Practical Tips for Two-Photon Microscopy

Appendix 1 Practical Tips for Two-Photon Microscopy

Mark B. Cannell, Angus McMorland, and Christian Soeller

INTRODUCTION blue and green diode lasers. To provide an alignment beam to which the external laser can be aligned, light from this reference As is clear from a number of the chapters in this volume, 2-photon laser needs to be bounced back through the microscope optical microscopy offers many advantages, especially for living-cell train and out through the external coupling port: studies of thick specimens such as brain slices and embryos. CAUTION: Before you switch on the reference laser in this However, these advantages must be balanced against the fact that configuration make sure that all PMTs are protected and/or commercial multiphoton instrumentation is much more costly than turned off. the equipment used for confocal or widefield/deconvolution. Given Place a front-surface mirror on the stage of the microscope and these two facts, it is not surprising that, to an extent much greater focus onto the reflective surface using an air objective for conve- than is true of confocal, many researchers have decided to add a nience (at sharp focus, you should be able to see scratches or other femtosecond (fs) pulsed near-IR laser to a scanner and a micro- mirror defects through the eyepieces). The idea of this method is scope to make their own system (Soeller and Cannell, 1996; Tsai to cause the reference laser beam to bounce back through the et al., 2002; Potter, 2005). Even those who purchase a commercial optical train and emerge from the other laser port. To do this, select multiphoton system find that it helps to understand a bit more about filter settings that will allow some of the light from the internal how to optimize the performance of the fs laser system.1 This laser to exit the chosen coupling port. In order to bring two laser Appendix has been added to the Handbook to provide the basic beams to co-linearity, a beam-steering device is essential. A single- alignment and operating information that such people need. mirror beam steerer provides angular control while changing the First, the safety announcement... separation between the mirrors of a 2-mirror steerer provides beam translation (Fig. A1.1). It is also possible to achieve beam translation with a second LASER SAFETY angular control mirror. After adjusting the incoming near-IR beam to an intensity where it can be viewed without totally overwhelm- 2 Light sources for multiphoton microscopy are almost without ing the reference beam, adjust one mirror to make both laser spots exception very powerful pulsed lasers (laser class IV). It is vital merge at the surface of the other (angle-adjustable) mirror. Then that any personnel who perform alignment or other operations that that mirror is adjusted to bring the beams to co-linearity. We find carry a risk of beam exposure are familiar with and follow laser it useful to use a piece of light-blue paper as this shows the dimmed safety regulations. During routine operation one MUST ensure infrared beam well. If the laser has been tuned to the far part of that accidental exposure to the pulsed laser beam is prevented by the spectrum, you may have to use an IR viewer or viewer card to providing proper shielding and interlocks. visualize the beam. During alignment, protective eyewear is not an option — it is essential! See http://www.osha.gov/SLTC/laserhazards/ for US TESTING ALIGNMENT AND guidelines. SYSTEM PERFORMANCE

On a regular basis and particularly subsequent to laser alignment, LASER ALIGNMENT the performance of the multiphoton microscope should be tested. The prime indicator of proper alignment of an imaging system is Just as in any other type of microscopy, correct optical alignment its point-spread function, as measured by using a sample contain- is crucial for achieving optimal, diffraction-limited performance in ing sub-resolution ﬂuorescent beads. A test slide can be prepared 2-photon microscopy. The alignment of external lasers such as the by letting a drop of diluted beads dry onto a coverslip. The beads Ti:S or similar 2-photon sources into a laser scanning microscope are then embedded in a drop of Sylgard elastomer (Dow Corning, can be simpliﬁed if a well-aligned “internal” or reference laser is USA) with a microscope slide placed on top. We usually use available. In commercial confocal microscopes, typical candidate 0.2mm beads from Molecular Probes (Eugene, OR). These are lasers include Argon-ion or green HeNe lasers or, more recently, available in a range of colors suitable for 2-photon microscopy. It

1 The Multiphoton Users Group e-mail network at ·mplsm-users@ 2 As is explained below, this can be achieved by over-closing the slit and/or yahoogroups.comÒ, operated by Steve Potter at Georgia Tech, enrolled its reducing pump power, because mode-locking is not required. We typically 500th member in 2003. use <20mw @ 800nm and <10mW at 720nm.

Mark B. Cannell, Angus McMorland, and Christian Soeller • Department of Physiology, FMHS, University of Auckland, New Zealand

900 Handbook of Biological Confocal Microscopy, Third Edition, edited by James B. Pawley, Springer Science+Business Media, LLC, New York, 2006. Practical Tips for Two-Photon Microscopy • Appendix 1 901

AB C

FIGURE A1.1. (A) 2D simplification of the beam alignment process using a conventional beam-steerer. A vertical translation of a tilted mirror is used to bring the two beams to a common point on a second, tiltable mirror. (B) Rotation of the second mirror at the point of the common spot makes the two beams co-linear. (C) The co-linear beams after alignment. takes only about 30 minutes to prepare 10 slides in this way. Once ticularly if the system is to be used for 2-photon flash photolysis the elastomer has set, these slides will last for months if kept in a or combined confocal and multiphoton co-localization studies. dark drawer. As a result, they provide a good standard to check the In our laboratory we perform a basic system test with a pre- microscope sensitivity and resolution provided you have recorded pared bead sample on a daily basis. This check (usually conducted microscope and laser settings (including center wavelength, laser following system startup) is well worth the ~5 minutes it takes, power and bandwidth/pulse length) with each reference image. especially if it helps avoid debugging signal problems later when With proper alignment, the beads should blur approximately a precious biological sample is on the stage. evenly as you focus above and below them. Asymmetric blurring above-and-below best focus indicates spherical aberration while motion of the centroid of intensity means that the objective LASER SETTINGS AND OPERATION aperture is filled asymmetrically. The spatial resolution (without a pinhole) should be similar to confocal performance, values Historically, the mode-locked lasers used for 2-photon imaging between 0.2–0.4mm in plane, full-width at half maximum could be quite temperamental and ensuring that proper laser oper- (FWHM) and 0.5–0.8mm out of plane (in the z direction) should ation was a large part of the challenge of running a multiphoton be attainable when using a high-numerical-aperture (NA ~1.3) microscope. With the advent of fully computer-controlled turn-key objective. laser systems, this has become less of an issue. In any case, as the A very weak and noisy signal can have a number of causes. If most versatile source for 2-photon imaging is still the tunable there is no problem with the detectors or emission filters (most of Ti:S laser in the femtosecond configuration, we will focus on it which would also be apparent when operating the microscope with here. Regardless of whether you are using a fully automated or a conventional [1-photon] laser excitation), check that the laser manually adjusted Ti:S system, it is important to monitor and beam fills the objective rear aperture fully and evenly by rotating optimize the laser output before imaging. the objective turret to an empty position, placing a lens tissue over The choice of center wavelength is generally determined by the opening and inspecting the pattern of illumination (using an IR the fluorochromes to be excited. As a general rule of thumb you viewer if necessary). The beam should be accurately centered in should try to use the longest wavelength compatible with the dyes the empty socket and should form a uniform circle of light that in your sample as this will help minimize photodamage and also will cover the rear aperture (~8–10mm wide) of a typical objec- reduce scattering of the excitation light. Data on excitation spectra tive lens. If the light intensity at the rear aperture is low (<10mW) is now available from many sources in the literature and, if in make sure that no IR-opaque optical items are obstructing the illu- doubt, there are mailing lists where one can ask other researchers mination path.3 It is also possible that the beam is so badly mis- for advice (see http://groups.yahoo.com/group/mplsm-users/ and aligned that only scattered light is being observed. You can check http://listserv.acsu.buffalo.edu/archives/confocal.html). for this by ensuring that adjustments of the alignment mirrors have the expected effects on the spot in the BFP. If the microscope is a combined confocal/multiphoton system, MONITORING LASER PERFORMANCE the bead slide is also a useful tool to disclose alignment offsets between the 2-photon laser system and any other lasers. In partic- During tuning and imaging, laser operation can be very conve- ular you should check for any axial offsets (i.e., focus shifts), par- niently monitored using a spectrum analyzer. We use a system made by Rees Instruments (currently available models include the Rees E200 series laser spectrum analyzers by Imaging and Sensing Technology Ltd., Alton, UK) to monitor a secondary beam con- 3 If little light is coming out of the objective, it may be the anti-reflection coatings that are at fault. Coatings used to reduce reflection losses in the visible taining only a small fraction of the total output power. During laser may become mirrors in the near-IR. See the transmission tables in Chapter 7 tuning, this device allows one to measure the center wavelength and its Appendix. and, more importantly, the width of the spectrum. The spectral 902 Appendix 1 • M.B. Cannell et al. width of the beam, as displayed by the analyzer, provides the feed- approximately Gaussian-shaped output spectrum which may have back for optimizing the slit width and position to obtain mode- a spike (Fig. A1.2B) indicating CW breakthrough. Optimal closure locked operation (with manually tuned laser systems). The start of of the slit leads to a smooth Gaussian-like spectrum (Fig. A1.2C) mode-locked operation is indicated by the change of the spectral which, in this case, is ~5nm wide (FWHM). At 750nm this spec- shape from one or a small number of sharply defined lines which tral width implies a 120fs pulse. Closing the slit further can lead indicate continuous wave (CW) operation, see Figure A1.2A, to an to an oscillation of pulse amplitude (Q-switching), which is shown

