS in far -red, 206n from tracking error in VTR, 2S6n specifications of, 194t vs. VTR adJustments, 296 spectral sensitivity of, 210f, 2llf, 415 Noise characteristics stereoscopy with, 452 of camera tubes, 217-219, 230! target material of, 194t of intensifier camera tubes, 230t, 397 target voltage of, 209 of low-light-level video image, !Of, IS4f, 336f, 401 , see also Camera tube specifications; Microscopy 402f, 409f Nicol prism, 4S3; see also Polarizer; Prism, calcite of monitor picture, IS2, IS4f Nipkow disk, 2-3, 5, 37g see also Dark current in confocal microscope, 416-417 Noiseless Nitrocellulose, birefringence of, 49In frame-by-frame advance Noise, 37g, lSI with OMDR, 304 camera: see Noise (video camera) with VCR, 2S2t, 437n caused by digital sharpening, 371 still-frame display vs . dynamic range, 214 from OMDR, 303, 306f, 307f electrical: see Electrical noise from VTR, 27S, 279t, 2S2t guard-band: see Noise bar Noise level harmonic: see Harmonic noise, removal of and bandwidth, IS0--1S4 optical: see Optical noise effect on choice of optimizing MTF, 123 rms, lSI, ISin, IS2t Noisy blank VTR frames, 29S sacrificing spatial or temporal information to reduce, Nomarski-type DIC, 410, 412; see also D!C microscope IS2, 233 Non-interlace, 37g; see also Interlace, I: I from VTRs, lSI, 274 Nonlinear sweep, vs. distortion, 227, 24S, 252f see also Glitch; Hash; Noise characteristics; Noise (video Non-uniform illumination camera); Snow; SiN ratio effect on diffraction pattern, 114n, 115 , llS Noise (video camera), 9f, IOf, 37g, 402f, 409f effect on intensity profile measured, 356, 35Sf from AC line interference, 65f, 67f, 157, lSI, 233 , 235 , see also Shading 254 Notch filter, 3Sg, 260 bandwidth, vs. resolution, 233 NTSC signal, 3Sg, 149-150 vs. bandwidth, temperature, gain, 315-319 bandwidth of, 260 character of, 230!, 396--397 camera for, 237f, 240-242 in chilled CCDs, 197-19S color bars, 240, 241 f in intensifier cameras, !Of, lSI, 196, 215, 2IS, 317- color bursts, 161 , 163f 319, 31Sf, 396--397, 402f, 409f color monitor, 237 from preamplifier, lSI, 195, 215, 217 also accepting RGB, or PAL, SECAM, 260n photon statistics as source of, 70, 1S2, 1%, 21S, 415 compatibility with monochrome video, 149-150, IS4- radio frequency, 67f IS5 vs. responsivity, 217 encoding system, applied to varifocal 3-D, 42lf in vidicon, vs. intensifier camera, 195-1% field rate for, 161 white, in intensifier camera tubes, IOf, lSI , IS3f, IS4f, exact, 240n 217-219 high-frequency details in, 260 see also Dark current format, 161, 163f, 237f Noise averaging (spatial) encoded signal, 23S-242, 24lf, 260 analog, 314-319, 31Sf IRE scale, 163f by automatic bandwidth suppression, 233 limitation of bandwidth, 260 by bandwidth compression, DIC image of, 317, 31Sf nations adopting, 164, 166t by convergence of rod cells in retina, SOn picture cycle for , 161 with convolution filter, 31Sf, 36S-369, 369f vs. RGB , 237-240, 237f, 242, 260 by summation, in SIT, 220 in Super Slo-Mo systems, 295 see also Noise averaging (temporal) and VTRs, 242, 277 , 279t, 2Sit, 2S2t, 290t Noise averaging (temporal), IS2-IS4, IS3f, IS4f, 335, Nucleosome, 3-D image of, 422 336f Nucleus by beam hold-off anaxial illumination, in sea urchin egg, 412, 412f and chilling SIT, 31S-319 brightfield, in human testis, 414f, 415 in Newvicon, 319 fluorescence digital, 9f, IOf, IS4f, 306f, 409f, 511 Drosophila polytene, 375 , 376f in human vision, SO, Sl , Sin Trypanosoma cruzi, 6, 6f by target integration, 193 image segmentation, from cytoplasm, 3S6 see also Frame summation; Image averaging; Image Numerical aperture: see NA integration; Noise averaging (spatial) Nyquist frequency , 3Sg, 337; see also Aliasing Noise bar, 37g effect of monitor and VTR controls on, 291 Objective lens influence of skew control on, 296 aberrations of: see Aberration from paused VCR picture, elimination of, 424 centering of, I 06n 564 INDEX Objective lens (cont.) Oculars (cont. ) cleaning oil off, 140, 14lf, 142f zoom correction collar for, 22g, 1311, 134, 135t balancing magnification and brightness with, 136 depth of field of, 23g, 118; see also Axial resolution; powered, 136-138, 137f, 145n, 147f, 496f Depth of field, microscopy rifle scope as, 138, 138f, 145n depth of focus of, 23g, 68, 69f uncoated, hot spots from, 67 diffraction pattern formed by, II 1-118, I lin, 112f, for video, 138, 146f 113f, 114n, 115n see also Ocular in 3-D, 115-118, ll6f, 117f; see also Diffraction OEM, 39g, 389, 512 pattern Offset, DC: see Pedestal control effect on plane wave from condenser, 102, 102f 75-ohm cable, 45g, 56, 57f flare in, 133 phase delay in, 185 image planes of, 98f, 104 75-0HMiHI-Z control, 63, 63f light-gathering power of, 130, 13 It 75-ohm termination, 45g, 56, 63 , 63f; see also Termination for epifluorescence, 128 Oil immersion: see Immersion medium magnification of, 100 OMDR, 38g MTF of, 122-125, 123n, 124f, 125f with audio capability, 303n, 304t numerical aperture of, 109f, I 14; see also NA input to digital image processor, 304 ocular to correct aberration in , I00 noiseless, single-frame display from , 289, 304 rear focal plane of: see Rear focal plane of objective lens pictures played back from, cover, 306f, 307f, 402f, reflection loss in, 132 409f, 414f resolving power of, 113-115, 113n, 113f, 114f price of, 303, 304, 304t sine condition in, 95n, 133 recording and playback functions, 303-304, 304t as source of optical noise, 69, 139-141 playback at video rate, 303n tube length and coverslip effects, I 34 schematic of, 305f see also Objective lenses single-frame recording, 303, 424n Objective lenses used with time-base corrector, 304 apochromatic, 15g as time-lapse recorder, 289, 304 for fluorescence microscopy, 13 It, 135t One-line delay circuit: see Circuits fluorite , 27g; see also Fluorite objective lens Ophthalmoscope, field-scanning, 418 , 419f with high light-gathering power, 13It Optical anisotropy: see Anisotropy, optical with long-working distance, 134, 135t Optical axes, 480-492, 480f-490f as long-working-distance condensers, 139t goniometer for viewing, 448f parafocal, for UV and visible wavelengths, 401 reference books (crystal optics), 404, 500 plan achromatic, 136 Optical bench microscope, inverted, 145-148, 495f, plan apochromatic: see Plan Apo objective 496f quartz, 401 with dual video camera, 147f quartz-fluorite, 40 I Optical calibration, internal, 323 rectified, 484f; see also Rectified optics Optical copying, 38g; see also Copying video optically reflecting, 401 Optical density Ultrafluar, 401 as function of gray value, 363-365 for video microscopy, 13 It, I 36 transformation to linear function of, 365f, 366f see also Microscopy; Objective lens integrated, in densitometry, 341 Oblique illumination: see Illumination, oblique Optical digitizer, 330f Ocular ideal, 332-333 adjustment of image distance with, 135 Optical disk: see OMDR arrangement in video microscopy, 60, 60f, 61f; see also Optical noise, 38g Oculars, zoom in high-extinction microscopy, 69, 193 balancing magnification and image brightness with, 130- location of source of, 139 131, 136-138, 472-475, 475f on ocular, 69 cleaning of, 139-141, 143-145, 144f sources of, 66-69, 139 field stop in, 99f, 103f, 104, 108 in immersion oil, 69 function of, 97-100, 134-135, 142f on slide and coverslip, 68-69, I 41 vs . intermediate image plane, 97-100, 98f, 99f, 104 subtraction, in digital image processing, 9f, !Of, 70, magnification by, I 00 410, 511; see also Background subtraction objectives designated with, 100, lOOn, 134--135 in video microscopy, 66, 70 source of optical noise on, 69 see also Blemishes; Hot spot; Shading; Shading projection distance with, 68, 69f, 100 distortion in projection mode, 98f, I00 Optical path difference: see Phase retardation of light viewing through, 97-99, 99f Optical rotation, 488 , 500 see also Eyepoint; Oculars; Ramsden disk Optical scrambler, 38g; see also Scrambler Ocular muscles, 80 Optical sectioning Oculars vs. axial resolution, I18 amplifying, 135 with confocal microscope, 5, 418 flat-field, 135 and Fourier filtering, 375, 376f projection, 100, 135 Koehler illumination and, I 0 I INDEX 565 Optical sectioning (cont. ) Out-of-focus image (cont.) video-enhanced of muscle A-band, 413n by anaxial illumination, 412, 412f of Siemens Test Stars, 115, 116f, 117f in brightfield microscopy, 414f subtraction, to sharpen image, 371 to determine cell outline, 410 visibility of, 121 in DIC microscopy, 410 and wide-field specular microscope, 418 in fluorescence microscopy, 375, 376f, 418 see also Circle of confusion; Depth of field, microscopy to measure areas, volume changes, 418-419 Overlay see also Depth of field, microscopy; Optical sections analog: see Keying Optical sections, 38g digital of buccal epithelial cell, 412f for area-selective manipulation, 383-384, 387 of chromatin, 376f, 414f to image cell fluorescence, 381f, 382f of cornea, 419f in image processing, 391 of diatom, cover, 306f, 412f masking, at video rate, 387 of sea urchin egg, 412f Overscan, 39g, 167, 173f, 258 stereo pairs generated from serial through-focus, 455n Overshoot, 174, 175f; see also Ringing see also Optical sectioning Optical surfaces, cleaning of, 139-145, 141f, 142f, 144f Pairing, of scan lines, 39g, 172, 172f Optical train of light microscope, 93, 94f, 102f, 103f PAL, 39g, 238 Optical transfer function, 38g, 123 525160, 626150, 164 vs. MTF and phase response, 467-470 monitors accepting, 260n Optical tube length, I 00 nations adopting, 166t Optics video recorder accepting, 279t, 28 It cleaning method for, 140-145 Pan, 39g cleanliness of, 139-140, 299, 492; see also Optical noise and scroll digital image, 389t-390t, 391 physical, reference books on, 116n, 119, 404, 500 Parallax Optimum binocular: see Binocular parallax magnification: see Video microscopy, optimum magni• error, in measuring distance on monitor, 319 fication for reduction of, 426 viewing distance for video, 442 Particle counting, 334 0-rays, in calcite, 481, 482f Pattern recognition Organization of text, v-vii, II classification of cells by, 386 Orthoscopic image, 39g, liOn, 493f history of, 5 vs. conoscopic image, liOn involving Fourier transform, 386 of crystals and fibers, 405 using computers, 416 use of color video cameras for, 405 PAUSE button/function, 39g, 277 see also Polarization microscopy assemble editing using, 286, 436-437, 438f Oscillating mirror, in slit-scanning microscope, 418, 419f; automatic exiting from, 297, 437 see also Vibrating mirror freezing a video field with, 425 Oscillator, master for smooth recording transition, 298, 437 as separate sync source, 186 on VCR, 297 providing H- and V-scan frequencies, 156n PAUSE capability, in record mode, 433, 433f in video camera, 231 Paused picture, eliminating noise bar from, 424 Oscilloscope Pause indicator light, 298 to check signal voltage for VTR recording, 433, 434f, PbO vidicon: see Plumbicon 436-437. 