E F

G FIGURE A1.2. (A–F) show spectrum analyzer output during Ti:S tuning. The small gradations at the bottom indicate 1nm. (A) Before mode-locking, the spectrum consists of a few narrow spikes. (B) With mode-locking underway, the spectrum increases in width, but the spike indicates CW breakthrough. To cure this, the slit needs to be closed more. (C) Optimal operation, the slit has been closed just enough to stop CW but at the same time not so closed that Q-switching starts, a mode of behavior shown in (D,E). To stop Q switching, more prism must be inserted into the beam path (which will increase system bandwidth) and/or the slit needs to be opened (or even a reduction in pump power). (F) shows the shortest pulse that can be readily achieved with our Coherent MIRA 900F system. The FWHM of the spectrum is ~14nm at a center wavelength of ~750nm. (G) shows the relationship between the FWHM of the spectrum and the pulse width for a transform-limited sec2 pulse, with center wavelength indicated next to each curve. In this case, a 14nm bandwidth from our laser (F) implies a very short pulse width of 40fs. Generally, we use longer pulses (~120fs e.g., C) than this in imaging experiments. Practical Tips for Two-Photon Microscopy • Appendix 1 903 in the spectrum as oscillations (Figs. A1.2D, A1.2E) and should be laser beam as a reasonable compromise between filling the real removed by re-opening the slit or increasing the intra-cavity group aperture adequately and throughput. (We built a simple expander velocity dispersion by moving the intra-cavity prism further in. By from a plano-convex and a plano-concave lens which were single- suitable adjustment of the slit and the intra-cavity group velocity layer antireflection coated.) In addition, by focusing the beam dispersion, the pulse may be shortened and this will be reflected expander carefully, it is possible to minimize the axial shift of focal in an increase the width of the output spectrum (Fig. A1.2F). plane between visible light and the IR. With our laser, a 14nm FWHM bandwidth can be achieved corresponding to a ~40fs pulse at 750nm. During imaging, Q- switching manifests itself as a sudden increase in image noise due CHOICE OF PULSE LENGTH to aliasing between laser excitation and the pixel clock. A quick The dispersion of the pulse by the microscope optics is typically look at the spectrum should indicate if the laser needs tuning to >2000fs2 at 800nm. This suggests that the shortest pulse width that remove this source of image noise. can be delivered to the sample would be >100fs unless group Typically, pulses leaving a commercial Ti:S laser, as used for velocity dispersion compensation is performed to “prechirp” the 2-photon microscopy, are ~100fs long. Pulse length is an impor- pulse (Soeller and Cannell, 1996). Shorter pulses increase the ratio tant variable that is most accurately determined with an optical of 3- to 2-photon excitation and, since 3-photon excitation at autocorrelator. However, from a practical point of view, a spec- 800nm would correspond to hard UV, such excitation is generally trum analyzer is easier to use than an autocorrelator and gives suf- undesirable. We therefore suggest that for routine operation ficient information on laser performance. The length of the laser ~120fs pulses are probably optimal. Perhaps paradoxically, in the pulse is inversely proportional to the spectral FWHM during absence of GVD compensation, a shorter pulse at the laser is trans- mode-locked operation. Figure A1.2G shows this relationship for lated to a much longer pulse at the sample. As it is hard to run a various center wavelengths. conventional Ti:S laser with pulses longer than ~150fs, longer pulses at the sample may be produced by making very short pulses (e.g., 40fs) at the laser. See Chapters 5 and 28 for further discus- POWER LEVELS AND TROUBLE-SHOOTING sion on pulse broadening. In our experience illumination power levels at the sample should be kept <20mW in living cells to minimize the risk of cell damage, CONTROLLING LASER POWER although that figure is dependent on the nature of the experiment, the 2-photon absorber, the objective NA, and sample scattering. Being able to control laser power electronically is useful because Problems with mode-locked lasers in 3D microscopic imaging it permits rapid suppression of the beam at the end of each scan most often arise from: line where the beam slows and stops before retracing its path. This slow movement subjects the parts of the specimen at either side of 1. Pump laser noise (amplitude noise or beam-pointing the raster to very high integrated excitation which is very damag- instability). ing. Unfortunately, the acousto-optic modulators (AOM), which 2. Pump laser alignment. are commonly used for this purpose in visible light microscopes, 3. Dirt on mirrors. are less suitable for 2-photon because heating and birefringent 4. Poor alignment within the cavity. effects in the crystal reduce beam intensity stability. The square- 5. Stray reflections from surfaces that reflect energy back into law dependence of 2-photon excitation on input power amplifies the cavity. this instability at the sample. Additionally, because the mode- 6. Poorly trained personnel changing the alignment between the locked laser beam has significant bandwidth (compared to a CW pump and the prisms of the Ti:S cavity over time. laser) the beam will be dispersed if it is diffracted in the AOM. As 7. UFM (unidentified fingerprints on mirrors!). the rear aperture of the objective must be overfilled, this disper- 8. Air currents that affect beam-pointing stability. sion results in a loss of bandwidth and therefore a longer and mis- 9. Loss of alignment of laser to microscope. shapen pulse. This effect can be avoided if one uses the zero-order 10. Poor matching of laser beam profile to microscope aperture. (i.e., undiffracted) beam of the AOM for microscopy and the first To address problems 1–4, the manufacturer generally provides order beam is used simply to extract energy from it. However, as troubleshooting advice that should be consulted. Problem 5 can be only about 75% of the beam can be diffracted out, this approach avoided by using an optical isolator, i.e., a device which allows only reduces the beam to 25% of the input power. light to pass only in the forward direction but blocks back reflec- A better alternative is to use a Pockels cell. While more expen- tions. A simpler workaround (that has worked well in our hands) sive, these devices are much faster and more controllable than an involves slightly tilting strongly reflecting surfaces (e.g., neutral AOM, but they also suffer from some problems: density filters — see below) with respect to the optical axis. For 1. The Pockels cell has a limited lifetime that is dependent on the laser safety you should provide an appropriate beam dump for any time spent in the energized state. strong reflections off the optical axis. Problems 6 and 7 should be 2. Alignment is critical: the full power of the beam must pass resolved by the system manager. Problem 8 can be reduced by sur- cleanly through the free aperture and not touch the interior of rounding all beams with plastic tubes. Problem 9 can be ascer- the cell under any circumstance or damage will result. tained using a reference laser, especially a laser built into the 3. High voltages are present. microscope itself. Problem 10 arises from the laser beam being too small to fully fill the objective rear aperture (so a loss of resolu- It should be noted that, for ~120fs pulses, dispersive broadening tion occurs) or too large, in which case there is a loss of intensity by the Pockels is generally small and should therefore be of no at the sample. In both cases, the problem can be fixed using laser concern when it is used in a 2-photon imaging setup. beam expansion (or compression) with a telescope (Galilean beam If rapid beam modulation is not needed, laser power can be expander). In our microscope we use ~4x expansion of the Ti:S controlled by neutral density filters or a polarizer. Such neutral 904 Appendix 1 • M.B. Cannell et al. density filters need to be of the reflecting type as high powers LASER POWER ADJUSTMENT FOR IMAGING destroy absorbing filters. The beam reflected from the filter needs AT DEPTH to be absorbed by something for safety and we use a “beam dump” made of black anodized aluminum with a machined recess so it is Although 2-photon excitation penetrates deeper into scattering hard to see the dumped beam. Since the output beam of the laser samples (such as brain), the loss of peak excitation power at the is polarized, beam intensity may be modulated by rotating a polar- focus caused by scattering and spherical aberration still leads to a izer in front of it. Glass Glan-Thompson polarizers can be used but loss of signal at depth. The solution to this problem is to alter the plastic polarizers are quite unsuitable for typical power levels as illumination power as a function of depth and this is where the they melt (see also Attenuation of Laser beams in Chapter 5, this intensity modulation provided by the Pockels cell may be used volume.)! to advantage. There are alternative ways to achieve changes in illumination power but all assume that the maximum power available from the 2-photon laser is higher than is needed for AM I SEEING TWO-PHOTON EXCITED normal operation. Thus, a wheel of reflective neutral density (ND) FLUORESCENCE OR . . . filters may be placed in the beam path, providing intensity control to quantized levels appropriate for different imaging depths. Sometimes it is unclear if a detected signal is due to multiphoton- A second option is to use a continuously variable reflective excited fluorescence or if it is due to optical bleed-through of the neutral density filter, which allows more precise control over laser (much more intense) near-IR excitation light. Such bleed-through power, but requires either manual rotation during imaging or a can occur, for example, if one uses filters with an unknown motorized filter wheel. We suggest that the ideal solution is to response in the near-IR region. A simple test to distinguish between automatically attenuate the laser beam. using a Pockels cell these possibilities can be made by taking a control image with the supplied with a varying drive voltage controlled by the focus multiphoton laser source running in CW mode (at similar power). position. When using a mode-locked Ti:S laser with manually operated slit In our experience, the laser power needs to increase (roughly) this can easily be achieved by over-closing the slit until mode- exponentially with depth (e.g., see Fig. A1.4 in Soeller and locking is lost and then reopening the slit with the starter mecha- Cannell, 1999) but the exponential factor is highly dependent on nism disabled. If the signal in question disappears when using CW the sample. Thus a control experiment may be needed where a illumination, it must be due to some sort of multiphoton excitation similar sample is labeled with fluorescent beads (~2mm in diame- (2- or 3-photon fluorescence, or second- or third-harmonic gener- ter). For brain slices, or other tissues which can be perfused, this ation). However, this simple test does not replace the more complex can be achieved by injecting the beads into a blood vessel before illumination-power vs. signal-intensity measurements needed to slicing. By imaging the beads at different depths, the depth depen- fully characterize each of these high-order excitation processes. dence of the excitation may be determined and used in subsequent experiments. (Using beads will give more reliable results than simply staining the entire specimen with a dye as this avoids prob- STRAY LIGHT AND NON-DESCANNED lems arising from non-uniform staining.) It is important to note that not all the signal loss is due to reduced excitation as emitted DETECTION light is also lost by scattering and adsorption. Thus, even if complete compensation of signal loss with depth can be achieved by One of the attractions of 2-photon microscopy resides in the raising excitation power, it is better to err on the side of caution as improved penetration depth obtained when imaging in strongly delivering too much power into the preparation at any depth may scattering biological samples such as brain slices (see Soeller and lead to other concerns — for example, heating and other higher- Cannell, 1999). Central to this advantage is the need to collect order effects (see Chapter 38, this volume). If 100mW were deliv- emitted photons that are also scattered and so may not be focused ered and (eventually) absorbed within 10mm3 of tissue, the by the microscope optics and are therefore lost at intermediate average rate of rise of temperature would be 2.5oC/s. Although this apertures. This problem can be overcome by using a photomulti- power is close to the maximum that may be achieved by typical plier tube that is mounted close to the sample (so that the emitted 2-photon microscopes, it is clearly in the range where heating light does not pass through the scanning system) to create a “non- effects could become a serious problem. descanned detector.” Such detectors are arranged so that any photons of the right color, regardless of where they originate, are directed onto the photocathode. As a result, non-descanned detection is also far more likely to pick up stray light from the SIMULTANEOUS IMAGING OF microscope surroundings than conventional confocal optics. For MULTIPLE LABELS example, in a normal laboratory, light from computer screens and equipment LEDs can cause a strong background signal even when Another advantage of 2-photon excitation is that the 2-photon exci- the room lights are turned off. To shield your setup from this stray tation spectra of fluorochromes are wider than their 1-photon coun- light, you may need to fabricate suitable shields around the sample terparts. Multiple labels may therefore be imaged simultaneously from black material. Alternatively, you may shield the whole by using a single excitation wavelength and multiple detectors with microscope from the surroundings by enclosing it in a completely appropriate optics to isolate each different emitted wavelength. light tight box. This can be conveniently combined with electrical This approach has several benefits: (1) Removal of offset problems shielding by providing a Faraday cage around 3 sides and the top caused by non-confocality of different lasers; (2) reduction in of the instrument and fully closing it during imaging by drawing imaging time (which may be important for imaging of live-cell a black curtain or blind across the fourth side. For safety reasons, processes); (3) reduction in the total amount of laser exposure to this cage and any blinds or curtain should be made from fire-proof the tissue and (4) avoidance of chromatic aberrations. Care must materials. be taken to ensure that bleed-through from one channel to another Practical Tips for Two-Photon Microscopy • Appendix 1 905 is minimized by the use of the optimal beam-splitters (see also and size of UV lasers. 2-photon excitation of UV dyes does not Chapter 3, this volume). If some bleed-through is unavoidable then suffer from these problems because the excitation wavelengths are an accurate measurement of the amount of bleed-through can be near-infrared, in a range that is compatible with normal optics. made by imaging, in all channels, control slides that contain the The ability to use UV dyes allows more labels, and colors, to be individual fluorochromes. From these measurements, contribu- used in multiple-labeling experiments. In addition, combining a tions from bleed-through from one channel to another can be esti- UV-excited probe emitting in the blue part of the spectrum mated and removed by subtraction during post-imaging analysis allows greater spectral separation from a yellow-red label. UV (so-called “spectral unmixing”). dyes, in general, may be excited by 2-photons at wavelengths £750 nm. For example, the AlexaFluor 350 fluorochromes (Molecular Probes, Eugene, OR) come in a range of forms. The near- MINIMIZE EXPOSURE DURING ORIENTATION UV-excited nucleic-acid probes DAPI and Hoechst are often so AND PARAMETER SETTING well excited using 2-photon illumination that it is necessary to use very low concentrations to prevent bleed-through into other In most applications, imaging parameters need to be established channels. by trial and observation prior to the commencement of image acquisition. Common examples are scanning across tissue looking for “that cover image” and then establishing the upper and lower limits of a volume of interest. While the use of 2-photon excita- ACKNOWLEDGEMENTS tion prevents photobleaching above and below the focal plane, in- plane photobleaching can be severe and care must be taken to We would like to thank Tim Murphy (University of British avoid over-exposure of samples to illumination light during these Columbia, Vancouver, CA) for helpful comments on the adjustment procedures. manuscript. The key is to think before imaging. For example, if the sample needs to be located in focus, is full power really necessary or will the detection of just a few photons be sufficient? It follows that during setup, the detector gain should always be set high and laser REFERENCES power as low as possible. Single scans should be used in prefer- ence to continuous scanning. Can the sample be moved to an unex- Potter, S.M., 2005, Two-photon microscopy for 4D imaging of living neurons. posed region once the acquisition parameters are set? Once the In: Imaging in Neuroscience and Development. A Laboratory Manual, (R. correct settings have been determined, then laser power can be Yuste, and A. Konnerth, eds.), pp. 59–70, Cold Spring Harbor Laboratory increased for actual imaging and focal-plane bleaching indicates Press. that the maximum amount of information available has been Soeller, C., and Cannell, M.B., 1996, Construction of a two-photon microscope extracted from the dye in the sample. and optimisation of illumination pulse width. Pflugers. Archiv. 432: 555–561. Soeller, C., and Cannell, M.B., 1999, Two-photon microscopy: Imaging in scattering samples and three-dimensionally resolved flash photolysis. Microsc. ULTRAVIOLET-EXCITED FLUOROCHROMES Res. Tech. 47:182–195. Taal, P.S., Nishimura, N., Yoder, E.J., White, A., Doluick, E., and Kleinfeld, The use of ultraviolet (UV) excited dyes in 1-photon imaging is D., 2002, Principles, design and construction of a two-photon scanning restricted by the opacity of conventional optical components at UV microscope for in vitro and in vivo studies, In: Method for in vivo Optical wavelengths as well as by chromatic aberrations and by the cost Imaging, (R. Frostig, ed), CRC Press, pp. 113–171. Appendix 2 Light Paths of the Current Commercial Confocal Light Microscopes Used in Biology

James B. Pawley

INTRODUCTION chance that such data would go out of date with their next product announcement. However, I tried to apply the same criteria to all Since biologists became aware of the confocal microscope in the the contributors and this is as good a place as any to thank the man- late 1980s, numerous optical designs have been introduced by ufacturers for their splendid cooperation. manufacturers to try to meet the often-contradictory requirements To assist the reader, some of the optical information consid- of the biological microscopist. Although many of these designs are ered most relevant to the optical performance of these instruments discussed at greater length in other chapters of the Handbook, it has been collected in Table A2.1. Although such a table cannot was thought that it might be both useful to the reader, and fairer contain all of the relevant information about such complex instru- to those designs not discussed elsewhere, to provide the reader ments, the headings have been chosen to reflect those specifica- with a concise compilation of all the designs now available. tions indicated to be of prime importance in the other chapters of To that end I requested optical diagrams and tabular informa- the Handbook. Abbreviations are explained in the footnote. tion from all of the major suppliers of the instruments used by biol- Of course, the manufacturers are correct about this informa- ogists for 3D microscopy1 and the items that they provided make tion going out of date. Fortunately the WWW is now there to bring up the bulk of this Appendix. Often manufacturers were hesitant you up to date. Even when the models are all different, we hope to provide specific information about details such as PMTs or scan- that the you find the column headings in the table of optical param- ning speeds etc., because they realized that there was a good eters useful as the basis of questions you might ask about future models. There has been no effort to compare the computer operating systems used to control these instruments. I wish to emphasize that 1 We have neglected to include any information on the systems for widefield/ this is not because I think such details unimportant, but rather deconvolution only because the optical paths of such systems are fairly because software systems tend to change with great speed and, straightforward, and not in need of explanation. in addition, operating systems are probably best assessed in person.

James B. Pawley • University of Wisconsin, Madison, Wisconsin, 53706

906 Handbook of Biological Confocal Microscopy, Third Edition, edited by James B. Pawley, Springer Science+Business Media, LLC, New York, 2006. Light Paths of the Current Commercial Confocal Light Microscopy Used in Biology •Appendix 2 907

FIGURE A2.1. Schematic of the BD-CARV II light path. The variable intensity light from a Hg/metal halide light source passes through an excitation filter before being defleted by a dichroic mirror towards the sample. The excitation light passes through a Nipkow spinning-disk containing multiple sets of spirally-arranged pinholes placed in the intermediate-image plane of the objective lens. The column of excitation light is projected through 1000 pinholes to simultaneously scan the entire field once every millisecond, thereby creating a full image of the focal plane in real-time. The emitted light passes through the dichroic mirror and the emission filter before either entering the CCD camera or the binoc- ular eye-piece. The pinhole disk can be moved in and out of the light path to produce a confocal or a widefield fluorescence image. A variable slit at the image plane can be used to selectively illuminate an area of the sample allowing Fluorescence Recovery After Photobleaching (FRAP) to be performed. All movable parts including the filter wheels, spinning- disk shutters, and mirrors are automated and are controlled via touchpad or third-party software. Figure kindly provided by BD-Biosciences, (Rockville, MS).

FIGURE A2.2. Schematic of the LaVision-BioTec TriM-Scope light path. Multifocal multiphoton microscopy using a beamsplitter built with flat optics. Light from a fs, near-IR, pulsed laser first passes a polarizing attenuator and a beam-expander before entering a pre-chirp compensator. It is then formed into as many as 64 beams of equal intensity and spacing by being reflected from an array of sliding, planar, optical elements. The linear array of beams is then deflected by 2, closely-spaced galvanometer mirrors and fed into the microscope by being reflected off a high-pass beam-splitter. Two-photon-excited fluorescence from any dye located at the focus plane of the objective passes through the short-pass dichroic, and barrier filters to a CCD camera or other photodetector.2 Because of the large number of parallel beams and the high-QE of the CCD camera, it is possible to obtain useful, optical-section images at up to 3.5k frames/second and, because the system relies on 2-photon excitation, bleaching is restricted to the focal plane. For more discussion see Chapter 29, this volume.

2 T. Nielsen, M. Fricke, D. Hellweg, P. Andresen, (2001), High efﬁciency beam splitter for multifocal multiphoton microscopy, J. Microsc., 201:368–376. 908 Appendix 2 •J.B. Pawley

Table A2.1. Optical Parameters of Current Commercial Confocal Microscopes Scanned Retrace Fastest ﬁeld, Largest protect/Laser Fiber Beam line scan, Pixel diam. int Raster Company Model Lasers/Arc atten optics Pre-optics expander Scanner Hz times im. plane Pixel

BD- CARV II X-Cite 120 NA/intensity Liquid- NA NA Single-sided 1 k fps ~1 ms21mmCCD Biosciences Hg/halide controlled by ﬁlled light Petran disk 5 k rpm arc, 8-place aperture guide ﬁlter wheel Lavision- TriMScope2 Ti-sapph NA/ Laser is NA Yes 1–64 beams 3.5 k fps > 500 ns 20 mm, CCD Biotech 750–100 nm, Attenuator coupled scanned by 2 adapted to 100 fs pulse 0.1–100% directly galvos CCD used

Leica TCS SP2 AOBS Many, Yes/3 SM-PP Laser-merge Adjustable Rotatable k-scan 1.4 k or >500 ns 22 mm 4096 ¥ 4096 351–633 nm AOTFs 2.8 k in bi-direct

MP RS Ti-Sapph Yes, EOM SM-PP Laser-merge Adjustable Rotatable k-scan 4 k, or 8k >500 ns 22 mm 4096 ¥ 4096 in bi- direct

Nikon C1-plus Up to 3, AOM (opt) SM-PP Laser-merge Fixed 2 close galvos 500, 1k >1.68us 17 mm 2048 ¥ 2048 408–638 nm in bi- @512 ¥ direct 512

C1si Up to 3, AOM SM-PP Laser-merge Fixed 2 close galvos 500, 1k From 17 mm Up to 408–638 nm laser input in bi- 4.08us w/scan 512 ¥ fiber. MM direct at rotation 512 in emission 512 ¥ 512 spectral fiber in mode spectral mode Olympus FV 300 Many, Yes, AOTF SM-PP Laser-merge Fixed 2 close galvos 1 k or 2 k >2 ms 20 mm 2048 ¥ 2048 405–633 nm bi-direct IR port FV 1000 Many, Yes, AOTF SM-PP Laser-merge Fixed 2 pair of close 2 k or 4 k >2 ms 18 mm 4096 ¥ 4096 active galvos, separate bi-direct stabilizer, image/bleach 351–633 nm, scanners, IR port circular bleach DSU Hg arc NA/intensity NA NA NA Interchangeable 3 k rpm, >1 ms18mmCCD controlled by single-sided slit- 15 fps aperture pattern disk, 3 k rpm Visitech VT-infinity Many, Yes, AOTF SM-PP Laser-merge NA Single galvo 2 kHz 2 ms17CCD 405–647 njm (opt) scans an array of point sources VT-Eye Many, Yes, AOTF SM-PP Laser-merge Fixed 1 galvo, 1 AOD 50 kHz 20–125 ns 1024 ¥ 1024 351–647 nm (opt)

Yokogawa5 CSU 10 2 or 3 lines6 NA/AOTF SM Laser-merge Fixed Double Petran 1800 rpm, ~1 ms 13 ¥ 9.5 mm CCD 3.5 mm Disk w/micro- 360 fps core lenses

CSU 22 3 or 4 laser NA/AOTF SM Laser-merge Fixed Double Petran Variable, ~1 ms 13 ¥ 9.5 mm CCD lines 3.5 mm Disk w/micro- to 5 k core lenses rpm, 1 k fps Zeiss LSM510META Many, Yes, AOTF SM-PP Laser-merge Adjustable 2 close galvos 1.3 k or 640 ns– 18 mm 2048 ¥ 351–633 nm 0.05–100% 2.6 k in 2.3 ms 2048 (Ti : Sapph) (AOM) bi-direct

LSM 5 Pascal Many, No/ SM-PP Laser-merge Adjustable 2 close galvos 1.3 k or 640 ns– 18 mm 2048 ¥ 2048 405–633 nm Mechanical 2.6 k in 2.3 ms attenuator bi-direct 0.05–100% LSM5-LIVE Many, Yes, AOTF SM-PP Laser-merge Adjustable 1 galvo (>60 k) 16 ms– 18 mm 1024 ¥ 1024 405–635 nm 0.05–100% Cylindrical line-scan 120 fps, 20 ms 512 ¥ 512, 1010 fps, 512 ¥ 50

1 Record transmitted light through disk. 2 As the TriMScope is actually a multi-focus multiphoton ﬂuorescence illuminator with wideﬁeld detection onto a CCD, its performance depends a great deal on the performance of this device. 3 These numbers assume that the tube mag is 1¥. Light Paths of the Current Commercial Confocal Light Microscopy Used in Biology •Appendix 2 909

Table A2.1. (Continued)

Zoom Tube Beam Beam- Pinhole Spectral Reﬂected/ range mag dump splitter alignment Pinhole range selection Photodetector Channels transmitted Digitizer z-motion

NA 1.1¥ NA 5-place Self- Fixed 70 mm, 8-place ﬁlter CCD or EM- 2-camera port No/yes1 CCD Piezo dichroic aligning 180 m spacing wheel CCD ±100 nm wheel (opt)

ROI for 1¥ NA Short-pass NA 8-place filter CCD or EM- 3-camera port Yes/no CCD, Stepper CCD mode dichroic (multiphoton wheel, CCD, 1–32 (opt ion, motor, excited spectrometer PMT array PMT, (peizo only) (opt) 12-bit) opt) 32 : 1 Yes Acousto- Preset Common Prism, 4 PMT, 8 Yes/yes 12-bit Galvo, Optic pinhole, adjust motorized cooling ±40 nm 20–800 mm mirrors option, APD option 32 : 1 Yes Acousto- Fixed Common Prism, 4 PMT, 8 Yes/yes 12-bit Galvo, optic pinhole, adjust motorized cooling ±40 nm 20–800 mm mirrors option, +2 non- descanned infinite 3.8 Yes Dichroic, Fixed Common Replaceable 3 side-window 4 Yes/yes 12-bit Stepper, changes w/focusable, pinhole, filter cubes fiber-coupled ±50 nm with cube alignable 30, 60, 100, PMTs pinhole lens 150 mm3 infinite Dichroic, Fixed Common 3 diffraction 32 element 32 acquired Yes/yes 12-bit Stepper, changes w/focusable, pinhole, gratings for multianode simultaneously 50 nm with cube alignable 30, 60, 100, 2.5 nm, 5 nm, PMT increments pinhole lens 150 mm4 and 10 nm channel width