440f Peaking circuit, 176 use of, reference books on, 256n Peak-to-peak, 39g see also Cathode ray oscilloscope voltage, of composite video signal, 158f, 159-160, 159f, Oscilloscope trace 185, 186, 186f of chromosome bands, 5 Pedestal, 39g; see also Pedestal control; Pedestal level montaging with video image, 440, 440f, 441f Pedestal control multiburst pattern, 257 to adjust DC offset, 345 of NTSC color signal, 241f in anaxial illumination, 412 Out-of-focus image, 115-118 automatic, 16g background subtraction to eliminate noise in, 9f in fluorescence microscopy, 5 causing blemishes, in video microscopy, 299 to reduce background luminance, 405 as 3-D diffraction pattern, 115-118, 116f role in raising picture contrast, 232 and 3-D image reconstruction, 418 of video signal, 311-312 Fourier filtering, fluorescence of, 375, 376f Pedestal level intruding, at low condenser NA, 414f of composite video signal, 159f, 160 minimal influence of optimized, during tape-to-tape copying, 433, 436 in anaxial illumination, 412f in polarization microscopy, 406f in brightfield microscopy, 414f Perfect lens, 93-97, 95f, 96f in confocal microscopy, 5, 418 diffraction pattern formed by, Ill, 113f in DIC microscopy, 410 Periodic structures see also Optical sections diffraction pattern of, 115-118, 117f 566 INDEX Penodic structures (cont.) Photocathode, 40g enhancement of by Ronchi grating, 428n and camera-tube performance, 194t, 203 Perpendicular magnetic recording: see Vertical-field mag• comparison of sensitivities, 213f netic recording of image intensifier tubes, 195-1%, 195f, 196f Persistence, 193; see also Lag of image isocon, 197 , 198f Phase large diameter, 199t of chrominance signal, vs. hue , 240, 24lf microscope image focused on, 100 of light waves , 94-95, %f responsivity, of SIT, 2II f of spatial frequencies, vs. image, 469 of SIT and SEC tubes , 1%, 197f of VTR scanner rotation, control of, 269 Photoconductive layer, 192 Phase-contrast image Photoconductivity of selenium, 2 edge mapping of, 379 Photodiode of eosinophil, 385f in laser disk player, 303 of PtK-2 cell, 337f, 370f silicon, in ceo, 198 subtraction, of slightly offset, 358, 359f Photodiode array, 40g, 194t, 198, 236 Phase-contrast microscopy, 5, 39g, II9-122 as image-scanning device, 227, 333, 416 annulus, 122, 139t physiological measurements with, 416 cell-tracking with video, 406-408 Photoelectric cell, behind Nipkow disk, 3, 416 condenser immersion in, 127 Photoelectrons, 3 diffraction pattern in, 115n in image intensifier, 193 effect of non-uniform background on, 356-358, 358f statistics, limiting intensifier resolution, 208 gray level histogram Photographic emulsion expansion of, 352, 352f, 354 vs. chilled ceo' limit of sensitivity' 198 typical in , 339, 339f, 354f dynamic range of, 125 as high-spatial-frequency pass filter, 408 vs. human visual system, response of, 92 vs. interference microscopy, 409 magnification of microscope image on , I00 MTF curve for, 123, 125f MTF of, 125 Polanret, variable, 407 UV sensitivity of, vs. photocell, 417 reference books on, II9, 12ln, 122 vs. video video cameras appropriate for, 2301, 395t characteristic curve of, 334 video enhancement in, 406 exposure time, 126 see also Microscopy wavelength response of, 229 Phase delay, in coaxial cable, 185n Photographic integration, 425 Phase difference, slow and fast waves, 485-487, 487f Photographing Phase errors the monitor, 423-429, 425f, 427f, 463 and aberrations, 469 macro lens for, 425f, 426n and electrical response, 471 use of Ronchi grating for, 427f, 428f Phase halo, 408 pictures for publication, 426 Phase-handling quality of amplifier, 231 Photography Phase-locked loop, 39g, 269 adjustment of field diaphragm for, 107 amplifier, 186 CRT phosphor for , 248 Phase-locking, to AC line frequency, 156 ghosts in, 430 reducing camera noise by , 157 image-averaging by, 70, 182, 319 Phase objects, visibility of, 121 adjustment of monitor contrast for, 426 in- and out-of-focus, 115n, 413n reference books on, 170, 425n Phase plate, 40g, 121 vs. video Phase relationship for fluorescence microscopy, 400 among different frequency components, 176 in light microscopy, 309-310 between H- and V-sync, 187 in low-light-level microscopy, 6f Phase retardation of light for polarization and ore microscopy' 405, 405n, 406f diffracted by absorbing object, ll9-120 video monitors for, 259 diffracted by transparent specimen, 121 Photometric unit, 40g, 77 measurement of specifying video imaging devices, 205 with interference microscopy, 409 Photometry by internal optical calibration, 323 of microscopic areas, reference book on, 415n small amounts of, 407 by video, 323, 324f, 325f, 381 with video, 363-365 vs. camera controls, 232 Phase telescope with ceo camera, 235, 236, 416 inspecting objective rear focal plane with, 110 shading compensation for, 227 used for centering condenser, I06 effect of shading distortion on , 363-366, 366f visualizing Fourier plane with, 373 and S/N requirements, 182-184 Phase-transfer function, 468-469 sulfide vidicon not suited for, 397 Phosphor screens unity gamma required for, 209 characteristics of, 248, 249t-250t see also Microspectrophotometry in image intensifier tubes, 195, 195f Photomultiplier noise reduction in , 220 dynamic range of, 415, 427 of picture tubes, 242-246, 243f in intensifier tubes, 195 INDEX 567 Photomultiplier (cont. ) P1xel (cont.) vs. microchannel plate, I 95 histogram of brightness, 512 in microphotometers, 4 I 5 locations, vs. mapping functions, 360 reference books on, 415n number of, as area of gray value histogram, 340 response of, 415n overlapping of, 245f used in slow-scan display, 427n size and resolution of, 335-33S, 471-472 vs. video camera, as video digitizer, 332-334 vs. spatial frequency , 36S Photon damage to specimen, 5, 416, 417n white, as edge elements, 3S5 reduced see also Picture element with flying spot, 417 , 417n Plan achromatic objective, 136 in video, 397, 401-403, 415 Plan Apo objective, 7f, 41g Photon statistics aberrations corrected in, 133, 133n integration in chilled CCD camera, 235; see also Target high light-gathering power of, Sf, !Of, 13lt, 136 integration use in video microscopy, 136 as limiting noise, in intensifier tubes, 40g, 196, 20S , with anaxial illumination, 412f 21S, 415 with brightfield microscopy, 414f photon-counting camera, 197, 230t with fluorescence microscopy, IOf, 412f Photopic vision, 40g, 74 with rectified DIC optics, 145n, 306f, 40Sf vs. scotopic luminosity, 76f, 77n, 205 with rectified polarization microscopy, 7f, Sf, 136, and scotopic vision, transition of 306f, 405, 409f CFF vs. illumination, 83, 85f Plane-polarized light, 4S2-4S3, 4S7; see also Polarized intensity discrimination, 81 , 81 f light threshold modulation contrast, 80, SOf Planes, conjugate: see Conjugate planes, points sensitivity, and wavelength response, 74-77, 77n Plane wave, 95, 96f, 101 , 102f, 119f, 120f Photoreceptors Platelet, form birefringence of, 491-492, 49ln of frog , excised, 403, 415 Playback of human eye, 78f, 79f OMDR: see OMDR Pickup device, 40g VCR/VTR: see VCR, playback; VTR, in playback early proposal for, 3 Playback head, VTR, 41 g photoelectric cell and Nipkow disk as , 3 Playback signal, VTR, and time-base corrector, ISSf reference books on, 4, 197f, 332 Pleochroism, 404 solid-state, specifications, 202t PLL: see Phase-locked loop see also Camera tube Plumbicon (PbO vidicon), 41 g Pickup head: see VTR build-up lag, vs . bias illumination, 223f Picture (video), 3, 40g, 55, 149 decay lag, vs. bias illumination, 220, 222f breaking-up of, 29lf SIN ratio, vs. faceplate illuminance, 217f flagging in, 26g, 167, lSSf, 291-293, 292f specifications for , 194t, 199t interactive pointing device for, 392 spectral response, 213f quality of see also Camera tube specifications; Microscopy vs. camera resolution needed, 23 I PLZT, 4lg recoverable from VTR, 2S3-2S5 shutter goggles for stereo, 451-454, 45ln, 454f recovered from OMDR, 303, 306f, 307f P-n junction diode, 41g; see also CCD; Silicon vidicon; Picture area, l-inch vidicon tubes, 194t SIT Picture blemishes: see Blemishes Pointing device, 41g, 392; see also Digital image pro• Picture diagonal, monitors, 243 , 24Sf, 442 cessors (equipment), interactive dev1ces for Picture element, 40g Point operation, 4lg, 342-356 size of, in vidicon tube, 169, 192, 330; see also Pixel vs. digital filtering , 367 Picture signal, 40g, 154f, 156, 15Sf Points, conjugate: see ConJugate planes, points stripping sync pulses from, IS5 Point-spread function, 41g, 33S termination and voltage of, IS5, IS6f vs. MTF, 33S , 470, 47lf proper voltage, for VTR recording, 24S Polanret variable-phase-contrast system, 407 voltage level for , 15Sf, 159f, 160 Polarizability, molecular, 490f, 491 , 49ln; see also Picture tube, 40g Dielectric polarizability, anisotropy of flat -screen type , 259 Polarization microscopy; see also Polarizing microscope resolution at corners of, 170, 252f (instrument) see also Cathode ray tube analog enhancement in, 405 Pinch roller, 26S, 269f, 277f of beating cilia, Sf, 405 Pincushion distortion, 24S of diatom frustule , 7f in intensifier camera tubes, 227, 253 of microtubule slurry, 9f see also Distortion; Geometrical distortion of rotating bacterial flagella , 407f Pinhole, in confocal microscope, 41S annotated bibliography on, 500-50 I Pinocytosis, of rhodamine-labeled protein, 397 birefringence distribution, line trace of, 323, 325f Pipeline processor, 40g, 391 compensation in, Sf, 9f, 407f, 409f, 4SSf, 489f, 493f, for logical operations between two images, 3S7 494f, 498f, 499f; see also Polarizing microscope Pixel, 40g, 169n, 310, 330, 471 (instrument), compensators in ceo, 19S compensation, optimum, for v1deo, 405, 405n, 406f gray values of, after digital manipulation, 360-363 condenser immersion in , 127 568 INDEX Polarization microscopy (cont.) Polarizer (cont.) condenser NA; see also Condenser NA electric vector direction in, 4SI-4S3, 4S3f, 4S7f for video microscopy, Ito, 136, 414f for high-extinction polarization microscopy , 405n, 492, .for visual observation, 110 495f conoscopic image with, 493f; see also Conoscopic im• transmittance through, 483f age; Conoscopic observation see also Polaroid; Prism, calcite contrast enhancement in, by video: see Polarization mi- Polarizing filter: see Polaroid croscopy, video enhancement in Polarizing prism: see Prism, calcite crystalline axes, detennination of, 4S5-4SS Polarizing microscope (instrument) dark-adaptation of human eye for, 492-493 aperture stops, vs. scattered light, 495f dichroism of retinal rods and cones in, 415 basic optical principles, 492-499 digital enhancement books and references on, 500 critical specimen, for testing, IS4f, 405, 409f cell fine structure studied with, 404, 492, 500 of diatom frustule, Cover, 306f compensators in, 4S6-4SS, 4S8n, 488f, 4S9f, 493f, of individual microtubules, 9f 495f; see also Compensator (polarization optics) of muscle, with noise reduction, IS4f, 409f conventional, 493f of DNA microdomains in living spenn, 412, 49Sf, 499 3-D diffraction pattern in, 115 and fluorescence microscopy, combined, 147f, 14S diffraction image errors in, 497f, 498, 498n generating edge maps in, 379 high-extinction: see Polarization microscopy, high-extinc- gray level histogram typical for, 339, 354, 354f tion; entries under Rectified high-extinction, 403-405, 405n, 492-499; see also en- high-resolution, 493-499, 495f, 496f tries under Rectified inverted, 145-148, 495f, 496f interference colors in, 49ln limits and refinements of, 403-406, 492-499, 500 measuring retardation in, 325f, 404 utility of, 477 monochrome vs. color camera for, 405 Polaroid photography for, vs. video, 405, 405n, 406 sheet, 492 use of Plan Apo objectives in , for video, 7f, Sf, !Of, stereo viewing glasses, 450, 452, 455 , 455f 136, 306f, 405. 