10 : 1 3.42¥ Yes Dichroic, Common 5 sizes Dichroic filter 3 PMTs, 2 3, 2 fl, 1 trans Yes/yes 12-bit Stepper, (infinity) cubes, 2 pinhole, cube fluor, 1 trans ±10 nm positions alignable 50 : 1 3.82¥ Yes Dichroic Common adjust 2 diff-grating 5 PMTs, 2 5, 4 fl, 1 trans Yes/yes 12-bit Stepper, (infinity) wheel, 6 pinhole, 50–800 or channels, spectral, 1 ±10 nm positions alignable 50–300 on motorized trans, Photon spectral slits counting mode

NA 1¥ No Filter cube Self- Vert & horiz Dichroic ﬁlter CCD or 1 No/yes CCD Stepper, aligning slits, cube EM-CCD ±10 nm 5 sizes

NA No Dichroic, preset, rect. ~1 k fixed, Dichroics/ CCD CCD Yes/no CCD Piezo, 4 positions array 50 mm filters ±100 nm adjustable 50 : 1 No Dichroic, preset/ 5 slits, Dichroics/ 4 hi-QE PMTs 4 Yes/yes 10 bits Piezo, 6 positions adjustable 10–100 mm filters ±100 nm

NA 1¥ NA 1 dichroic, Self- 50 mm, 20 k on Dichroics/ CCD or 2-camera port No/no* CCD exchangeable aligning disk, ~1 k Filters, EM-CCD by user /FOV 3 emisson, 3 barrier NA 1¥ NA Dichroic, Self- 50 mm, 20 k on Dichroics/ CCD or 2-camera port No/no* CCD 3 positions aligning disk, ~1 k Filters, EM-CCD /FOV 3 emisson, 3 barrier 0.7–40¥ 0.84¥ Yes Dichroic, 4 x,y,(z), 3, 200 steps, 3 dichroics, 3/4 ﬁltered 8 Yes/yes 8–12-bit Microscope 4 positions diameter 0.1–13 Airy 6 positions, + PMTs;&/or 10 nm, adjustable Units 10–1 k spectral diff. Grating Piezo, mm detector, 10 nm w/32 mPMTs ±5nm /channel array,7

0.7–40¥ 0.84¥ Yes Dichroic, 2 x,y, 1, 200 steps, 2 dichroics, 2 ﬁltered 4 No/yes 8–12-bit Microscope 2 positions diameter 0.1–13 Airy 6 positions PMTs, 10 nm, adjustable Units trans PMT Piezo, 10–1 k mm ±5nm 0.5–2¥ 1.18¥ Yes Achrogate, 2 17 slits, 0.5–10 detector 512 ¥ 1 linear 2 No/no 8–12-bit Microscope line-mirror adjustable Airy units dichroic, CCD 10 nm, on clear 12 positions, Piezo, ±5nm blank 8 position barriers

4 These numbers assume that the tube mag is 1¥. 5 Yokogawa scanners are manufactured by Yokogawa Electric (Tokyo, Japan), but retailed by a number of companies including, Andor Technologies (Belfast, UK), Solamere Technology (Salt Lake City, UT), PerkinElmer (Downer Grove, Il), Visitech (Sunderland, UK). 6 It is possible to use 4 lasers with a quad, dichroic beamsplitter. 7 Transmission PMT and 4-channel non-descanned PMT detector also available. 1. Detection channels with stepless tunable bandpass and PMT 2. Beam splitter or mirror for auxiliary emission outlet (optional) 3. Emission filter and polarization filter (rotatable) (optional) 4. Excitation pinholes (excpt. IR) 5. Merge module. Combination of up to 4 visible lasers 6. a) Multiline Ar-Laser (457 – 476 – 488 - 496 – 514) b) HeNe Laser 543 c) HeNe Laser 594 d) Kr Laser 568 e) HeNe Laser 633 f) IR Laser TiS for Multiphoton excitation g) HeCd Laser 442 h) Solid state Laser 430 i) Ar Laser 351 – 364 j) Diode Laser 405 7. EOM for intensity control of IR Laser 8. AOTF for intensity control on VIS and UV Lasers 9. Variable adaptation optics for UV / 405nm illumination 10. K-Scanning module for optically correct scanning method and field rotation 11. Scan lens 12. Beam splitter for non-descanned reflected light mode (optional) 13. Objective optics 14. Sample 15. Condensor optics 16. Detectors for non-descanned transmitted light (optional) 17. Secondary beam splitter for NDD transmitted light (optional) 18. Secondary beam splitter for NDD reflected light (optional) 19. Detectors for non-descanned reflected light (optional) 20. Beam splitter for UV illumination (optional) 21. Variable beam expander optics 22. Beam splitter for IR or violet illumination (optional) 23. Acousto Optical Beam Splitter (AOBS ) 24. Pinhole optics 25. Detection pinhole 26. Spectral detector prism

FIGURE A2.3. Schematic diagram of Leica TCS SP2 AOBS. The Leica TCS SP2 AOBS is an advanced confocal microscope in which all ﬁltering and beam-splitting functions are performed by either liquid-crystal or acousto-optical components. This makes the system extremely ﬂexible in terms of being able to add new lasers or adapt to new emission bands. The acousto-optical beam-splitter (AOBS) is essentially transparent except at exactly the laser wavelengths (see Fig. 3.23). The K-scan galvanometer mirror arrangement is capable of being rotated around the optical axis to change scan directions. There is one adjustable pinhole for all 4 prism/moving-mirror spectral-detection channels. Leica also makes the TCS SP5, which is similar but employes a tandem scan system which permits one to switch between a scanner employing a normal, analog galvanometer and one employing resonant galvanometer for high-speed, bi-directional scanning at up to 16k lines/s.

1. TiS Laser (pulsed IR) 2. EOM for intensity control of IR Laser 3. K-Scanning Module for optically correct scanning method and field rotation 4. Scan optics 5. Beam splitter for non-descanned reflected light mode (optional) 6. Objective lens 7. Sample 8. Condensor optics 9. Detectors for non-descanned transmitted light 10. Secondary beam splitter for NDD transmitted light 11. Secondary beam splitter for NDD reflected light (optional) 12. Detectors for non-descanned reflected light (optional) 13. Variable Beam expander optics

FIGURE A2.4. Schematic of the Leica MP RS Multiphoton Fluorescence Microscope. The Leica MP RS is a single-beam scanning ﬂuorescence microscope that uses a ps near-IR laser light source to produce optical-section images of suitable specimens. It is designed for viewing living cells and incorporates a variety of non-descanned detectors to record both transmitted and backscattered ﬂuorescence signal. This instrument uses a fs-pulsed, near-IR laser multiphoton excitation and a high speed galvanometer to provide fast imaging. Figures kindly provided by Leica Inc. (Heidelberg, Germany). Multi-anode PMT

SPECTRAL DETECTOR

Polarization Unpolarized Light P Multiple Gratings Rotator (2.5/5/10nm) Polarized Beam Splitter

SCAN HEAD Optical from Laser Fiber Module Galvanometer Primary SMA Connector Pair Dichroic Mirror Mirror

3-COLOR Lens Pinhole DETECTOR Lens Fixed Mirror Mirror CH1 Emission Emission Dichroic 1 Filter

Nikon EF-4 PMT 1 Filter Block Pinhole Scan Lens Turret

CH3 Emission Emission Dichroic 2 Filter

Nikon EF-4 Prism PMT 3 Filter Block CH2 Emission Filter

PMT 2 Mounting Adapter

to Microscope

FIGURE A2.5. Schematic of the Nikon C1si light path. The C1-Plus is a 3-channel fluorescence plus transmission, single-beam, galvanometer-scanned, confocal microscope. Because both the lasers and the PMTs are located externally and coupled through fibers, the C1 scan head is extremely compact and is very easy to move from one microscope to another. The standard unit includes laser module, in which a wide variety of gas and solid-state lasers can be installed. a scan-head and a DU-3 three-PMT detector module containing the collimating and focusing lenses, and photomultiplier tubes. The “si” version includes an additional sophisticated spectral detector that is also coupled to the scan head through a multi-mode fiber. The detector itself incorporates a Diffraction Efficiency Enhancement System (DEES) in which a polarized beam splitter separates the unpolarized signal beam into two parts (red and blue lines). One part passes through a prism polarization rotator so that all the light strikes the diffraction grating with the optimal (s-plane) polarization to be diffracted with maximum efficiency by one of 3 gratings (2.5, 5 and 10nm/channel). Both ray bundles are then focused onto a 32 channel micro PMT by a pair of reflecting lenses (A and B). Simultaneous readout is possible from all channels. The digitization system uses 2 sample-and-hold circuits to optimize signal integration. Figures kindly provided by Nikon Inc. (Tokyo, Japan). 912 Appendix 2 •J.B. Pawley

PMT4

Emimission bea stpl tser Etsmission fil er Laser from optional scanner Confocal Grating PMT3 pinhole

Grating Laser PMT2 port 3 Slit Laser port 2 Laser PMT1 port 1 Slit

Scanning mirrors

Csonfocal len

Psupil transfer len

Eaxcit tion beamsplitters Tco objeetsiv len Tube lens A

FIGURE A2.6. (A) Schematic of the Olympus Fluoview 1000. The Fluoview 1000 is the most recent single-beam laser-scanning confocal fluorescence microscope introduced by Olympus. It offers 4 separate fluorescence detection channels, two of which incorporate diffraction gratings and adjustable slits to tune the passband. Besides the normal scanning mirrors there is a second independent SIM-scanning arrangement (not shown in the figure) to control lasers used for photo-uncaging or for intentionally bleaching the specimen. To keep the signal up when the light dose to the specimen must be kept low, this new scanner not only incorporates dichroic elements employing “hard’ coatings to ensure the highest transmission, it also offers a photon-counting option to reduce PMT multiplicative noise. Figures kindly provided by Olympus Corp. (Tokyo, Japan). Light Paths of the Current Commercial Confocal Light Microscopy Used in Biology •Appendix 2 913

Monitor

CCD camera

Imaging lens

Camera adapter Light ND filter source DSU Fluorescent mirror unit Rotary disk Disk box DSU Illumination Imaging lens tube Light illuminator

Objective lens

Specimen

W L

FIGURE A2.6. (Continued) (B, C) Schematic of the Olympus DSU disk-scanner. The Olympus DSU is a disk-scanning confocal ﬂuorescence microscope that uses a mercury arc for excitation. The optical system is identical to that used for normal epi-ﬂuorescence with the exception that an opaque disk is located in the intermediate image plane. Slits in this coating on this disk allow light to reach the focus plane and prevents light from this plane from reaching the CCD camera. To keep the light dose to the specimen low, this new scanner not only incorporates “hard” coatings to ensure the highest transmission of the dichroic elements, it also offers a photon-counting option to reduce PMT multiplicative noise. (C) Layout of one of several interchangeable scanning disks used in the Olympus DSU disk-scanner. The thickness and spacing of the slits varies on the 5 available disks have each been optimized for use with a particular objective. Figures kindly provided by Olympus Corp. (Tokyo, Japan). Micro lens array

Dichroic

Pinholes

Image plane

Galvo scanner A

FIGURE A2.7A. Schematic diagram of Visitech VT Infinity. The optical path starts with a stationary micro-lens array illuminated by an expanded laser beam. A galvanometer mirror (x) incorporating a piezoelectric micro-deflector (y) scans the array to cover the sample and then de-scans the returning fluorescence signal. This light is separated from the illuminating beam by a dichroic mirror, and passes through a stationary pinhole array to create confocal data. This data is re-scanned, in perfect synchronization, by being reflected off the reverse side of the galvanometer mirror onto a sensitive CCD camera. The galvanometer scanner is readily synchronized to the camera capture parameters, both exposure time and frame capture rate. Either multiple-line lasers or multiple lasers in any combination can be coupled through an AOTF that provides high speed (~ms) laser-line selection and intensity control. Laser excitation can be coupled in either by optical fiber or by direct coupling. Motor-driven filters change dichroic and detection bandpass. This system couples the advantages of high-brightness, laser illumination with multipoint scanning to keep the instantaneous intensity down while providing a data rate high enough for fast image detection, using a high- quantum-efficiency CCD camera. Figure kindly provided by Visitech Inc. (Sunderland, UK).

Primary wheel Fiber

Barrier Negative wheel cylinder

Slit Collimating

Positive cylinder

AOD input

FIGURE A2.7B. Schematic diagram of Visitech VT-eye. The VT-eye incor- PMT porates a novel acousto-optical deﬂector (AOD) scanner, that combines ultra- lens(e) fast horizontal scanning to provide high-resolution confocal imaging for Scan real-time, living-cell confocal microscopy. The AOD scans the X axis at up to 50,000 lines/s or 400 frames/s, fast enough to capture clear images of dynamic events such as Ca++ puffs, sparks and waves. Multi-wavelength imaging for multi-labeled specimens from UV through the visible to the near infrared is achieved by using a selection of motorized, primary multi-band dichroics. The system operates with almost any laser, or combination of lasers, and uses AOTF technology to provide fast laser-line selection. The VTeye comes with up to 4 AOD high-QE PMTs. The piezoelectric focusing system is capable of changing focus (astigmatism) positions at up to 100 slices per second. Although high-speed acquisition creates vast quantities of data in a very short time, hours of experiments may Field be recorded at the maximum capture rates on a range of parallel, hard-disk B modules. Figure kindly provided by Visitech Inc. (Sunderland, UK). Light Paths of the Current Commercial Confocal Light Microscopy Used in Biology •Appendix 2 915

Laser for excitation Camera port

Port select mirror

Barrier filter(triple)

Exciter filter(triple)

Eyepiece

ND filter & Shutter Microlens array disk

Pinhole array disk Dichroic mirror (triple) Microscope excitation laser Optical Path of CSU22 fluorescence A

Microlens Disk Pinhole Disk

Collimating Lens Fiber Input

Camera Detector Microscope Port

Relay Lens Relay Lens

Filter Wheel B

FIGURE A2.8. Schematic of the Yokogawa CSU 22. The Yokogawa scanner was the first disk scanner to offer both laser illumination and multibeam excitation. The mircolenses increase the efficiency of the illumination path from the 2–10% common to ordinary disks to almost 60%. (A) Laser light enters the scan head through a single-mode optical fiber, reflects off a mirror and through one of 3 exciter filters. After passing through an ND filter, and a beam expander, it illuminates the microlens array on the top disk of the rotating scanning assembly. The lenses focus the light through a short-pass dichroic and onto the array of pinholes in the lower disk. As this disk is in an image plane, the light passing each pinhole is focused into a point at the focus plane of the objective. Fluores- cent light returning from the focus plane passes up through the pinholes, and reflects off one of 3 dichroic mirrors located between the two disks and into the detection path. After passing through one of 3 barrier filters, a selection mirror sends this light either to the camera port or to the eyepiece. (B) Simplified ray optical diagram of the CSU-22. The pinhole disk resides in an image plane and the signal passing the pinholes is first made parallel by a relay lens, then passed through the emission filter before being focused onto the CCD chip by a second relay lens. Other details shown in Figure 10.9. Figures kindly provided by PerkinElmer Corp. (Shelton, CT). FIGURE A2.9A. Schematic diagram of Zeiss LSM-5-LIVE Fast Slit Scanner. The LSM-5-Live is a line-scanning confocal microscope using line illumination and a linear detector. Because it illuminates about 100x more points than does a single-beam instrument, the LSM-5 Live can acquire data at a much higher speed while still keeping the peak light intensity low enough to avoid singlet-state saturation. In addition, the quantum efficiency of the linear CCD is about 10x greater than that of most PMTs. Laser light enters the scan head through optical fibers (1) where it is combined by a series of mirrors (2, 3) and then passes to beam shaper (an expander and a cylindrical lens that converts the collimated Gaussian beam into laser light with a rectangular cross-section) (4) and also focuses it precisely onto the AchroGate beam splitter (5), reflects all wavelengths but only along a reflective line across its center. As a result, no matter what the wavelength, it reflects 100% of the laser light but passes >95% of the signal light to the detectors. The size of the raster on the specimen is controlled by a 0.5–2x zoom optic (6), that feeds the light to the y-scanning mirror (7), through the scan lens (8), the objective lens (9) and on to the specimen, (10). Returning signal follows the same path but mostly misses the reflective strip in the Achrogate and proceeds through a wheel of secondary dichroic beam-splitters (11) to one of 2 tube-lenses (12) that each focuses the line illuminated in the specimen onto a 17-position, slit aperture plate (13). Light passing the slits is first filtered by emission filters (14) and then detected by a 1 ¥ 512 linear CCD detector (15) (see also Fig. 9.6).

M Spectral Imaging L laser CL collimator lens M mirror PMTA BC beam combiner MDBS main dichroic beam splitter G PH PMT Imaging SCXY scanner X/Y EF O objective PH FO/ S sample EPD PH variable pinhole EF PH DBS PMT DBS DBS dichroic beam splitter EF emission filter Excitation PMT photo multiplier tube L MDBS G grating BC PMTA PMT array (META) CL SCXY

L M NDD non-descanned detector NDD O FO fiber out EPD external photodetector S B

FIGURE A2.9B. Optical beam path of the Zeiss LSM 510 META. A unique scanning module is the core of the LSM 510 META. It contains motorized dichroic mirrors and barrier filters, adjustable collimators, individually adjustable and alignable pinholes for each of 3 (or even 4) detection channels, as well as scanning mirrors, and highly sensitive PMT detectors including the 32 micro-PMTs of the META spectral detector. All these components are arranged to ensure optimum specimen illumination and efficient collection of reflected or emitted light. The highly optimized optical diffraction grating in the META detector provides an innovative way of separating the fluorescence emission spectrum to strike 32 separate, micro-PMTs, each of which covers a bandwidth of ~10 nm. Thus, a spectral signature is acquired at each pixel of the scanned image. Such a dataset can subsequently be digitally “unmixed” to separate signals from dyes with overlapping emission spectra. The Beam Path: (1) Optical Fibers, (2) Motorized collimators, (3) Beam combiner, (4) Main dichroic beamsplitter, (5) Scan- ning mirrors, (6) Scanning lens, (7) Objective lens, (8) Specimen, (9) Secondary dichroic beamsplitter, (10) Confocal pinhole, (11) Emission filters, (12) Pho- tomultiplier, (13) META detector, (14) Neutral density filter, (15) Monitor diode, (16) Fiber out. Light Paths of the Current Commercial Confocal Light Microscopy Used in Biology •Appendix 2 917

VIS Fiber T-PMT Mirror HAL Condensor Collimator Specimen UV Fiber DBC Objective LSFNDF Plate Monitor Diode Fiber HBO Scan Pinhole Coupler Tube Lens Lens Scanner Optica Fiber AOTF x Coupler y Shutter MDBS VP4 AOTF EF4 Eyepiece DBS2 Shutter Tube Lens PMT3 DBS1 VP2 DBS3 VP3 PMT4 Inverted Microscope EF3 EF2 λ-selective Element Collimator VP1 Spectral Ar-UV Laser or 413 nm Ar/ArKr Laser HeNe Laser HeNe Laser MDBS PMT2 EF1 Detector PMT1 Pinhole Optics Scan Module on Side Port Laser Laser TV Module UV Module VIS

DBS1 APD1 EF1 VP2 VP1 EF2

APD2

AP D U nitFCS on Base Port APD UnitFCS C

FIGURE A2.9C. (Continued) Schematic diagram of Zeiss LSM FCS showing how the ﬂuorescence-correlation spectroscopy (FCS) unit is attached via the base port of the Axiovert 200M microscope while the LSM 510 META is attached to the side camera port. All ﬁgures kindly provided by Carl Zeiss Inc. (Jena, Germany). Appendix 3 More Than You Ever Really Wanted to Know About Charge-Coupled Devices

James B. Pawley

INTRODUCTION are kept electrically separate from their neighbors by additional layers of SiO2. Every third stripe is connected together to form The electronic structure of crystalline Si is such that electromag- three sets of interdigitating strips that we will refer to as Phases 1, netic waves having the energy of light photons (1.75–3.0 electron 2 and 3 (f1, f2, f3, Fig. A3.1). Taken together, all these phases con- volts) can be absorbed to produce one free or “conduction” elec- stitute the vertical register (VR) and, after the assembly has been tron. If an image is focused onto a Si surface, the number of the exposed to a pattern of light, they are used to transfer the photo- photoelectrons (PE) produced at each location over the surface is induced charge pattern downwards, one line at a time. The pixels proportional to the local light intensity. Clearly, all that is needed along each line are separated from each other by vertical strips of to create an image sensor is a method for rapidly converting the positively doped material injected into the Si. These positive local PE concentration into an electronic signal. After almost 40 “channel blocks” create fields that prevent charge from diffusing years of NASA and DOD funding, the slow-scan, scientific-grade, sideways without reducing the active area of the sensor. charge-coupled device (CCD) camera is now an almost perfect Any photon that passes through the stripes and the SiO2, is solution to this problem. absorbed in the Si, producing a PE. If a small positive voltage (~15 Success in modern biological light microscopy depends to an volts) is applied to the f1 electrodes, any PE produced nearby will ever-increasing extent on the performance of CCD cameras. be attracted to a location just below the nearest f1 strip (Fig. A3.2). Because such cameras differ widely in their capabilities and are As additional PEs are produced, they form a small cloud of PEs also items that most biologists buy separately, rather than as part referred to as a charge packet. The number of PEs in the packet is of a system, some knowledge of their operation may be useful to proportional to the local light intensity times the exposure period those practicing biologists who have not yet found it necessary to and the problem now is to convey this packet to some location be particularly interested in “electronics.” Although the basics of where its size can be measured, and to do this without changing it CCD operation are described in many other chapters (particularly, or losing track of the location from which it was collected. This Chapters 4, 10 and 12) this Appendix describes the operating will be achieved by using the overlying electrodes to drag the principles of these devices in greater detail and also discusses the charge packet around in an orderly way until it is deposited at the ways that they “don’t work as planned.” It then covers the opera- readout node of the charge amplifier. tion of the electron-multiplier CCD (EM-CCD), a new variant that reduces the read noise almost to zero, although at the cost 1 Charge Coupling of reduced effective quantum efficiency (QEeff). The second section, How to choose a CCD, is a review of CCD specifications The dragging mechanism operates in the following way: First f2 with comments on the relevance of each in fluorescence is also made positive so that the cloud diffuses to fill the area microscopy. underneath both f1 and f2. Then f1 is made zero, forcing the packet to concentrate under f2 alone (Fig A3.2). So far, these 3 steps have succeeded in moving the charge PART I: HOW CHARGE-COUPLED packets that were originally under each of the f1 electrodes down- DEVICES WORK wards by one phase or 1/3 of a “line” in the x-y raster. If this sequence is now repeated, but between f2 and f3 and then again The first step is to imagine a rectangular area of the Si surface as between f3 and the f1 belonging to the next triplet of strips, packets being divided into rows and columns, or more usually, lines and will have moved down by the one entire raster line. PEs created pixels. Each pixel is between 4 ¥ 4mm and about 24 ¥ 24mm in within a particular pixel of each horizontal stripe remain confined size and the location of any pixel of the surface can be defined in by the channel stops as they are transferred to the next line below. terms of it being x pixels from the left side, on line y. A pixel of the image is therefore defined as the area under a To construct an actual system like this, start with a smooth Si triplet of overlying, vertical charge-transfer electrodes and surface; cover it with a thin, transparent, insulating layer of SiO ; between two neighboring channel blocks. The pixels on scientific 2 CCDs, are usually square, 4 to 30mm on a side while those on deposit onto the SiO2, a pattern of horizontal strips, made out of a transparent conductor called amorphous silicon (or poly-silicon), commercial, video CCDs are likely to be wider than they are high, so that the strips cover the entire image sensor area. Although, to conform with the reduced horizontal resolution of commercial viewed from the top, these strips partially overlap each other, they video standards. Only square pixels can be conveniently displayed in a truly digital manner. Larger pixels have more leakage current (dark-current), but are also able to store more charge per pixel (see 1 This loss can be avoided if the system is used in photon-conting mode. Blooming, below).