409f sunglasses, 481-483, 483f, 485n reference books on, 122, 500-501 transmittance of, 13 3 scan-line removal, 464f, 465f, 466 Polars: see Polarizer sensitivity, for detecting DNA molecules, 404 Pollen mother cell, living, birefringence in, 494f theory on image fonnation in, need for, 125 Polyiodide crystals, 485n video cameras for, 193, 230!, 394t, 405 Polymer structure, and physical properties, 500 video enhancement in DNA, 404, 491 , 501 with Chalnicon, 9f, 405n nitrocellulose, 49Jn with Newvicon, 7f, Sf, 306f, 325f, 405n, 407f, 464f, polyvinyl alcohol: see PYA 465f, 466 Polyvinyl alcohol: see PV A with SIT, JS4f, 409f Position sensors, linear, 227 see also Birefringence; Dichroism; Microscopy; Polarized Power line, AC , vs. camera noise: see Noise (video light; Polarizing microscope (instrument); Video camera) microscopy PPH, 41g, 178, 231, 246, 473; see also Horizontal Polarization rectifier, 4S4f, 493-499, 495f; see also entries resolution under Rectified Preamplifier, 181, 274; see also Electrical noise, from Polarized light preamplifier birefringence: see Birefringence Price cosine-squared law, 4S3f of digital image processors, 329, 511-513 dichroism: see Dichroism of hard-copy devices, 331 elliptically, 4S6, 4S7, 4S7f, 4S9f of image storage, compared , 304, 331 interaction of, with matter, 404, 477-4S5, 4SS-492 of magnetic recorders, 331 microspectrophotometry in, 40 I, 415 of minimum setup, 55-56 plane, 4S2-4S3, 487 of OMDR, 304t reflected by water, 482 disks, 304 transmitted by calcite, 481, 482f, 483 , 483f, 484f of monitors transmitted by Polaroid, 481-4S3, 483f vs. distortion, 251 transmitted by tounnaline, 484-485, 485f, 486f vs. flagging, 291 polarizer: see Polarizer; Polaroid; Prism, calcite of software development, 513 Prado Pol demonstration kit, 405n of varifocal systems, 422n rotation of, 4S8 of VCR slow and fast waves, 4S5-4S8, 487f, 4SSf, 4S9f high-resolution, standard-speed, 284t use in SSEE microscopy, 412 112-inch, standard-speed, 282t see also Polarization microscopy; Polarizing microscope 3/4-inch, standard-speed, 281t (instrument) with insert edit capability, 437n Polarized-light microscopy: see Polarization microscopy tape, 287 Polarized UV microbeam, 4S3, 4S4f time-lapse, 290t analyzing arrangement of DNA bases with, 49Sf, 499 of video projectors, 444 Polarizer, 4S4f, 493f, 495f ofVTR books and references on, 500 high-speed, 294t INDEX 569 Price (cont. ) Pupil (cont. ) of VTR (cont.) human eye (cont. ) l-inch, reel-to-reel, 279t diameter and Stiles-Crawford effect, 74n tape, 287 diffraction by, 74, 78 Primary colors, combinations of, 238, 238f eyepoint partially occluded by, 449n Principal plane, 4Ig, 97f muscles of, 72 Prism relation to microscope eyepoint, 102, 102f Ahrens, 483 PYA, 488-491, 489f calcite, 483, 495f dried gel, isotropy of, 488-489, 490f astigmatism from, 483n, 495f stretched sheet commercial source of, 145n anisotropy of, 489-491, 490f high-extinction, 405n, 495f as substrate for sheet Polaroid, 485n reference books describing, 500 Pyroelectric vidicons, 397n transmission through, 133, 483f see also Calcite crystal Quantitation (video) Dove, for image rotation, 420f of autoradiographic grains, 364 erecting, in dissecting microscope, 447f digital, 5, 512, 513 Glan-Thompson, 483, 495f of dynamic changes of fluorescence, 401 Nicol, 483 of optical density, 363-365 Nomarski/Wollaston of physiological events, 323 in DIC optics, 410, 412 precautions, in using tape recording for, 274 directional effect on MTF, 123 see also Line scan; Measurement Probability density function, 341 Quantitative analysis Processing amplifier, 42g, 185, 243 by flying-spot UV microscope, 5 Program, computer of imaging systems, by MTF: see Modulation transfer for histogram matching, 351 function; MTF for image processing, 512, 513, 513n Quantum efficiency for stereo pair, from optical sections, 455n of CCDs, 197-198, 212f Projected image, copying of, with video camera, 429-430 of human eye, 81n of movie, 430 Quantum-limited mode, 230! Projection distance, of ocular Quarter-wave retardation, 121 optimum, 100 Quartz vs. size of Ramsden disk, 68, 69f crystal oscillator, 186 Projection ocular objectives, 401 adjustment of image distance with, 135 optical rotation in, 488 reduced image distortion with, 100 Quinacrine mustard, 5 Projection, video, 5, 442-445, 443f, 444f Propidium iodide, fluorescent stain for DNA, 6f Protein, molecular polarizability of, 491 ; see also Actin; Radio frequency, 42g, 270 Flagella, bacterial, flagellin polymerization; in dark Radio frequency spikes, 61-62, 181, 296 field; Microtubules Radiometric unit, 42g, 77 Pseudocolor display, 42g Ramsden disk, 42g, 68, 69f, 103; see also Eyepoint analog, 314 Random access, 42g vs. detectable steps of gray, 356 OMDR, 263, 302-304 of fluorescence intensity, xxv, IOf, 26f Random interlace, 42g, 56n, 161, 172, 185 change with time, xxvi, 382-383, 382f Random interlace camera, 56n, 256, 290, 293 real-time transformation, 381 Random noise: see Noise; Noise averaging (spatial); Noise with ITF, in look-up tables, 348, 348f, 381, 382f, 387, averaging (temporal) 512 Raster, 42g multispectral analysis with, 348 dimensions of, 245f recording of, on VTR, 283 display devices representation of image feature with, 149 liquid crystal, 242 transformation of taped video images, 381, 382f reference books on, 24 2n use of monochrome camera and color monitor for, 237 and Nipkow disk, 2f, 3 of very-low-light-level fluorescence, xxv stabilized, by external sync, 259 PSF: see Point-spread function video, 152-156, 153f, 154f, 155f Pulse-cross display, 42g, 164-169, 168f, 169f Raster line: see Scan lines vs. correction of time-base error, 188f Ratioing, 364, 366f showing flagging, 292f Ray diagram, 95, 96f; see also Light rays of VTR output, 187, 188f Rayleigh criterion for resolution, 43g, 113f, 114, 470; see Pupil also Resolution (microscope), limit of circular, MTF of microscope with, 470f Read/write head: see VTR, head entrance, effect on OTF of microscope, 469 Real time, 43g, 242 exit, of optics, 469 conditional image manipulation in, 390f human eye, 72f, 99f, 102, 102f, 103f digital image processing in , 330- 331, 381-384, 388t, area, for defining retinal illuminance, 72 390! , 512 diameter of, 72-74, 72f interactive, 458t, 511 570 INDEX Real time (cont.) Reference books concerning (cont.) image convolutions in, 372 spatial filtration, 463, 470 intensity transfonnation with LUTs in, 347, 512 stereoscopic vision, 9ln, 420n, 446, 446n logic operations in, 512 video pseudocolor transfonnation in, xxvi, 381 , 382f, 512 circuits, v(n) response of video camera corrected in, 364-365 color encoding, 240n, 260n, 261f stereo video images in, 451, 452f display devices, 242n, 245n, 246n, 25ln tracking of cell movements in , 384 image pickup devices, 4, 197f, 332 Real-time display, 242 principles, v(n), 149n, 167-169, 3lln of birefringence distribution, 325f projection, 443f of fluorescence distribution, xxvi, 324f, 381, 382f projection, stereoscopic, 446 of luminescence changes, 400 pulse-cross display, 167 Real-time optical digitizer, video camera as, 333-334 standards, 169 Rear focal plane of objective lens, 98f, 99f, 101, 102f, test equipment, 256n 109f, 110 VTR, 266f, 267n, 273f, 300n, 30ln absorbing mask at, 119, 119f Reference color subcarrier, 163f, 240-242, 241f diffracted and background waves at, 121 exact frequency of, 242n as Fourier plane, 373 Reference edge (video tape) , 263f, 266f, 267, 276, inspection of, with phase telescope, 106, 110 301 Recam, 274, 275f Reference lists, vi, 515-528; see also Reference books Receiver-monitor, 43g, 242 concerning Recommended standards: see entries under RS• Appendix JII, 500-5 10 Reconstruction, image Reflected-light interference microscopy, 410 3-D: see 3-D image reconstruction frustrated total internal reflection, 122, 410 transfonnation during, 419 Reflecting obJective lens, 401 Recorder/recording: see OMDR; VCR; VTR Reflection, 122 Rectified condenser, 145n, 484f, 495f; see also Polarization from coated surface, 132 microscopy Fresnel coefficient of, 132n, 482 used with Plan Apo objective loss, of polarized light, 132n in video DIC microscopy, 306f, 408f, 410 off monitor faceplate, 425f, 426, 431 in video polarization microscopy, 7f, 306f, 407f, 409f see also Mirror Rectified optics, 43g, 145n, 484f, 495f; see also Polarizing Refractive index, 480f microscope (instrument) of anisotropic medium, 480f for detecting weak birefringence, 404, 493-499 vs. dielectric constant, 479-483, 480f effect on diffraction pattern, 115n, 404, 404n-405n, vs. edge birefringence, 498n 497f, 498 in human eye, 72f, 97, 99f, 104 Rectified polarizing microscope, 496f, 497f, 498f of imbibing medium, 491, 49ln display of DNA packing arrangement with, 488f matching of, 409f, 49ln, 498 and chromosomes in living spenn, 499, 499f of rodlets and platelets, 49ln Red, green, and blue: see RGB of specimen and medium , 12ln Reel-to-reel VTR , 268, 274 vs. velocity of light, 479-483, 480f portable, l-inch, 279t Refractive index ellipse: see Index ellipse, axes of see also VTR Refractive index ellipsoid, 480-481, 480f, 482f Reference black and white levels, 159f, 162f, 163f Registration, 43g Reference books concerning in color video, 240, 444 .circuit diagrams, v, 149n, 167-169, 311n convergence, 2lg, 262 crystallography, 404, 500 of serial sections, blinker, 420 digital image/signal processing, 184, 352, 368, 372, Relay optics, 474, 474f; see also Magnification, micro• 373, 457n, 513 scope, vs. camera tube sensitivity human vision, 71, 72f Reporter dyes, 416 light microscopy, vi, 93n, 457, 500 Resolution (human eye) aberrations in, 93n, 133n, 134 angular, 77-80 contrast generation in, 122 axial, 101, 110, 118 darkfield, 122 limit of, 77-80, 203 diffraction pattern vs. lens aberrations, 133n role of retinal cones in, 79f fluorescence, 122, 457 vs. threshold detectability, 79 laser microbeam irradiation, 417 Resolution (microscope) phase-contrast, 119, 121 n, 122 vs. aperture and field planes, I 04, 108 photometry, 415n vs. coherence, 113n polarization, 122, 404, 500-501 vs. condenser NA, 108-110, 114- 115, 124f, 414f scanning, 418 considered as linear systems, 374 stereoscopy and 3-D reconstruction, 446n and contrast, vs. NA, 124f; see also Modulation transfer MTF, 170, 176, 470 function; MTF optics, 119, 500 and diffraction pattern, 113-118, 113f, 114f, 116f, 117f photography, 170, 425n doubled photomultipliers, 415n with confocal microscope, 418 physical optics, 116n, 119, 404, 500 m fluorescence microscopy, 418 INDEX 571 Resolution (microscope) (cont.) Response (cont. ) improving on, 377 logarithmic, of eye and photographic emulsion, SOn, with video, 110 405, 406f limit of low-frequency Abbe theory, 13g, 470 of human visual system, S3 Rayleigh criterion, 43g, 113f, 114, 470 of v1deo circuits, 17S, 246 Sparrow criterion, 47g, 471 radiance, of human eye, 