James B. Pawley • University of Wisconsin, Madison, Wisconsin 53706

918 Handbook of Biological Confocal Microscopy, Third Edition, edited by James B. Pawley, Springer Science+Business Media, LLC, New York, 2006. More Than YouBASIC Ever Really CCD Wanted ARRAY to Know About Charge-Coupled Devices • Appendix 3 919

Vertical One pixel phase Control electrodes Φ1 Φ2 Φ3 Drive pulse connections

Image Channel stop section Parallel channel Serial channel

Readout section Output

Readout node

Φ4Φ5 Φ6

Horizontal phase Drive pulse connections

FIGURE A3.1. Layout of CCD array, viewed en face.

FIGURE A3.2. Charge coupling: Three stages in the process of moving a charge packet initially beneath phase 1 (A), so that it first spreads to be also under phase 2 (B) and finally is confined to entirely under phase 2 (C). These 3 steps must be repeated 3 times before the charge packet has been moved downwards (or in the diagram, to the right) by one line of the CCD array. C 920 Appendix 3 • J.B. Pawley

At the bottom of the sensor, an entire line of charge packets is ferred past bright features in the image, producing vertical streak- simultaneously transferred to the adjacent pixels of the horizontal ing. This problem is more important when the exposure time is register (HR, also sometimes called a shift register). Like the VR, short relative to the readout time. the HR is composed of a system of overlying poly-Si electrodes In frame transfer readout, at the end of the exposure, the and channel stops. Each column of pixels in the VR is eventually entire charge pattern is rapidly (0.1–3 ms) transferred by charge- transferred directly into the same specific pixel on the HR. The coupling to a second 2D storage array. The storage array is the three phases of the HR (f4, f5, f6) work exactly like those in the same size as the sensor array and is located next to it but it is phys- VR, except that they must cycle at a much faster rate because the ically masked with evaporated metal to shield it from light. The entire HR must be emptied before the next line of packets is trans- charge pattern is then read out from the storage array while the ferred down from the bottom line of the VR. In other words, in the sensor array collects a new image. Because vertical transfer can be time between one complete line-transfer cycle of the VR and the much faster if the charge packets do not have to be read out, this next, the horizontal register must cycle as many times as there are system reduces streaking by up to 1000¥ but does not eliminate it pixels in each line. and the need for a storage register reduces the fraction of the Si At the right-hand end of the HR is a charge amplifier that mea- surface area that can be used for sensing by 50%. sures the charge in each packet as it is transferred into it from the In interline transfer, the masked storage cells are interlaced last pixel of the HR. The first pixel to be read out is that on the between the sensor cells (i.e., each pixel is divided into sense and extreme right-hand side of the bottom line. The last pixel will be read areas). After exposure, all charge packets can be moved to the that on the left side of the top line.2 readout array in less than a microsecond. This ability can be used The entire charge-transfer process has the effect of coding as an electronic shutter to eliminate vertical smearing but, because position as time. If we digitize the signals from the charge ampli- at least half of the area of each sensor must be masked, and any fier, and store the resulting numbers in a video memory, we will light striking a masked area is lost, the “fill factor” of the sensor is be able to see a representation of the light intensity pattern strik- reduced, proportionately decreasing QEeff. A solution to the “filling the sensor on any monitor attached to this video memory. Alter- factor” dilemma is to incorporate an array of microlenses, aligned natively, as long as the dimensions of the CCD array match those so that there is one above every pixel. With such a system, most of of some video standard, such as NTSC or PAL, the time sequence the light striking any pixel will be focused onto the unmasked area.3 of charge-packet readout voltages can be smoothed and, with the Although microlenses restore the QEeff somewhat, the full-well addition of synch pulses, turned into an analog video signal. While signal possible is still limited by the smaller sensitive area. this latter process is often convenient, it is a poor plan if the analog signal must then be re-digitized. The necessity to digitize twice can reduce the effective horizontal resolution of the CCD sensor by WHAT COULD GO WRONG? about a factor of 2 and because the process is AC coupled, photometric accuracy is severely compromised. When I first heard the CCD story, it struck me as pretty prepos- It is important to understand the relationship between the terous! How could you get all the correct voltages (9 different charge-transfer electrodes and the charge packet. The electrodes voltage combinations per pixel shift, ~3.6 million for each TV do not somehow “connect to” the charge packet, and “conduct” it frame, 108 million/s for video rate!) to the right charge-transfer to the amplifier. Such a process would be subject to resistive losses, electrodes at the correct times? How could you get all of the charge charge would be lost and a lot of “wires” would be needed. The in a packet to stay together during a transfer? Wouldn’t Poisson charge-coupling process is better thought of in terms of a ball statistics apply, making even one transfer imprecise and the 2000 bearing “dragged” over the surface of a loose blanket by moving transfers needed to read out the top, right pixel of a 1000 ¥ 1000 a cooking pot around underneath the blanket. The weight of the pixel array impossibly inaccurate? How long would the PEs stay ball and the lip of the pot create a dimple and gravity keeps the free to be dragged around the lattice? Wouldn’t the charge packets ball in the dimple as the pot is moved. The voltage on the charge- decay with time? transfer electrode creates an electronic “dimple.” Changing the In fact, many of these problems did occur, but remedies to most voltages on nearby electrodes moves the dimple. In this way, have now been devised. The difference between a $300 commer- groups of charged particles (electrons) can be pushed around cial CCD camera and a $65,000, top-of-the-line scientific CCD can without actually “touching” or losing them. often be measured in terms of how many of these remedies have been implemented. Therefore, it is worthwhile trying to understand some of them so that one can buy what one needs. The following Readout Methods discussion will define and discuss some of the more important There are three distinct methods for reading out the charge pattern CCD technical specifications. of a CCD: full-frame, full-frame transfer and interline transfer (Fig. A3.4). Most early scientific CCDs used the first method, which operates as has just been described. Although full-frame Quantum Efficiency readout provides the largest sensitive area for a given area of Quantum efficiency is the ratio of the number of impinging silicon, the lowest level of readout noise and the greatest photo- photons to the number of PEs produced.4 Any photon with energy metric accuracy, it also has some disadvantages. One cannot both in the range of 1–100 eV striking crystalline Si has a very high collect and read out signal at the same time. Unless some sort of probability of producing a PE. However, reflections and absorp- shutter is used to prevent light from striking the sensor during ver- tion by the overlying polysilicon electrodes,5 reduce the QE of tical transfer, signal will be added to any packets that are trans-

3 This occurs only as long as the initial angle of incidence is near to normal, a condition met when CCDs are used for light microscopy. 2 This may seem backwards until one remembers that any image of the real 4 In the visible range, each absorbed photon makes only one PE. world is usually focused onto the CCD by a single, converging lens, a process 5 Kodak had pioneered the use of charge transfer electrodes made out of In and that always inverts the image. Sn oxides that scatter less light than do those made of poly-Si. More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3 921

FIGURE A3.3. Four CCD readout patterns: Full-frame, frame-transfer, interline transfer and gain register (EM- CCD).

front-illuminated CCDs especially in the blue end of the spectrum. amplifier noise. One can also see that at readout speeds higher than To reduce this effect, some UV-enhanced sensors are coated with 1MHz (or 1 second to read out a 1024 ¥ 1024 CCD), the read fluorescent plastics, which absorb in the blue and emit at longer noise increases with the square root of the read speed. wavelengths. Others have their backs etched away and are turned over to permit the illumination to reach the light-sensitive area from the back side.6 Figure A3.4 shows the intrinsic QE of differ- Charge Loss ent types of CCD (not Qeff, which would take into account the light The lifetime of a PE (before it drops back into the ground state) lost if some of the sensor is covered by charge storage areas). The depends on the purity and crystalline perfection of the Si and on effective QE can usually only be determined by actual measure- other factors such as temperature. Generally it is long enough that ment or by very careful evaluation of the published specifications little charge is lost during the exposure times commonly used in

(QEeffective = QEintrinsic ¥ fill factor). fluorescence microscopy. If necessary, it can be increased by cooling the detector, something often done to reduce dark charge. Edge Effects In early CCDs, PEs were often “lost” in the crystalline imperfec- Leakage or “Dark Charge” tions that are always present at the Si/SiO2 junction. To avoid this, Dark charge is the charge that leaks into a pixel during the expo- ion implantation is now used to make an N-doped, sub-surface sure time in the absence of light. It can be thought of as the dark layer called the buried channel about 1mm below this surface (Fig. current7 deposited into one pixel. Many processes other than A3.2). This channel attracts the free PEs, keeping them away from photon absorption can add PE to the charge packet. The magni- the edge of the Si crystal. Any serious CCDs will have a buried tude of this dark charge depends on the length of the exposure, and channel but the need for ion-implantation keeps CCD chip prices is substantially reduced by cooling. The rule of thumb is that for high! Figure A3.5 shows the readout noise, in root-mean squared every 8°C of cooling, the dark charge is halved. As noted above, (RMS) electrons/pixel, for surface and buried-channel CCDs dark charge is principally a problem because it produces Poisson having two different pixel sizes. From this you can see that small noise equal to the square root of its magnitude, and if this is pixels (here ~5.5 ¥ 5.5mm) have lower read noise than larger ones left unchecked, it can significantly increase the noise floor of the (~17 ¥ 17mm), mostly because the larger ones have higher capac- CCD. itance and capacitance is the most important parameter of read- Since ~1987, a process called multipinned phasing (MPP) has been available to reduce dark charge build-up by about a factor of 1000, making it immeasurable in exposures up to a minute or so. 6 Back-illuminated CCDs have to be thinned to 7–10mm so that conduction This feature should be specified if one expects to use exposures electrons created near what would have been the back surface can respond to longer than a few seconds without deep-cooling. the fields created by the buried channel and the CC electrodes. Thinning increases cost and also reduces QE at longer wavelength where the absorption distance of the photons becomes comparable with the actual thickness. Back-illuminated CCDs are also more expensive because it is difficult to 7 A current is a flow of charge measured in charge/time. The unit of charge is create electrical contacts with electrodes, etc., that are now on the bottom side the Coulomb (c). The unit of current is the Ampere (A). One Amp represents of the chip. a flow of one Coulomb/s or 6.16 ¥ 1018 electrons/s. 922 Appendix 3 • J.B. Pawley

FIGURE A3.4. Intrinsic QE as a function of wavelength for a front-illuminated CCD (blue), a visible- enhanced, back-illuminated CCD (green) and a UV-enhanced CCD (red).

It should also be remembered that, while dark charge is never the number of electrons/pixel it represents. CCDs should always good, its average value can be measured and subtracted on a pixel- be operated such that the noise on the dark charge is less than the by-pixel basis, by subtracting a “dark image” from each recorded readout noise. On conventional CCDs this condition can usually image as part of flat-fielding. However, because, by definition be met quite easily by slightly cooling the sensor (0°C or about “dark” images contain very few photons/pixel, they have relatively -20°C from ambient). The use of lower temperatures is compli- high Poisson noise and low S/N. Therefore, a number of such cated by the risk of condensing atmospheric water, a process that images must be averaged to produce a correction mask that is sta- can be avoided only by enclosing the sensor in a vacuum chamber. tistically defined well enough that subtracting it from the data does Generally, a vacuum-hermetic enclosure, combined with good not substantially increase the noise present in the final, corrected outgassing prevention, carries with it the significant benefits of image. more effective cooling, long-term protection of the sensor from This is not a problem when there are many counts in each pixel moisture and other degrading organic condensates as well as because the subtractive process of dark-charge normalization the prevention of front-window fogging. At video rate, where involves a change that is small compared with the intrinsic noise exposures are short, dark charge is only a problem when the present in a large signal. It can be a problem when the black mask readout noise is reduced to <1e/pixel, as it is when an “electron- image is subtracted from a faint image that also contains only a multiplier” (EM) charge amplifier is used (see below and also few counts/pixel. Chapters 4 and 10). In EM-CCDs the read noise is so low that dark What cannot be removed by flat-fielding is the Poisson noise current becomes the main source of noise and cooling to -80°C associated with the dark charge. This is equal to the square root of becomes necessary.

Readout noise, electrons, rms 100

Measured 50 Surface channel 2 30 A = 300 µm

Surface channel 10 A = 30 µm2 Buried channel (T<200K) A = 300 µm2 5

3 Buried channel (T<200K) 2 A = 30 µm2 FIGURE A3.5. CCD ﬁeld effect transistor (FET) noise as a function of pixel dwell time for large and small pixels and when using buried channel vs. surface 1 channels. Smaller pixels have less read noise because 10 ns 100 ns 1 µs 10 µs 100 µs Clamp-to-sample time they have less capacitance. Buried channels have almost 100 MHz 10 MHz 1 MHz 100 kHz 10 kHz Pixel readout rate 10¥ less read noise than surface channels. More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3 923

TABLE A3.1. Typical Performance of Various Types of CCD Cameras. The “Sensitivity” Column Is a Reasonable Estimate of the Relative Suitability of the Camera for Detecting Very Faint Signals. It Spans a Very Large Range of Performance! All CCDs are not equal Type Grade QE % (effective) Noise (e/pix) Sensitivity (relative) Bit depth Dynamic Range Video commercial color 10 200 1 10 1,000 monochrome 20 200 2 10 1,000 Digital 1Mhz, color 15 50 12 12 4,000 1Mhz, mono 30 50 24 12 4,000 Back. Illum/ 90 5 720 15 40,000 slow-scan LLL-CCD 45 0.1 (18,000)* ?* 200,000 (EMCCD) *Because the gain of the electron multiplier ampliﬁer is unknown and large, it is not simple to measure, or even deﬁne, the sensitivity and bit depth of the EM-CCDs.

Blooming scientific applications), operate with much (100¥?) less perfection. In microscopy today, we find CCDs that span this range of per- As more photons are absorbed, the charge packet clustered around formance (Table A3.1). the buried channel grows and mutual repulsion between these All CCDs are not equal!! electrons renders the field imposed by the charge-transfer electrode ever less successful in keeping the packet together. The maximum charge packet that can be stored without it overflowing into nearby CHARGE AMPLIFIERS pixels can be estimated by multiplying the pixel area (in square micrometers) by 600PE/pixel (i.e., 27kPEs for a 6.7 ¥ 6.7mm So far, I have described an image sensor in which up to 90% of pixel, 540kPEs for a 30 ¥ 30mm pixel). This overflow problem is the impinging photons make free PEs and explained how the referred to as “blooming” and, in CCDs for the home-video market charge packets that result from many photons hitting a given pixel it is limited by the presence of an n-layer, deeper in the Si. When can be conveyed to the charge amplifier, in a time-labeled manner the charge packet gets too big, mutual repulsion between the PEs and almost without change. Clearly the performance of the entire forces some of them into this overflow layer, through which they image detector will depend crucially on the capabilities of this are conducted to ground. amplifier. While this anti-blooming feature is convenient for removing the effects of the specular reflections found in images from everyday life,8 it is not incorporated into many full-frame or frame- What Is a Charge Amplifier? transfer scientific CCDs because it reduces QE for long- Although most scientists have had some exposure to electronic cir- wavelength light. As this light penetrates farther into the Si crystal cuits that amplify an input voltage or current, they may be less before being absorbed, much of it reaches the overflow layer where familiar with the operation of the type of charge amplifier found any PEs produced are lost. in a CCD. The following outline is presented to enable the reader to understand enough about the process to appreciate some of the Incomplete Charge Transfer important differences between the various types of CCD. Because of the pulsatile nature of the CCD charge delivery Sometimes, an imperfection in the Si will produce a pixel that system, the optimal way to measure charge packet size is to deposit “leaks” charge. Charge deposited into, or transferred through, this it into a (very) small capacitor (the “read node”) and then measure pixel will be lost, producing a dark vertical line above it. In addi- the voltage on this capacitor with a high impedance amplifier. As tion, if one pushes the pixel clock too fast, some PE in the packet a field-effect transistor (FET) has an almost-infinite input imped- will not move fast enough and they will be left behind. In general ance, it is ideal for this purpose and in fact, its existence makes however, on a slow-scanned, scientific CCD, fewer than 5PEs out charge-amplification possible. of a million are lost (or gained) in each, slower, vertical transfer There are two basic types of conventional CCD readout and only 50 (0.005%) are lost during each, faster horizontal trans- amplifier, non-destructive and destructive.10 Both employ FET fer. In such devices, the main noise term is Poisson noise for any amplifiers. 9 signal level above ~20PE/pixel, and it seems hard to imagine Non-destructive (“skipper”) amplifiers use an FET with a doing much better than this except for signal levels <16 PE/pixel. “floating gate” to sense the size of a charge packet by responding On the other hand, it is also true that the vast majority of CCDs to the moving field that is produced as the packet is transferred made (those for camcorders, surveillance cameras and even many along a charge-coupled register. Because the charge packet itself is not affected by this process, the process can be repeated hundreds or even thousands of times. If the results of all these mea- 8 Features such as the image of the sun reflecting off a shiny automobile can surements are averaged, very low readout noise levels (>±1 be over 1,000¥ brighter than the rest of the scene. Fortunately, such extremely electron/pixel) can be obtained, but at the cost of a substantial bright features are seldom found in microscopic images unless a crystal of fluorescent dye occurs in the field of view. 9 This calculation assumes that the read noise is 4e/pixel, and this will be less than the Poisson Noise for any signal >16 PE. However, as many CCDs used 10 The “electron-multiplier” amplifiers mentioned previously, act essentially as in microscopy have >4e/pixel of noise, this cut-off point should not be con- pre-amps to the conventional FET amps described here. They will be covered sidered inflexible. later in this Appendix. 924 Appendix 3 • J.B. Pawley

Reset would, itself, produce a random electronic noise signal larger than trigger this, and electronic noise increases with readout speed, read-node capacitance and, to a lesser extent, temperature. VReset Supply voltage The success of the CCD in overcoming this limitation depends on two factors: Reset • The extremely small capacitance of the read node compared transistor to that of any other photosensor such as a photodiode. Readout transistor, Gain G • Special measurement techniques such as correlated double- ∆V = Q sampling Charge Cn input Output, G∆V = GQ Clearly there are a lot of tricks to making the perfect CCD Cn ampliﬁer and not all CCDs employ them. Table A3.1 lists typical Q Load performance for a variety of common camera types.

CCD Register Substrate and ground NOISE SOURCES IN THE CHARGE-COUPLED DEVICE Readout node capacitance Fixed Pattern Noise FIGURE A3.6. Destructive read-out amplifier for a CCD chip. When exposed to a uniform level of illumination, some pixels in a CCD array will collect more charge than others because of small differences in their geometry or their electrical properties. Conse- (¥100 or ¥1,000) increase in readout time and logical complexity. quently, it can be necessary to use stored measurements of the rel- This approach might make sense on a Mars probe but it has not ative sensitivity of each pixel to normalize, or “flat field,” the final been used in microscopy to my knowledge. dataset on a pixel-by-pixel basis. This is accomplished by first Destructive readout amplifiers are more common, probably recording an image of a featureless “white” field. This is often because they can operate more rapidly (Fig. A3.6). As imple- approximated by a brightfield transmission image with no speci- mented in a scientific CCD, the charge amplifier consists of the men, a process that will also record “inhomogeneity,” or mottle, following components: in the optical system. Differences in gain between pixels are evident as visible as nonuniformities in the digital signal stored in overlying charge transfer electrodes to drag the next charge • the memory and these are used to derive multiplicative correction packet into the “read node” coefficients.12 the read node itself: a 0.03–0.1 picofarad capacitor • Unfortunately, one can only preserve the high precision of the the sense FET • CCD output if the coefficient used to normalize each pixel is the reset FET • equally precise. In any event, these correction coefficients vary In operation, fields from the overlying charge-coupling elec- with both the photon wavelength and the angle at which the light trodes force a charge packet into the read-node capacitor, creating passes through the polysilicon electrodes on its way to the buried a voltage, Vc, that is proportional to the amount of charge in the channel. This, in turn, depends on the details of the precise optical packet. This voltage is sensed by the sense FET and the output is path in operation when an image is recorded and may even change passed, via additional amplifiers, to the analog-to-digital converter with microscope focus! As the intrinsic noise of a pixel holding (ADC) where the signal is converted into a digital number. Finally, 360k, PEs is only ±600 electrons or 0.16%, pixel-to-pixel nor- just before the next charge packet is coupled into the read node, a malization for changes in sensor gain is seldom perfectly effective reset FET discharges the capacitor, forcing Vc to zero, and allow- and consequently there is usually some level of “Fixed-pattern ing the read-FET to sense it again. noise” superimposed on the final data. In addition, the “white” image that must be used for pixel-level FET Amplifier Performance sensitivity normalization is itself subject to intrinsic noise (±600 electrons for a signal from a pixel with a full well charge of 360k The signal current (signal charge/s) coming from a CCD sensor is electrons) and so multiplicative normalization may actually add very small. Suppose that there were, on average, 400 PE in every some noise to the raw, uncorrected signal! Fortunately, if the white pixel of a 512 ¥ 512 pixel sensor.11 Reading this out in one second image can be defined by a multi-frame averages of several, nearly would constitute a current of only 10-11 Amps. The current through full-well “white” images, this normalization noise should only be the bulb in a home flashlight is 1010 times more. A very good con- noticeable when the image data to be corrected is similarly noise ventional electronic amplifier designed to amplify this current free. Without details of the signal levels present or the optical system in use, it is difficult to estimate the magnitude of normalization noise but it will be comparatively less important for images of faint objects containing few counts/pixel because these mea- 11 Although this number may seem small, it is actually quite high compared to some uses in biological confocal microscopy. Many authors have found that surements are themselves less precise. in normal” use, a single-beam confocal microscope used to image a fairly faint stain will count 4–8 PE/pixel in bright areas of the image. Allowing that the effective QR of a good CCD will be ~10¥ higher than that of the photomultiplier tube used in the confocal microscope, this makes the expected 12 These correction coefficients are small and only needed when operating on peak CCD signal in an image from a disk-scanning confocal microscope only images involving large numbers of photons (and consequently having rela- 40–80 PE/pixel. tively low Poisson noise and good S/N). More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3 925