229 vs. MTF, 118 of video camera, to highlights, 66 vs. NA, 113-115, 114f see also Amplitude response; Spectral response curve and wavelength, 469 Response curve, effect of shading on, 332f need for Koehler illumination, 64 with and without ratioing, 366f vs. OTF, 469-470 see also Respons1vity; Sensitivity, of camera tubes raised, by sacrificing field of view' 418n Responsivity, 43g, 205 vs. spatial frequency response, 470-471 of ceo, 212f super-resolution, 418n of SIT, 199f, 2llf with confocal microscope, 41S vs. SIN ratio, of vidicons, 217 infinite aperture, 377 units for measuring, 205 vs. video camera, 474-475 of video pickup devices, 194t, 199t see also Limit of resolution Retardance of specimen, 403n Resolution (video) vs. image brightness, 492-493 definition, 170 Retina in encoded color video, 238, 241 and brain, image processing in, 79-80, SOn, S2-83, S4f, vs. frequency response, 174-175, 176t 90, 91-92 of gray values, 334-335, 337f, 338 and critical flicker frequency, S5f in high-resolution systems, 165t distribution of cone cells in, 7Sf line pair, vs. TV lines, 474 ganglion cells in, 73f loss of, by color video encoding, 238 IR insensitivity of, 403 vs. microscope resolution, 231, 474-475, 475f isolated elements, dichroism of, 415 vs. noise, trade-off between, 318f light-sensitive elements, bleaching of, 415 of signal, 174-175, 174f microscope image formation on, 97-99, 99f, 103f, 104 and system MTF, 231 processing of signals in, SO in video, 170 scanning of image on, 79 vs. VTR, 130, 424 see also Cone cells; Human visual system; Rod cells see also Horizontal resolution Retinex theory of color vision, 90 Resolution (video camera) Retrace, 152-153, 153f bandwidth, vs. noise, 233 Return beam, 197 of camera tubes, 204f, 205f; see also Limiting resolution RF converter, 43g, 242 factors affecting, 229, 231 portable VCR equipped with, 2S6 in instrumentation camera, 231 RF spikes, 61-62, lSI, 296 limited by photon statistics, 208f ROB, 43g limiting, at very-low-light levels, 424 ROB camera, 237, 237f Joss of, as trade-off for brightness, 475 ROB color video, 237-240, 237f, 242 vs. microscope optics, 130-131 recording onto tape, 430-432 monochrome vs. color video camera, 229 ROB monitor, 237, 237f vs. MTF curve, 473 resolution of, 260, 424 vs. noise, in intensifier tubes, 397 ROB-sync, 44g, 262 ROB vs. NTSC color, 241-242 ROBY, 44g, 237, 23S-240, 240n solid-state, 202t Rhodamine 60, 3S0f, 381, 3S2f TV lines across full width of vidicons, 473 Ringing, 44g, 174, 175f, 17S vertical, and Kell factor, 170-172, 171f, 172f, 231 in camera circuit, 233 in vidicons, 193, 203 vs. tube orientation, 235, 235n see also Limiting resolution in video monitor, 254 Resolution (video monitor) Rise time, 44g at comers of, 170, 252f vs. horizontal video resolution, 176, 176t factors affecting, 244-246 relation to bandwidth, 176, 176t vs. picture size, 243 required for H-scan, 173, 175f of ROB monitor, 260, 424 rms Noise, 181, 1S2t test chart for, 255-256, 255f, 257f, 25Sn measuring, !Sin Resolution (VTR) Roaming, in digital image processing, 392, 512; see also in edited tapes, 2S3 Pan; Scrolling of frozen field, 425 Roberts operator, 372 limited by, 231, 424 Rod cells monochrome vs. color, 424 convergence to ganglion cell, 79-80, SOn Response critical flicker fusion in, S3 high-frequency distribution of magnetic tape, 270-272, 273f in fovea, 77-79, 7Sf, 79f of video circuits, 178, 246 in retina, 73f, 7S, 78f 572 INDEX Rod cells (cont.) Scan lines (cont. ) excised, 403 removing isolated, dichroism of, 415 with Ronchi grating, 427, 428-429, 428f scotopic vision, function in, 7 4 by spatial filtration, 463-466, 464f, 465f loss of color sensation in, 90 seeing, 256n spatial resolution in, SOn vs. vertical resolution, 171-172, 17lf, 471 threshold sensitivity of, SOn see also Interlace, 2:I, exact types of, 9{) Scan rate, 45g see also Retina 525/60, 153-157, 155f, 158f, 164, 166t, 172, 175 Rolling, 44g, 58, 62, 167 625/50, 153, 158t, 164, 166t Roll-off (frequency response) vs. flicker, 153-154, 452-455 in audio and video signals, 179f for high-resolution video, 165t in VTR recording, 273f special, 256-258 Ronchi grating see also Incompatibility enhancing periodic structures with, 428n Scanner (VTR), 264, 264f rtozen-field image, with and without:'407f control of speed and phase of, 269 full-frame image, without, 307f direction of spin, 268n prominence of scan lines reduced with, 427f, 428, 428f four-head, 276, 282t, 285 spatial filtration without, 463 Scanning aperture, 229 RS-170, 44g, 159 Scanning-beam hold-off, 318-319 compatible solid-state cameras, 202t Scanning electron microscope: see Electron microscope H-sync pulse detail in, 161, 162f Scanning light microscopy main features of, 159f aperture, 419-420 monitor response to, 185, 256 field, 417-418 VTR compatibility for, 277 focal: see 3-D display; 4-D display RS-170A, 44g reference books on, 418 color bursts, field rate for, 161 Scanning spot (video), 172, 173, 174f monitor response to, 256 Schlieren optical system, 443f, 444 NTSC color standards, 161, 163f, 240n Schmidt correction plate, 444, 444f VTR compatibility, 277 Schwann sheath, form birefringence of, 491 RS-232C, communications link, 44g, 303 Scotopic vision, 45g, 74 RS-330, 44g, 159, 161 function of rods in, 74 H-sync pulse detail in, 160f, 161 and natural illuminance levels, 75f main features of, 159f vs. perception of color, 90 monitor response to, 185, 256 wavelength response, 76f standards for, 160f factors limiting, 74n VTR compatibility, 277 see also Dark adaptation; Photopic vision RS-343A, high-resolution video, 165t Scrambler RS-420, for random interlace, 161 attenuating diaphragm in, 126n, 241 optical, 38g, 127n, 495f, 496f Scratch, on video tape, 296, 297f Sampling Scrolling, 45g, 58 bar patterns, 471-472, 472f to display hidden portions, 167, 391 interval, 44g, 335-338 due to equipment incompatibility, 164 vertical, 4 71 Search, binary, for centroid location, 384-386 Saticon, 44g SEC, 45g, 197f amplitude response curve for, 204f lag, vs . faceplate illuminance, 219f light-transfer characteristics of, 215f with large-diameter photocathode, 199t noise character, microscopy applications, 230t, 394t- limiting resolution, vs. faceplate illuminance, 208f 395t SiN ratio, vs. faceplate illuminance, 217f specifications of, 194t; see also Camera tube specifications of (25-mm, 40-mm), 199t-200t specifications use in astronomy, 319n Saturation, 44g SECAM, 45g, 238 and blooming, 221, 224f monitors accepting, 260n of color, xxv, 89-90, 238, 239f, 240; see also 625/50, nations adopting, 164, 166t Chrominance VTR accepting, 2811 of signal, 179 Sector wheels, aperture-occluding, 451 , 453f of video camera, 125, 214 Segmentation: see Image segmentation Sb2S3 vidicon: see Sulfide vidicon Segmented scan recording, 45g Scan, 44g, 153f video tracks in, 264, 264f Scan line removal, 44g; see also Scan lines, removing see also VTR Scan lines, 151, 338, 463 Selenium, photoconductivity of, 2 distracting from fine detail, 307f, 322f, 407f, 426, 463, Sensitivity, 45g 464f of camera tubes, 193, 194t, 199t, 203-209 Gaussian distribution of, 17lf, 245f vs. microscope magnification, 130 pairing of, 172, 172f, 426 of ceo, 197-198, 202t, 203, 212f INDEX 573 Sensitivity (cont. ) Shading distortion, 333f, 334, 363 of color video camera effect of contrast enhancement on, 333f and dynamic range, 240--241 in Newvicon, 332f, 363 quantum-limited, 2301 reduction of in far-red: see Far-red sensitivity by convolution operation, 384 of human eye: see Human eye, spectral sensitivity of with sharpening filter, 384 of intensifier camera tubes: see Image intensifier of video camera, 334, 364, 366f vs . noise Shadow-cast appearance character of, and microscopy applications, 2301 in DIC microscopy, 410 in intensifier camera tubes, 397 from oblique illumination, 90, 412, 446 in video cameras, 217, 217f, 233 Shadow detail, analog expansion of, 314f spectral Shadow mask, in color video picture tube, 46g, 260, 261f of CCD, 212f Shape of Chalnicon, 210f of cell, binary mask in, 380f, 383 of eye, vs. video camera tube, 415 Fourier descriptors of, 379 of Newvicon, 210f, 415 Sharpening filter, 46g of photocathodes, 213f analog, edge crispening by, 314, 317f of Plumbicon, 213f as convolution mask, 369-371, 369f of SIT, 211f produces noise, 371 of sulfide vidicon, 210f reducing shading distortion with , 384 of Ultricon, 210f Siemens Test Stars, 115, 497f of UV vidicon, 213f out-of-focus, 116f, 117f of video imaging devices, 205 Signal threshold amplitude loss of, at high frequency, 256, 257f of human eye, 74; see also Photopic vision; Scotopic EIA standard: see entries under RS- vision from time-lapse VTR , syncing to: see Automatic hori• of intensifier cameras, 397 zontal frequency control of retinal rods, SOn for VTR recording; see also VTR see also Light transfer characteristics; Responsivity adjustment of with video processor, 431-433, 433f, Sensor size, in video cameras, 199t-200t, 231, 232t 434f Serial sections, 413 de-emphasis, in playback, 274 blinker registration of, 420 see also Color signal; Composite video signal; FM; see also Optical sectioning Picture signal; Sync signal; Video signal Serrations, in V-sync pulse, 45g, 161, 164f Signal averaging Servo controls, in VTR, 269-270 in human vision, 79-80, 80n Setup, in video signal, 45g, 159f, 160 to remove random noise, 183f Shading, 9f, 45g by scanning with a slit, 183 caused by poor low-frequency response, 178 see also Frame summation; Image averaging; Image in microscope, 64-66, 69-70 integration; Noise averaging (temporal) in monitor, 227n, 251 Signal current, 192f low-frequency response, 246 vs. dark current, 217 in Newvicon, 332f vs . faceplate illumination percentage of, in vidicon tubes, 194t intensifier camera tubes, 215f reduction, by digital image processing, 511 vidicons, 214f, 215f in SIT camera, IOf, 253f, 409f vs. lag sources of, 224-227 of Chalnicon, 223f uneven field, 105 of Plumbicon, 223f in video imaging devices, 225-227 of SIT, 218 visual response to, 83, 84f of video pickup devices and cameras, 209 as three-dimensional clue, 90; see also Shadow-cast Signal degradation, 178-184; see also Distortion; Electrical appearance noise; Noise see also Shading distortion Signal electrode, 192-193, 192f Shading compensation (analog), 46g Signal plate, 3, 3f dynamic, in video camera, 227 Signal processing in high-resolution monitor, 258n analog, 310-319 for photometry, 227 VS. digital, 309310, 5)3 wave form added for, 312, 316f edge sharpening by , 314, 317f Shading correction (digital) image averaging by, 70; see also Target integration by background subtraction, 9f, IOf, 69-70; see also keying with, 313, 440-441, 441f Background subtraction noise reduction by , 314-319, 318f line trace of, 183f shading correction by, 312, 316f effect on photometry, 363-366, 366f in video microscopy, 310-311 with Laplacian filter, 371 see also Analog enhancement; Image processing, need for, prior to edge detection, 356, 358f analog preprocessing, for feature extraction, 377-378 in human vision, 79- 80, 82- 83, 84f, 91 by ratioing gray-value histogram, 366f in tape-to-tape copying, 433, 439f 574 INDEX Signal processing (cont.) SIT (cont. ) in video, II shading in, 253f see also Digital image processing; Image processing SIN ratio, vs. faceplate illuminance, 200t, 217f Signal-processing circuits specifications of (25-mm, 35-mm, 40-mm), 199t-200t in cameras, 231-235 square-wave response curve of, 207f in monitor, 244f, 246 for very-low-light-level fluorescence microscopy, 324f, in VTR, 270-274 397-401 see also Circuits in video-intensification microscopy, 397 Signal-to-noise ratio, 46g; see also SIN ratio Si vidicon: see Silicon vidicon Silicon, 46g, 194t; see also Silicon vidicon Skew Silicon diode, 196 causes of, 286 in CCD, 198 in VTR playback, 46g Silicon-intensifier-target tube: see SIT SKEW control Silicon target vidicon: see Silicon vidicon influence on noise bar, 296 Silicon vidicon, 46g, 221, 224 on VTR, 46g, 280f blooming in, vs. Ultricon, 221 in VTR recording, 298 burn in, 224 Skip-field recording/playback, 46g, 289 gamma of, 221 Slave camera, 47g lag, vs. faceplate illuminance, 219f synchronizing of, 186, 186f, 439, 440f light-transfer characteristics, 215f Slide: see Microscope slide SIN ratio, vs. faceplate illuminance, 217f Slit-scanning mirror microscope, 418, 419f specifications for, 194t, 199t; see also Camera tube Slow replay, 294 specifications Slow scan, 47g spectral response curve for, 2\0f, 213f bandwidth compression and noise reduction by, 310n, see also Ultricon 317 Silicon wafer, 403 high resolution achievable with, 417, 417n Sine condition, 95n, 133 photomultiplier used in, 427n Sine wave, 175n Smear, reversed polarity: see Streaking fundamental harmonic, 174-176, 174f Smith-type DIC microscopy, 320, 322f, 410 modulated grating, visual response to, 81-82, 82f Smoothing convolution, 368-369 square wave made up of, 175n noise reduction and blurring by, 368, 368f, 369f, 37 lf see also Sinusoid; Sinusoidal object, contrast of see also Image averaging; Noise averaging (spatial) Single-sideband edge enhancement microscopy, 46g; see Smoothing filter, 47g also SSEE microscopy analog, noise reduction with, 317, 318f Single-tube color camera, 240, 242 convolution mask for, 368-369, 368f Sinusoid, 467-470, 467n as part of sharpening filter, 371, 37lf Sinusoidal object, contrast of, 81, 82f, 467; see also size, vs. noise and blurring, 369f Modulation transfer function; MTF; Optical transfer SMPTE, 47g; see also Time code, SMPTE function Snail, Hermissenda, statocysts in, 44lf SIT, 46g, 187f, 196, 197f Sniperscope, in low-light-level microscopy, 396 analog noise reduction in, 318f Snow, 47g, 181 , 2 17, 230!, 296; see also Noise blooming in, 221 SIN ratio, 46g, 47g, 181-184 burn in, 224 vs. bandwidth, 181 , 182, 183f, 184f chilling, to reduce target leakage, 319 of camera, improved by pedestal control, 232 comet tailing in, 219, 220! of clean video image, 182 coupled to darkfield microscope, 40 I expressed in dB, 181-182, 182t coupled to four-stage intensifier, 396n vs. frequency characteristics, 124 dark current of frozen field, vs. frame, 425 vs. operating temperature, 216f, 217 in human eye vs. target voltage, 216f, 217 and storage time, 81 n digitally processed images from , xxv(f), !Of threshold, modeled for, Sin frame averaging in, 184f of image isocon, 410 frame summing, 402f, 409f improved by summing and averaging, 335, 336t lag of intensifier camera tubes vs. faceplate illuminance, 219, 219f vs. faceplate illuminance, 217f vs. signal current, 218f at low faceplate illumination, 200! with large-diameter photocathodes, 199t for line scans at different levels of. 183f light-transfer characteristics (16-mm, 40-mm), 215f and MTF, 124 limiting resolution, vs. faceplate illuminance (40-mm), of OMDR, 303, 304t 208f of solid-state cameras, 202t in low-light-level microscopy, 396-397 of VCR/VTR noise averaging high-resolution VCR, 2 84t by beam hold-off and chilling, 317-319 improved by FM recording, 273 line scan showing, 183f l /2- inch, standard-speed VCRs, 282t noise character, microscopy applications, 230!, 394t 3/4-inch, standard speed VCR, 28\t pickup of fluorescence, on dual video camera, 147f and overall picture quality, 285 in polarized-light microscopy, 409f time-lapse, 290t INDEX 575 SIN ratio (cont.) Spectal effects generator (cont.) video image at various levels of, 182 wipmg video scenes with , 313, 439f fluorescence microscope image, 402 Specimen polarized-light image, 184f and contrast in microscope image, 119 of vidicon tubes plane of, in Koehler illumination, 101-104, 102f, 103f vs. faceplate illuminance, 217f Specimen damage: see Photon damage to specimen vs. responsivity, 217 Specimen rotation, as 3-D cue, 91 see also Electrical noise; rms Noise; Video signal Spectral output Sobel operator, for edge detection, 372 of CRT phosphor, 248, 249t-250t Software, for image processing incandescent lamp at low voltage, 415 C-library for, 47g, 512 lamps with high luminous density, 128t, 129t cost of development, 513 of xenon arc lamp, 129t, 418 FORTRAN program, for ITF transformation, 350-352, see also Color temperature; Lamps; Light source 351f Spectral response curve, 47g, 206 Solid-state cameras, 20 If for ceo, 212f high-speed, shuttered, 294n of human eye, 74-77, 76f specification, sample, 202t vs . video imaging devices, 205, 415 vendors of, 203n, 236t for Newvicon, 210f, 415 see also CCD camera of photocathodes, 213f Solid-state laser, 457 for SIT, 21lf in OMDR, 305f for video imaging devices, 203-206 Solid-state sensors, 47g, 197-198, 203 of vidicons, 210f-213f blooming in, 221 , 226f factors determining, 205 chilled CCD, 197-198, 235 vs. fluorescence microscopy, 206 linear, with low geometrical distortion, 227, 236, 417 Speed search, in VTR , 277 see also CCD Sperm, living Solvent acrosomal process growth kinetics, 319-320, 322f, 323f benzene, 140-141, 141f calcium ion release by entry of, 396, 398f-399f ethanol, 143n, 144, 144f cave cricket, in polarized light ether, 140 birefringence and DNA microdomains, 498f, 499 , 499f water, 140, 142 by SSEE, 412 xylene, 140 sea urchin , in DIC Sonoluminescence, 396n showing lag, 219, 221f Sorting: see Classification stereo video images in real time, 451 Sound track: see Audio track Spherical aberration, 48g, 133, 133n SP, LP, SLP, 47g, 282t; see also Extended-play modes of apochromatic objective, 133 SpageGraph system, 422, 422f; see also 3-D display of condenser, 10 I, 139 Sparrow limit, 47g, 471; see also Resolution (mtcroscope), of lamp collector lenses, 105n, 138 limit of of Plan Apo objective, 133n Spatial convolution, 47g; see also Convolution operation Spherical wave , 95, 96f, 102, 102f, 104, 120 Spatial filtration , 47g Spindle high-frequency pass, phase-contrast microscopy as , 125f, birefringence distribution in , 325f 408 DIC image of 292f reference books on, 184, 368 , 373 , 457n, 463, 470 Spindle fibers to remove video scan lines, 427-429, 427f, 428f, 464- birefringence in, 271f, 404, 494f 465. 464f, 465f and chromosome movement, 500 see also Convolution kernal/mask; Filter (optics); Filter Square wave, made up of sine waves, 175n (video); Fourier filtering; Spatial frequency Square wave response, 468; see also Contrast transfer Spatial frequency, 47g function components, vs. edge sharpness, 469 SSEE microscopy, 46g, 122 vs. digital processing, 368- 371, 369f, 370f, 372f MTF curve of, 123, 125f limiting, 123 sensitivity, and potential of video, 412 and MTF, 118-119, 122-125, 467-475 Stage micrometer vs. OTF, and out-of-focus image, 115-118, 116f, 117f to determine exact magnification, I 00 response of eye to, 81 - 82 , 82f, 83f for video measurements, 319- 320 vs. temporal frequency , 124 Stage, microscope vs. video resolution, 173-176, 174f motorized, 384, 386 digital, 471-472 universal, 447 , 448f see also Amplitude response curve; Convolution opera• Stain tion; Fourier filtenng ; Spatial filtration FITC , protein fluorescence , 6f Spatial resolution: see entries under Resolution fluorescent antibody, microtubules, 402f, 41lf, 447-449, Special effects generator, 47g, 439f 449n CRO trace, and statocyst, 44lf Golgi , neuron in stereo, 449n hookup of, 438-439, 440f haematoxylin, lightly-stained chromatin , 4!4f mixing signals with , 429 proptdium iodide, DNA, fluorescence , 6f montaging with, 431 , 438-441 quinacrine mustard, DNA , UV and fluorescence , 5 use during video editing, 431 , 438-441 Rhodamine 60, fluorescence , vital , 380f, 381 , 382f 576 INDEX Stain density , calibration curve for , 364 Stretching (cont.) Staircase effect, 322f, 360, 380f, 427 of VTR tape, 301 Standards, video: see EIA pre-stretchmg, to minimize, 293 Statistics, of image features, 379 Strobe flash, for highspeed video recording, 294 Statocyst, action potential from cilia in , 441f Stroboscopic record, with video camera, 126n Stentor , cilia in, 8f, 405 Substage ntirror and illuminator adjustment, )05-107 Stepping motor, 48g, 384 Subtraction of background: see Background subtraction Stereo microscopy, high-resolution Sugar solution, optical rotation in, 488 by aperture scanning, 420 Sulfide vidicon , 48g confocal microscope for , 418 amplitude response curve for, 204f vs. 3-D reconstruction, 420, 420n bum in, 224 reference book on, 420n, 446 dark current in, 214f with conventional compound microscope, 446-450, dynamic range of, 209, 215 448f, 449n, 449f gamma of, 209, 219f, 246 of Golgi-stained neurons, 449n lag, vs. faceplate illuminance and gamma, 219f of fluorescent microtubules, 447, 449n light-transfer characteristic curve for, 214f, 215f simultaneous stereo pairs with, 449-450, 450f luminance level, nticroscope contrast modes, 394t-395t see also Stereoscopic video; Stereoscopy sensitivity, noise character, microscopy application, Stereoscopic acuity, 48g, 90-91, 90f 230t vs . luminance, 9lf SIN ratio, vs. faceplate illuminance, 217f vs. visual acuity , 91 specifications for, 194t, 199t Stereoscopic video spectral response curve for, 2\0f, 213f flicker-free projection from tape , 455n target voltage microscopy , 451-455, 452f, 453f, 454f, 455f vs. dark current, 217 from serial optical sections, 455n vs. light-transfer characteristics, 209 of swimming sperm, 451 variable, 209, 216, 217 see also Stereo microscopy unsuitable for photometry, 397 reference books on , 446 used in PV, PV, 397 Stereoscopy Sunglasses, Polaroid, 481-483, 483f, 485n cues in microscope for , 90-91, 446 Super-resolution, 418n and human vision, 90-91, 90f, 9\f; see also with confocal microscope, 418 Stereoscopic acuity infinite aperture, 377 using dissecting microscope, 446, 447f Super Slo-Mo system, 295 with video , 451, 452f Surveillance camera, 55-56; see also Random interlace reference books on, 9\n, 446 camera Sticking, 197, 221, 224 Swab stick, 143-145, 144f dissipation of, 224 Sweep, nonlinear, distortion arising from, 155, 227; see see also Bum also Geometrical distortion Stigmatizing lenses, 495f Symposia, 457; see also Courses, short, related to video Stiles-Crawford effect, 74n nticroscopy Still-field display Sync, 48g; see also External sync; H-sync pulse; Syn• clean, in VTR playback, 289 chronizing; Sync pulse; V-sync pulse for motion analysis, 285 Sync formats, 160-161, 164f photographing, 423-429, 425f, 427f, 428f CCTV vs. broadcast standard, 185 VCR tape for, 267n see also entries under RS- see also Freeze-field image; Freeze frame; Still frame Sync generator, 48g, 156, 187 Still frame , 48g Synchronizing, 48g capability of VTR, 277 an array of video equipment, 186-189 variable-rate advance, with OMDR, 304t camera outputs, using delay circuit, 185n see also Freeze-field image; Freeze frame ; Still-field cameras, 186f, 187-188, 187f display copy camera to monitor, 431 Stops motion-picture camera with time-base corrector, 189, vs. flare in polarizing microscope, 495f 189n, 189f vs . hot spots in microscope, 67f, 68, 68f using genlock, 187f; see also Genlocking ocular field, 99f, 103f, 108 Sync level, 48g Storage device, video tube target as, 193 , 319; see also vs. terntination, in color monitors, 185n Target integration volts, vs. IRE scale, 158f, 159-160, 163f Storage time , and SIN ratio, in human eye , Sin Sync negative/positive, 159 Straightened image, of polytene chromosomes, 363, 364f Sync pulse, 48g, 156-157, 159f, 164f Strain-free lenses, 492- 493 adding to picture signal, 158f Strain-free slides/coverslips, 492 display in pulse-cross, 168f, I69f Streaking, 48g, 234, 251, 254f, 435f driving the monitor, 258-259 Stretching duration of, 157f of gray scale external, voltage level for , 186, 186f, 259 analog, 312f, 313f, 314f, 315f polarity of, 159 with digital image processor, 345, 346f, 349f, 352f, recorded on blank tape , 436 353f, 512 separation of H- and V-, in monitor, 243 INDEX 577 Sync pulse (cont.) Temporal resolution standard voltage, in composite video, 159 vs. noise, trade-off between, 397 adjusted by video processor, 433 as trade-off for spattal resolution, 182 stripping of, with nonlinear amplifier, 180f see also entries under Resolution timing precision Tension roller, 268, 269f and video resolution, 229 TensiOn, on VTR tape, 298, see also Skew; SKEW control between H-and V-, 160 Termination, 49g, 63-64, 185 wave forms, 162f, 163f, 164f of coaxial cable, 63-64, 63f, 64f, 185 see also H-sync pulse; Sync formats; Synchronizing; for composite video and sync signal, 262 Sync signal; V-syn c pulse distortion, caused by improper, 185, 185n Sync-select switch, on monitor, 62 echo, caused by improper, 63, 185 Sync signal, 48g; see also Sync pulse 75-ohm/Hi-Z, 29g, 45g, 63-64, 63f Sync source, 49g, 186 in VCR recording, 64f, 431f, 432f, 433f, 434f, 436, master oscillator as separate, 186 440f special effects generator as, 439, 440f of video monitor, 58, 58f, 62-64, 63f, 64f VTR as, in playback, 431 color, 185n Sync stripper, 49g, 185-186 Test chart: see Test pattern driving video processor, 434f Test equipment, video, 256 in helical-scan VTR, 187 reference books o n, 256n synchronizing two cameras, 185f VTR, 289 Sync tip, 49g, 159f Testis, human, opttcal section of lightly-stained, 414f, 415 voltage, for internal and external sync, 186f, 259 Test pattern Synthalizer, 420-421, 420f; see also 3-D display alignment, 153f System MTF, 125, 177 gray scale, 256 of microscope-video, 474-475, 474f, 475f horizontal resolution, frequency response, 172-175, phase response of, 468-469 174f, 175f Systems analysis, linear, 373- 377, 374f, 375f, 472-475 reference books on, 256n Systolic array processors, 422n resolution and distortion, 248-251, 252f, 253f, 255f, 307f Ball chart, 256, 258f Tape multiburst, 256, 257f drafting, 429 stepped gray wedge, 312f-315f; see also Calibration video: see Video tape curve (video densitometry) Tape-slack indicator, 296 vertical resolution, 170- 171 , 171 f Tape-to-tape copying/ editing, 432-433, 433f, 434f see also Test signal generator Target, 49g Test signal generator, 256 and amplitude response curve, 203 for checking camera, monitor, VTR, 255f, 256 in iconoscope, 3 -4 color bars from, 256 in image isocon, 197, 198f for checking distortion, 248, 255f non-uniform sensitivity of, 225; see also Shading Test specimen, critical, for microscopy, 184f, 405, 409f distortion line scans, with various SiN ratios, 182, 183f of vidicon tube, 191-193, 192f Text, overlays of, 391 microscope image focused on, 59f, 60, 60f, 61f, 100 with special effects generator, 429, 438-441, 440f Target control, automatic, of video camera, 16g, 216-217, Third-field lag, 219-220; see also Lag 233 Three-stage image intensifier tube, 196f; see also !'Vidicon Target electrode, 192-193, 192f Three-tube color camera, 49g, 237, 237f; see also Color Target integration, 49g, 193 camera in iconoscope, 3-4 Threshold, 49g by scan beam hold-off of human eye for in Newvicon, 319 contrast detection, 80-83, 80f, Sin, 8lf, 203 in SIT, 317-319 delectability, vs. resolution, 79 Target material, of vidicons, 193, 194t flicker modulation, 86f Target voltage sensitivity to light, 74, SOn effect on usable light range, 215-216 SIN ratio, modeled for human eye, Sin vs. dark current, in SIT, 216f, 217 Thresholded image vs. light-transfer-characteristic curve, 209 of diatom frustule, cover, 254f of sulfide, vs. other, vidicons, 209; see also Sulfide of eosinophil, crawling, 384-386, 385f vidicon fluorescent microspheres, xxv(f), !Of Tearing, 164; see also Flagging of myocardial cell, fluorescent, 380f, 381 Technology in retrospect, and science, an NSF study, 295, of PtK-2 cell, 355f, 370f 295n Thresholding (analog image), 312, 314f; see also Keying Television: see entries under TV Thresholding (digital image), !Of, 32g, 254f Temperature, 216f of bimodal histogram, 340, 340f vs. dark current, 214f, 215f, 216f, 217, 217f of gray level vs. noise, in electronic circuit, 315, 319 effect of background on, 356, 358f vs. VTR tape, magnetic print-through, 301n for generating binary image, 378- 379 see also Chilling generating edge map by. 380f, 383 578 INDEX Thresholding (dig1tal 1mage) (com.) Time-lapse recordmg (com.) of gray level (com.) track angle for record and playback in, 288 by multidimensional histogram, 379 U-matic cassettes for , 297f in real time , 383 Time resolution Through-focus optical sections of frozen video field , vs. frame , 425 stereo pairs synthesized from, 455n in time-lapse recording, 288 Synthalizer, 420-421 , 420f see also High-speed video vibrating mirror, 421-422, 42lf, 422f Timing pulse/signal, 156-169, 186f Tilting specimen, stereo microscopy by, 447, 448f reference, external, for VTR, 187n Time-base corrector, 49g, 189f in VTR, 187 effect on pulse-cross display, 188f Tissue culture cell: see Living cell (video microscopy) effect on time-lapse recorded video, 189n, 291-293 Titles, video used with broadcast-quality VTR, 293 editing of, 433-436 used in copying video tape, 433 production of, 435-436, 435f used with OMDR, 304 stating conclusions with , 433 see also Time-base error video copying of, 429-432, 43lf Time-base error Tomography shown in pulse-cross display, 187-189, 188f computerized, digital filtering in, 367 in time-lapse recording, 187, 288-289 with linear imaging device in , 227 and VTR recording , 270, 291-293 in electron microscopy, 419 Time code generator, 50g; see also Time code, SMPTE in light microscopy, 418-422 Time code, SMPTE with X-rays, 419 recorded on sound track, 275f Tourmaline, dichroism in, 484-485, 485f, 486f used in video editing, 285 Track angle for locating scenes on tape, 287 on helical-scan recording, 265f, 267 Time constants indicated by scratch on tape, 297f of AHFC , 14g, 187, 259 , 291 for moving and stationary tape, 267-268, 268f of photomultiplier, vs . video camera, 415 in time-lapse recording, 288 Time course on U-format recording, 265f of acrosomal process growth, 319-320, 322f, 323f Trackball, 392 of bioluminescent flash , 396n, 400f Tracking of diffusion of fluorescent dye , 324f automatic, of cell reproduction, 407 of fluorescent flash, 380f, 381 of cell migration, 384-386, 385f, 407 , 417 see also Living cell; Living cell (video microscopy) dynamic, 24g, 285 Time-date generator, 50g proper, in VTR, 286 E-to-E hookup through, 64 Tracking control, VTR, 291 , 29lf, 296, 298 use in VTR recording, 293 , 298 Tracking error, 286n Time-date record Trade magazines/shows, 457, 459t as cue for editing, 437 Transferring video to motion picture, 445-446; see also V• inclusion in VTR recording, 298, 423 to-F transfer Time-lapsed scene Transformation of gray values: see ITF effect of time-base corrector on, 189n Transmittance of light flagging in playback of, 292f through calcite, 133, 483 guard-band noise in playback of, 271f; see also Noise in human eye, 77, 77f bar low, in ground-glass diffuser, 13 2, 138-139 optical copying of, 290, 431 through microscope, 132-133 Time-lapse recorder, 50g, 281 , 289 through polarizing filters, 133 compatibility of, 286, 291 between polars, and cosine-squared law, 483f l-inch, animation, 279t Transparent specimen mode of operation, 288 phase shift by, 121-122 and monitor AHFC, 259 visibility of, 121 noise bar, in playback of, 270, 27lf, 296 Troland, 50g, 77, 8lf, 85f OMDR as , 304 Trypanosoma cruzi, 6, 6f picture breakup, 291 , 291f Tube: see Camera tube; Cathode ray tube; Microscope tube; VCRs, 288-289, 290t Picture tube cassette compatibility, 432n Tube length, 50g partial specifications of, 290t effect on high-NA objective, 134 recording formats for, 296n mechanical, 134, 134f Time-lapse recording, 288 optical, 100, 134n of recorded video tape, 290n reference book on, 134 reducing time-base errors in, 291-293 Tubulin: see Microtubules single field/frame advance in, 279t, 289 Tungsten illuminator: see Lamps special tape for, 297f, 299n, 300 TV broadcast quality, 293 bandwidth limitations in, 154n, 242 time-base error in, 187 vs. human visual system, 92 time resolution in, 288 invention of, 1-4, 5 INDEX 579 TV (cont.) UV (cont.) see also Broadcast TV image converter tube, 5 TV lines PPH, 4lg, 178, 231 , 246, 473g microbeam irradiation, 417, 500 video resolution expressed in , 130, 170 polarized, 483, 484f, 498f, 499 see also Amplitude response curve; Horizontal resolution output of, vs. lamp age, 132 TV set UV dichroism, in DNA , 491 vs. monitor, display on, 242, 259, 260 UV-emitting CRT, flashing of, 417 VTR with RF converter displayed on, 286 UV and IR microscopy, 401 TV standards, for display signals, 158t applications of video for, 401-403 international, 166t UV microscopy, 5 Two images, algebraic manipulation of, 356-358 absorbance image, chromosomes, 401 , 417 Two-stage intensifier Newvicon catadioptric lenses for, 401 low-light-level microscopy, 396-397 flying-spot, 417 sensitivity, noise character, microscopy application, 2301, quantitative analysis using, 5 394t history of, 2, 4-5 luminance level, typical video cameras for, 394t microspectrophotometry, 40 I U format, 50g, 265f, 267n, 280f, 281-293 objective lenses for, l3lt cassette for, 297f Ultrafluar, 401 high-resolution VCRs, 284, 284t of quinacrine mustard-stained DNA, 5 specifications, 275-276 UV sensitivity standard-speed VCRs, 280f, 28lt of eye, 74n, 76f tape path in, 268-269, 269f photography vs. photoelectric, 417 time-lapse VCRs 288-290, 2901 UV vidicon fast search of scenes, 290n sensitivity to UV wavelengths, 40ln tracks on, 265f, 297f spectral response of, 213f, 397n see also U-matic; VCR; VTR UHF connector, 50g, 55-56, 57f; see also Connectors, BNC, UHF Varifocal mirror Ultrafluar objective lens , 401 for 3-D video display, 421-422, 42lf, 422f Ultra-high resolution price of, 422n video system, 283 V blank, 5lg, 153, 159f monitor, 427 and start of next field, 160-161, 16lf, 162f VTR, 2791, 284, 284t V-blanking intervals, 160f, 162f, 163f, 164f non-standard V-scan rates for , 165t, 284 in pulse-cross display, 164-167, 168f, 169f see also High-resolution video VCR, 51g Ultraviolet: see UV automatic playback of scenes, 287 Ultricon , 50g azimuth recording in, 266, 266f, 276 blooming in, vs. silicon vidicon, 221 introduction of, 5 IR sensitivity of, 206n Beta-format: see Beta format light-transfer characteristics, 215f care and maintenance of, 274, 295-297, 299-300 sensitivity, noise, microscopy applications, 230t cassettes for, 268, 269f specifications for, 194t; see also Camera tube capacity of, 276, 279t, 28lt, 282t, 284t, 286, 2901, specifications 294t spectral response curve of, 210f for time-lapse, 286, 290f, 297f, 299n, 300-301 see also Silicon vidicon write-protect button on, 296, 297f, 436 U-matic, 5lg, 267n; see also U format color-under recording used in, 276, 276n U-matic cassette, 297f compatibility, 164, 185, 187-189, 189n U-matic VCR, 280f, 281 of cassettes, 286, 433n high-resolution, 284t custom modification of, 289n specificatiqns, 28lt dew-indicator light on, 287, 296 time-lapse, 290t dynamic tracking in, 285 see also U format; VCR; VTR eject sequence in, 25g , 268 Underscan, 5lg, 167, 170n equipment charcteristics of, 288-289, 290t viewing hidden picture with, 258, 426 fast-forward sequence in , 268 Unterminated input, problems with, 185; see also Hi-Z; field-by-field analysis using, 8f Termination field-by-field playback with, 285 Uranium-glass block, 108f, 109n formats for: see VTR formats Usable light range, 5lg, 125, 215-217 four-head , 1/2-inch, 275f, 282f, 285, 437n in color video camera, 240-241 guard-band noise in: see VTR in intensifier camera tubes, 199t head cleaning: see VTR in photon-counting camera, 197 head gap: see VTR see also Dynamic range helical-scan recording in: see VTR uv high-resolution, 284, 284t aphakics able to focus in , 74n high-speed, 293-295, 294t damage to living cell, 417 history of: see VTR 580 INDEX VCR (cont.) Vertical retrace, 51g; see also Flyback 1/2-inch, standard-speed, 282t Vertical scan, Slg, 152-153, 153f with clean still-field display, 285 Very-high-resolution recording, 2791, 284t with insert editing capability, 437n custom modification of VTR for, 289n 3/4-inch, standard-speed: see U format; U-matic Very-large-scale integrated circuit, 5lg, 513 input to digital image processor from, 293 Very-low-light-level fluorescence microscopy insert edit capability, 437n digitaJ image processing of, !Of linear track, 294t, 295 noise reduction in, 402f PAUSE function in with SIT camera, 324f, 397-401 automatic exiting from, 297, 437 showing lag, 219, 220f editing using: see Editing, assemble V-to-F transfer, 445-446, 445f noise in, 424- 425 commercial, 445-446 for noise-free transition, 298, 437 vs. F-to-V, 430 playback V front porch, 161; see also V-blanking intervals noise as warning sign in, 296, 297f VHS format, 52g, 276, 281 servo control in, 269-270 blank tapes compared, 30 I variable-speed, 279t, 281t, 282t, 284t, 285, 290t dimensions of head gap and video track in, 276 see also VTR, in playback guard bands and azimuth of magnetic fields in, 266--267, portable, 3/ 4-inch, 280f, 286 266f pre-stretching of VCR tape, 293 high-speed recording VCR using, 294-295, 294t recording with standard-speed VCRs with, 282t formats for, 296n time-lapse VCRs with, 289, 290t master log for, 298 VCR operation of, 288n, 298-299, 436-437 with clean still-field, 285, 437n resolution in: see Resolution (VTR) with dynamic tracking, 285 see also VTR, recording v-HOLD control, Slg, 56f, 58 resolution of, 283-284 fine-tuning of, 255-256, 256n, 426 rewind sequence, 268, 296, 437 V- and H-sync signals, separation of, 243 "stand-by" lamps, 48g, 287, 296 Vibrating mirror, 3-D video display, 421-422, 42lf, 422f stopping 'sequence, 268, 437 Vibration, 55n, 145, 497 supply and take-up reel, 269f Video, 52g reversed in 3/4-inch, vs. 1/2-inch, 268n, 269f, 277f early history of, 1-4, 2f, 3f time-lapse, 288-289, 289n, 290t, 291-293 vs. photography monitor for, 259, 291 characteristic curves for, 334 troubleshooting, 299 for fluorescence microscopy, 400 U format, 50g, 265f, 281; see also U format in light microscopy, 309-310 U-matic, 51g, 280f in low-light-level microscopy, 6f variable-speed, clean field display, 285 vs. spot photometer, 381 very-high-resolution, monochrome, 279t, 284 reference books on VHS format: see VHS format color for, 240n, 260n, 261n for video microscopy, choice for, 281 , 284 display devices for, 242n, 245f, 246n, 25ln video/TV select switch ON, 436 principles of, v, 149n, 167-169, 31ln warning lights on, 287, 296 projection for, 443n write-protecting, 296, 297f, 436 test equipment for, 256n see also VTR Video amplifier Velocity bandwidth of, 231 acoustic, anisotropy of, 478, 478f characteristics of, 178-181 , !80f, 311-314 of electromagnetic wave, 479-480, 479f Video analysis, analog, 311, 319-326 flow , of red blood cells in capillaries, 320 of blood flow , 323 of light distance measurement by, 319/-320, 320f, 32lf, 322f, vs. dielectric constant, 479- 481, 480f 323f vs. refractive index, 479-481, 482f, 485-488 of motion, 319-320, 322f Vertical blanking: see V blank photometry by, 320--323, 324f, 325f Vertical field magnetic recording, 51g, 272n Video analyzers: see Image analysis; Image analyzing Vertical frequency, Slg; see also Field rate systems in 525/60 scanning system, 156 Video bandwidth, 17g exact value for NTSC, 161 , 240n vs. horizontal resolution, 175-176, 175n Vertical line scan: see Line scan Video camera, 55, 56f, 57f, 58f, 92, 20lf, 513 Vertical resolution, Slg aperture correction in, 231 vs. adJUStment of v-HOLD control, 426 automatic bandwidth suppression in, 13g, 13ln, 233n of digitaJ signal, 171n automatic iris in, 125, 240 of frozen video field, 425 choice of, for microscopy, 228-236, 230t, 394t vs. horizontal resolution, 170, 264n for contrast enhancement, 405, 410 effect of interlacing on, 172f low -light-level , 396-403 in random interlace, 161 color: see Color camera vs. scan-lines and Kell factor, 171-172, 17lf contrast-enhancement by, 7f, 231-232, 345-346; see vs. scanning spot size and shape, 172 also Contrast enhancement (analog) INDEX 581 Video camera (cont.) Video camera tube: see Camera tube copying projected image with, 430, 431f Video cassette: see VCR , cassettes for coupled to inverted microscope, 137f, 138f, 146f, 496f Video disk: see Disk dual Video editing: see Editing to combine microscope images, 147f, 148 Video enhancement: see Video image, enhancing for stereoscopy, 452f Video field : see Field (video) dynamic focusing in, 24g, 227 Video formats: see NTSC signal; PAL; RS-170; RS-170A; dynamic range of: see Dynamic range RS-330; RS-343; VTR formats dynamic shading correction in, 227 Video gain, 52g; see also Automatic gain control; Gain filtering circuit in, 233, 234, 243; see also Circuits; Video guard-band noise: see Noise bar Filter (video) Video head, 52g; see also VTR, head flare reduction in, IR region, 206n Video image flash exposure with, 126n clean, vs. SIN ratio, 182, 183f, 184f focal plane of, 59, 59f, 60f, 61f digitized, gray level in: see Gray level; Gray value gain control in, 232, 234, 240 displaying of: see 3-D display; 4-D display; Hard-copy AGC , 217, 234 device; Monitor; Pseudocolor display; Raster, dis• gamma compensation in, 209 play devices; Stereoscopic video; Video projector with genlock: see Genlocking enhancing: see Background subtraction; Contrast en• high-definition, 295 hancement (analog); Contrast enhance• high-resolution, 197, 231, 236t, 417 ment/stretching (digital); Digital image processing; high-speed, and recorder, 281, 294-295, 294t Digitally processed image; Edge enhancement; Im• and image processing: see Contrast enhancement (ana- age averaging; Noise averaging (spatial); Noise log); Image processing, analog averaging (temporal); Shading compensation; Shad• instrumentation-type, 32g, 55, 231, 235, 236t ing correction (digital) internal focus of, 59, 62 gray-level histogram of: see Histogram iris of lens, not usable for microscopy, 233n, 240-241 optimum viewing distance of, 442 IR-sensitive, 206n, 210f, 212f, 213f, 397f, 403 photography of: see Photographing PV, 201f pseudocolor transformation: see Pseudocolor display lens of quantitation of: see Measurement; Quantitation (video) removing, for microscopy, 56, 60, 60n, 66, 240-241 storage of special, for microscopy, 60, 6lf, 66, 98f, 137f on magnetic disk, 301-302, 331 vs. luminance levels of microscope images, 394t on OMDR, 302-307 master oscillator in, 231 on tape, 263-301, 432-441 mechanical design of, 235 Video image processor: see Digital image processors 35-mm macro lens, mounting to, 429, 430f (equipment); Image processing, analog, processor monochrome: see Monochrome camera Video imaging device: see Camera tube; Intensifier camera MlF of, 205f, 473, 473f; see also Contrast transfer tubes; Solid-state sensors function VIDEO-IN/ OUT COnnector, 52g, 57f, 58f, 63-64, 63f, 43lf, noise, nonrandom in, 181; see also Noise (video camera) 432f, 433f, 434f, 440f noise reduction Video intensification microscopy, 397; see also Low-light at low light level, 13g, 13ln, 233n level, video microscopy at at power line frequency, 157, 181 Video laser disk player, 303; see also OMDR non-linearity of, real-time correction, 364-365 Video micrographs for publication: see Photographing optical copying with, 430-432 Video micrometer, 320; see also Measurement as optical digitizer, 330f, 332-334, 332f, 363-365, 366f Video microscopy, 52g vs. photomultiplier, 332-334 choice of real-time, 333-334 cameras for, 55, 203, 228-236, 230!, 394t-395t, output, calibration of: see Calibration curve (video 396-403, 405-406, 410, 413-417 densitometry) microscope lenses for, 131!, 136-139 as photometer, vs. gamma of, 209 monitors for, 256-262 random interlace: see Random interlace camera VTR for, 277-295, 281!, 284t red sensitivity of: see Far-red sensitivity in color, 229; see also Color camera, in video response of, to highlight, 66, 234; see also Highlight microscopy sensitivity: see Responsivity; Sensitivity early applications, 4-6 shading distortion: see Shading; Shading distortion enhanced images: see Digitally processed images; Living solid-state, 197-203, 20lf, 235-236, 294n cell (video microscopy) stroboscopic record with, 126n, 294 geometrical distortion in; see Distortion surveillance, 565-56; see also Random interlace camera image lag in: see Lag target of: see Target image processing in: see Video image, enhancing time constants of, vs. photomultiplier, 415 image processors for: see Video image processor usable light range of, 125-126, 215-217, 233-234 inverted microscope for: see Microscope, inverted see also Camera (video); Camera tube; Camera tube journals, frequently with articles on, 457 specifications; Microscopy limited field-of-view in, 136-138; see also Field of view Video camera-microscope system mechanical stability of: see Microscope, inverted; analysis of, 472-475 Vibration combined MTF of, 474f, 475f noise in: see Electrical noise; Noise (video camera); optimizing, 130-131, 136-138, 472-475 Optical noise 582 INDEX V tdeo microscopy (coni. ) Video tape recorders: see VCR; VTR optical sectioning with: see Optical sectioning; Optical Video titles: see Titles, video sections Video tracks optimum magnification for, 130-131, 136-138, 472- in Beta format, 266, 266f, 276 475, 475f in C format, 275-276, 278, 278f zoom oculars for: see Zoom ocular in helical-scan recording, 265, 265f, 266f publication list on, 457n; see also Reference books linear, 264, 264n, 295 concerning; Reference lists in high-speed recorder, 295 shading in: see Shading; Shading distortion in M format, 275f stereoscopic, 420n, 451-455 in segmented recording, 264, 264f systems analysis of, 472-475 in U format, 265f, 268f, 276 see also Microscopy in VHS format, 266, 266f, 276 Video mixer, 52g, 313, 438-441, 439f, 440f, 441f; see Video waveform, 53g, 158f, 164, 167f, 247 also Special effects generator displaying noise, 183f Video monitor: see Color monitor; Monitor hookup for evaluating distortion, 255f Video photometry: see Photometry of multiburs.t pattern, 156, 157f Video picture: see Picture (video) of NTSC color signal, 240, 241f Video processors: see Image processing, analog; Digital for shading compensation, 312, 316f image processors (equipment) Vidicon (sulfide), 52g; see also Sulfide vidicon Video projection, 5, 442-445, 443f, 444f Vidicons, 52g Video projector amplitude response catadioptric, 444-445, 444f curves for, 203, 204f, 205f keystone correction in, 445 percent, at 400 TV lines, 194t light valve, 442-444, 443f, 444f blooming in, 221 , 224 prices of, 444, 444n dark current in, 194t, 199t Video recording: see OMDR; VCR, recording with; VTR, diagram of, 192f recording distortion, vs. deflection magnet of, 227 Video resolution: see Horizontal resolution; Resolution dynamic range of, 193, 214-217 (video) faceplate of, 25g, 191-192, 192f Video response, calibrated using gamma of, 28g, 194t, 209 sine-squared pulse, 256 general description, 191-193 window (broad white bar), 173-174, 175n, 175f, 256 geometrical distortion in, 227 Video scan lines: see Scan lines improvements in, 225f, 427 Video scan rate, vs. flicker, 153-154, 452-455; see also l-inch (25-mm), 38g, 231, 232t Critical flicker frequency introduction of, 4 Video signal, 55, 57f, 149-184 lag in, 220, 222f, 223f bandwidth of, 174-176, 176f, 178-179, 179f third-field, 194t, 219-221, 219f composite, 156-158, 158f, 159f light-transfer characteristics of, 209, 214f-215f EIA standard: see entries under RS- limitng resolution of, 194t, 199t noise in, 37g, 67f, 180-184, 182t, 183f, 184f MTF of, 473, 4 73f in microscopy, 149-158, 150f, 151f noise limitation, vs. intensifier camera tubes, 2 15, 217- Video signal level 219 composite noise from preamplifier of, 195, 217 IEEE scale, 160n percentage shading in, 194t, 225-227 voltage, 192f, 193 responsivity of, 194t, 199t, 203-206 picture signal: see Picture signal sensitivity of, 194t sync signal: see Sync level; Sync tip SIN ratio Video signals, compatibility of: see Compatibility; vs. faceplate illuminance, 217f Incompatibility vs. responsi vity, 217 Video tape specificauons of, 194t; see also Camera tube BR quality, 18g, 293, 301 specifications choice of, 300-301 spectral response of, 194t, i99t, 206, 210f, 213f vs. disk, recording on, 301 target of, 191-193, 192f guard bands on, 29g, 265f, 266f integration in, 49g, 193, 220, 319 KCA, KCS, 297f, 299n, 300-301 material of, 193, 194t, 199t pre-stretching of, 293 types of, 193, 194t, 199t price of, 287 yoke for, 192f, 193, 227 print-through, magnetic, 301n see also Camera tube; Chalnicon; Intensifier vidicons; searching for scenes on, 423n Newvicon; Plumbicon (PbO vidicon); Pyroelectric storage, precautions in, 30 I , 30 In vidicons; Saticon; Silicon vidicon; Sulfide vidicon; stretching of, 30 I Ultricon; UV vtdtcon; Vistacon pre-stretching to minimize, 293 Vignetting of microscope image, 64-66 tests on blanks, JOin Visibility, of phase objects, 115n, 121 , 413n time and date record on, 8 Vision: see Eye; Human eye; Human visual system time-lapsed copy of, 290n Vtstacon for time-lapse recording, 297f, 299n, 300-301 light-transfer characteristics, 215f see also VCR; VTR; VTR formats limiting resolution of, 194t INDEX 583 Vistacon (cont. ) VTR (cont.) specification, typical for , 194t; see also Camera tube in playback (cont. ) specifications clean still-fields in, 289; see also OMDR target material of, I 93, 194t de-emphasis in , 274 Visual acuity , 53g, 77-80, 78f, 79f difficult problems in, 187-188 in central fovea , 77-79 digital enhancement during, 424 vs. stereoscopic acuity, 48g, 91 dynamic tracking in, 285 Visual response, to modulated sine-wave grating, 81-82, external sync for, 285 82f fiducial marks added to signal, 438 Voltage spikes, from arc lamp igniter, 61-62 field-by-field, 285 !-volt peak-to-peak, 158f, 159-160, 159f, 185, 186, 186f field-sequential, 26g V scan: see Vertical scan of high-speed recording, 295 V-scan interval, 156; see also Vertical frequency locating a frame, 302 V-scan rate, 156, 185, 189n; see also Field rate noise in picture, 296; see also Noise bar V-sync pulse, 5lg noiseless still-frame, 278 , 282t, 303 , 306f, 307f added back, during VTR playback, 270 quality of picture recoverable from, 283- 285 , and control pulse, in VTR recording, 267 296 selective filtering of, 243 removal of high-frequency noise in, 274 see also Sync pulse repeated, automatic, of tape parts, 287 VTR servo control needed for, 270 animation skewing in, 46g, 270 , 286 customizing for, 289n speed and format of, 432 l-inch reel-to-reel, 279t and time-base error, 188f, 291-292, 292f; see also animation capability, 187n, 278, 279t, 285n Time-base corrector audio dubbing, 16g, 267, 287 , 298, 436 tracking in, 50g, 286, 291, 29lf and camera outputs, combining, 438 variable-speed , 285 clogged read/write head in, 296, 297f, 299n playback head: see VTR , head comb filter in , 260n portable, 279t, 280f, 286 copying from color to monochrome on , 431 rapid picture search in , 277 , 437n copying different formats on, 290 recording dropout compensation in , 272-274 A- and B-fields in, 274, 274n drum in, 265f, 267, 269, 277f color vs. monochrome, 270 guide rabbet on, 265f, 267, 301 FM, 272-274 editing capability of, 187n helical-scan, 24g , 265-274, 265f, 266f, 269f, 277f external sync in playback, 187n linear track, 264, 264n, 294t, 295 editing deck, 24g, 277, 285-286, 432, 437 of pseudocolor, 283 equipment characteristics of, 279t roll-off of frequency response in, 273f high-resolution, 284t segmented, 264, 264f effect of humidity on , 268 sequential, using pause function, 298 fast-search mode in, 290n , 437n signal level adjusted for, 248 formats for: see VTR formats SKEW control and tape tension during, 298 guard-band noise in, 29g, 270, 271f, 277 , 286n, 296, and time-base correctors, 293 424 time capacity of, 276 , 279!, 281!, 282t, 284t, 290t, guide rollers in, 265f, 267 , 277f, 300 294t head, 416 time-date records in, 293, 298 , 423 cleaning of, 299-300, 300n see also Magnetic recording; OMDR clogged, 296, 297f, 299n reel-to-reel, 268, 274 magnetic field induced in, 270-272, 272f, 273f reference books on , 266n, 267n, 273f, 300n, 30ln head gap , 266, 266f, 272, 272f, 276 reference timing signal, external for, 187n high-resolution, 284, 284t resolution of history of, 5, 295n vs. camera and monitor, 424 l-inch format, 275-276 color vs . monochrome, 270 C-format, 275, 279t limited, 283-284 l-inch, reel-to-reel, 278, 278f see also Resolution (VTR) high-resolution, 279!, 284t searching for scenes in, 423n insert editing: see VTR, editing deck speed search, 277 linear track, 294t, 295 servo controls in , 267 , 269-270 noise bar from, 37g, 270, 27Jf, 286n; see also Guard- signal-processing circuits in, 270-274 band noise SKEW control on: see VTR, in playback, skewing in and NTSC color, 277, 279t, 2811, 282t, 290t skip-field recording/playback in, 46g-47g output of, vs. distortion of highlights, 299 SMPTE time code on, 275 , 275t using PAL, 279t, 281! SIN ratio of, 284t, 285, 290t, 291!, 292t photographing from running, 425 vs. FM recording, 273 pickup head in: see VTR head still-frame capability, 277; see also Freeze frame ; Still pinch roller in, 268 , 269f, 277f frame in playback studio recorders , 277 blurring of digitized image, averaged from, 338 vs. system MTF, 475 584 INDEX VTR (cont.) White (as color), 89, 89n, 90, 238, 238f, 239, 239f, 24lf tape speed, 264, 267, 275-276, 279t, 28lt, 282t, 284t, White balance, 53g, 240 289' 290!, 294t, 295' 432-433 White-balancing filters, 240n vs. pitch of video track, 268f, 288n White clipping, 234 tape-slack indicator, 296 White level, 53g technology vs. science, NSF study, 295, 295n reference, 159f, 162f, 163f tension roller in, 268, 269f White noise threading, 287 in intensifier camera tube, !Of, 181, 184f time-lapse, 281, 288-289, 296n CRO trace, 183f l-inch, animation, 279t from Vidicon preamplifier, 181, 217 mode of operation, 288 White peak, 53g monitors for, 259; see also AHFC White punch, 53g, 234 signal breakup in, 291, 29lf Wide-field specular microscope, 418 sources of noise bar in, 27lf, 288 Wollaston prism: see Prism, Nomarski/Wollaston vs. time-base corrector, 189, 189f Wood, anisotropy in, 478, 478f tracking error in, 286n, 288-289, 291-293, 292f Working distance see also VCR, time-lapse of condensers, 139t see also VCR long, 139t VTR formats of objectives, 13lt, 134f, 135t l-inch; C: see C format recording Working NA of condenser, 109-110, 109f, 115 3/4-inch; U: see U format effect on image resolution, 114-115 112-inch in Koehler illumination, 101 Beta: see Beta format WRITE-PROTECT button, 296, 297f, 436 M: see M format VHS: see VHS format Xenon arc lamp VU meter, audio, 53g, 436 ground-glass diffuser in, 107n with high luminous density, 128t, 129t Warping, 358-363, 360f, 36lf in light-valve video projector, 442-444 straightened image, polytene chromosomes, 362f, 363f optical scrambler for, 127n Water in slit-scanning ophthalmoscope, 418 as immersion medium, 13lt, 134, 135t spectral output, 129t, 418 as solvent, 140, 142f voltage spikes from igniter, 61-62 Waveform monitor, 53g, 164, 167, 183, 325 Xylene, 140 use in VTR recording, 433 , 434f, 440f Wavelength, of magnetic record on VTR tape, 270n Y signal, 53g, 237, 239-240, 240n, 24lf Wavelength response Yoke, 53g, 192f, 193 of camera tubes: see Spectral response curve deflection magnets in assembly, 227 of human eye, 74-77, 76f, 403 far-red , 415 Zemike, F., 119 photopic sensitivity, 74-77, 74n Zoomed image of photographic emulsion, vs. video, 229 of diatom, 254f, 306f Wave optics, 94-95, 96f, 111-118 electronic, 512 and depth of field, 118 Zoom lens, in dissecting microscope, 446 and diffraction, 111-115, 112f, 113f, 114f Zoom ocular out-of-focus, 115-118, 116f, 117f fine-tuning of magnification with, 136 Wave propagation powered, 136-138, 137f, 145n, 147f, 496f electromagnetic, 479, 479f; see also Velocity rifle scopes as, 138, 138f, 145n vs. structural anisotropy, 478 uncoated, hot spots from, 67 Wave, vs. ray, diagram, 94-95, 96f, 102f, 103f, 112f for video, 146f Weber law, 80n, 406 Zoom range, 137, 138f, 146f