It should be also noted that the vignetting and “mottle” visible Where Is Zero? in images characteristic of video-enhanced contrast microscopy will produce small intensity errors in the data obtained by both A final important feature of the CCD readout is that, compared to widefield and confocal. However, this noise term will be more the photomultiplier tube (PMT), it is relatively difficult to deter- noticeable in widefield where more photons are used and hence the mine the exact output signal level that corresponds to a zero-light precision of the data is greater. Mottle is produced by dirt and signal. A properly operated PMT never records negative counts. surface imperfections on any optical components that are not However, as the electronic readout noise of a cooled-CCD is an located exactly at aperture planes, as well as by non-uniformities RMS function with both positive and negative excursions, there in the image sensor. Fortunately, to the extent that it is stable with will be some pixels that measure lower than the mean value of the time, mottle will be removed by the flat-field correction for CCD zero-light pixel intensity distribution. sensitivity just discussed. To ensure that no data is “lost,” scientific CCDs are usually set What will not be removed is any change in signal caused by up so that the zero-light signal is stored to be a few tens of digital stray light (room light, light that goes through filters designed to units (ADU) above zero. A histogram of numbers stored from a remove it, etc.). The simplest test of any CCD set-up is to record “black” image will show a Gaussian-like peak centered at the an image of “nothing” (i.e., room dark, no excitation, no specimen offset and with a half-width equal to 2¥ the RMS read noise (see etc.). Then do the same with 100¥ longer exposure time with the Fig. 4.20). This offset makes it more difficult to apply the gain and room lights at your normal operating level. Now adjust the display offset normalization procedures to images that record only a few look-up tables so that you can “see the noise” in both the images detected photons in each pixel, a factor that will become more on the screen. Although the only difference between the two important as CCDs are increasingly used to image living cells that images should be increased dark noise in the image with the longer cannot tolerate intense illumination and which therefore produce exposure, this is seldom the case. substantially lower signal levels.

Noise from the Charge Amplifier A NEW IDEA: THE GAIN REGISTER AMPLIFIER!! Noise is generated by both the readout and the reset FETs in the charge amplifier. Noise generated in the readout FET reaches the Early in 2002, a new type of readout amplifier was introduced by ADC directly. If thermal noise in the reset FET prevents it from Texas Instruments (Houston, TX) and E2V Technologies (Chelms- completely discharging the read-node capacitance, it produces a ford, UK). As only E2V makes back-illuminated sensors, I will random offset at each pixel (i.e., the read-node voltage is not reset describe their system but both work along similar lines. E2V orig- exactly to zero). This is referred to as Reset Noise and has the inally referred to their device as the “gain register” and its purpose effect that the dark charge seems to vary from pixel to pixel. For- is to amplify the size of the charge packet before it arrives at the tunately, Reset noise can be almost eliminated by employing the read node. Although the term gain register has recently been technique of Correlated Double-sampling (CDS) in the readout replaced by the term “electron multiplier”, it is important to amplifier. In CDS, the circuitry of the charge-to-voltage amplifier remember that these new detectors work on a completely different is modified so that the output is proportional to the difference principle from that employed in intensified-CCDs. between the value of Vc just after the reset pulse and its value after The gain register superficially resembles an additional HR, the next charge packet has been inserted. with two important differences: Although CDS essentially eliminates the effect of reset noise, There are 4 phases rather than the usual 3 and the new it also distorts the noise spectrum. On the one hand, this distortion • phase consists of a grounded electrode located between f has the beneficial effect of converting the low frequency, 1/F noise 1 and f . from the FET into broadband noise which is more easily treated 2 The charge transfer voltage on f , is now variable, between theoretically and which is less visually distracting than the short, • 2 +35 and +40 volts rather than the usual +15 volts. horizontal flashes characteristic of 1/F noise.13 On the other hand, it means that the input to the ADC must be carefully frequency- As a result, when f2 is excited, there exists a high electric field filtered. This filtering can be implemented either by employing RC between it and the grounded electrode. The high field accelerates circuits or by using dual-slope integration (DSI) in the ADC itself. the electrons in the charge packets more rapidly as they pass from

If there are large intensity variations between neighboring pixels, f1 to f2 with the result that each PE has a small (but finite; usually the use of RC circuits will effectively compromise the large in the range of 0.5% to 1.5%) chance of colliding with a lattice dynamic range of the CCD. Therefore, ADCs using DSI are electron and knocking it into the conduction band (Fig. A3.7). employed on most slow-scan scientific, cooled-CCDs. Assuming the 1% gain figure, this means that for every 100 PE in The fact that CDS and, in particular, DSI work best at low the packet, on average one of these will become two electrons readout speeds is a final reason why most scientific CCDs operate before it reaches the space under f2. Although this seems like a best at relatively low readout speeds (Fig. A3.5). The other two trivial improvement, after it has been repeated as part of the 400 reasons are improved charge transfer efficiency and the reduction to 590 transfers in the gain register, a total average gain of hun- in broadband electronic noise from the FETs (noted above.) dreds or even thousands is possible. If the voltage on f2 is reduced to normal levels, the sensor operates as a normal CCD. As a result, a single PE can be amplified sufficiently to be safely above the noise of the FET amplifier, even when it is operating at speeds considerably higher than video rate (35–50MHz, vs. 13MHz for video). As the amount of gain depends exponen- 13 In a CCD without CDS, noise features will seem to be smeared sideways, tially on the exact voltage on f2, it is possible to “dial in” the while in one with CDS, they will appear as one-pixel-wide stipple with no amount of gain needed to keep the signal level well above the noise directionality. of the FET amplifier. However, it is important to remember that 926 Appendix 3 • J.B. Pawley

FIGURE A3.7. Energy diagram of an electron-multiplier CCD amplifier. The high field region that occurs between f2 and fDC when f2 goes strongly positive (right) causes about 1% of the electrons passing this region to collide with a lattice electron with sufficient energy to boost it to the conduction band. Repeated over hundreds of transfers, this process is capable of providing an average amplification of hundreds or even thousands of times.

the use of high EM gain will tend to saturate the “full-well” capac- EM-CCDs have one other important form of “dark noise” ity of later pixels in the gain register, reducing intra-scene dynamic called Clock Induced Charge (CIC, also known as spurious noise). range.14 Although this effect can be reduced to some extent by CIC typically consists of the single-electron events that are present making each pixel in the gain register (and the read node) larger, in any CCD, and are generated by the vertical clocking of charge this approach is limited by the fact that one triplet of electrodes during the sensor readout. The process involved is actually the can control a band of silicon only ~18mm wide and because a same impact ionization that produces multiplication in the gain larger read-node capacitance increases the read noise of the FET register; however, levels are much lower because lower voltages amplifier. are involved. In conventional CCDs, CIC is rarely an issue as In sum, the gain-register CCD works like a normal fast-scan single-electron events are lost in the read noise. However, in EM- CCD with no read noise. The high scan speed makes focusing and CCDs where the read noise is essentially zero and dark charge has sample scanning quick and easy and the device preserves the full been eliminated through effective cooling, CIC is the remaining spatial resolution of the CCD because the charge packet from one source of single-electron, EM-amplified noise. If left unchecked, pixel is always handled as a discrete entity (unlike in an intensi- it can be as high as 1 event in every 7 pixels. Fortunately, it can fied-CCD). Of course, with fast readout, there is less time to accu- be minimized by careful control of clocking voltages and by opti- mulate much signal and the resulting image may have considerable mizing the readout process to cope with faster vertical clock speeds Poisson noise. But this is not the camera’s fault! (down to 0.4ms/shift). This leaves a detector with less than one Alternatively, the output of many frames can be summed to noise pulse in every 250 pixels: a detector extremely well adapted reduce Poisson noise or, if the signal is bright, one can turn off the for measuring zero! EM gain and have a fully functional scientific CCD.15 If the gain-register CCD is read out fast, there is so little time for dark charge to accumulate that cooling would seem unneces- Of Course, There Is One Snag! sary until one remembers that one can now “see” even one PE of The charge amplification process is not quite noise free because dark-charge above the read noise. Because multi-pinned phasing the exact amount by which each electron in the packet is ampli- (MPP) is less effective during the readout clockings, significant fied varies in a stochastic manner (i.e., some electrons are “more dark charge can be generated during readout. If the exposures are equal” than others.). The statistical arguments are discussed in a short, this source of dark charge becomes significant, and in an paper found at the URL listed below and in Chapter 4. In summary: EM-CCD, even one electron is significant! In practice, the best as the multiplicative noise inherent in the charge multiplication performance is obtained when the EM-CCD is cooled to between process creates noise that has a form very similar to that produced -80° and -100°C. by Poisson statistics, the easiest way to think of its effect is to assume that the amplifier has no noise at all but that the signal being fed into it is half as big as it really is. In other words, the camera will work perfectly but it will work as though it has a QE 14 If a register designed with enough pixel area to hold a normal full-well charge that is only half of what it really is. Back-illuminated sensors are of 30,000 electrons, is used with a gain of 100¥, then the pixels near the end now available with an intrinsic QE of about ~90% or ~45% when of the gain register will become full whenever the original charge packet has >300 electrons. used in the gain-register mode. This is 5–10¥ better than the per- 15 Because, as noted above, the read node of the FET amplifier at the end of formance available from most PMTs especially in the red end of the gain register in an EM-CCD has a relatively large capacitance, E2V offers the spectrum. two separate FET readout amps. The one mounted at the end of the gain reg- It is worth noting that one can use electron multiplication and ister is optimized for fast readout. The other is mounted at the end of the HR not connected to the gain-register, has low input capacitance and is optimized still maintain the full QE by using the detector in photon-counting to read out slowly with low noise. Signal is sent to the latter by reversing the mode, as is now being done by many astronomers. Photon count- charge transfer sequence applied to the HR. ing is only possible when one is able to confidently see a single- More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3 927 photon event as different from any dark event and when the on back-illuminated chips, it can reach 90% (with somewhat number of photons collected during an exposure is so low that higher fixed-pattern noise). there is little probability of >2 photons falling into a pixel. To The fill-factor is the fraction of the sensor surface actually sen- implement photon-counting, one records a sequence of short expo- sitive to light. On the best frame-transfer CCDs, it can be almost sures containing “binary”-type single-photon data, and combines 100%. On interline transfer CCDs it may be only 40%. Light not 16 them to generate an image that is free of multiplication noise. To absorbed in a sensitive area is lost, reducing the QEeff of the sensor be useful for recording dynamic events in living cells, an extremely proportionally. fast frame rate would be needed. This may be more possible with Factors affecting QE: some future EMCCD sensors. (More info on EM-CCDs at http://www.emccd.com and Front-illuminated chips http://www.marconitech.com/ccds/lllccd/technology.html) • Light is scattered by the transparent, polysilicon charge- transfer electrodes that overlie the photosensitive silicon PART II: EVALUATING A surface. • This scattering is more severe at shorter wavelengths. Light CHARGE-COUPLED DEVICE that is scattered is not detected. • As blue light is absorbed nearer to the surface than red light, A. Important Charge-Coupled Device Specs for and “deep electrons” may go to the wrong pixel, CCD resolu- Live-Cell Stuff! tion may be a bit lower than the pixel count at longer wavelengths, especially on chips with small pixels. Although in Part I, much time was spent discussing cooled, slow- Best QE: ~20% blue, ~35% red/green scan, scientific CCDs, in fact, these have not been much used in • Two efforts to improve the QE of front-illuminated chips biological microscopy since Sony introduced the ICX085, 1k ¥ • include “Virtual Phase” (one phase “open,” Texas Instruments, 1.3k, micro-lens-coupled, interline-transfer chip in the late 1990s. Houston, TX) and the use of indium oxides for the transfer Although initially developed not for the scientific market but to electrodes (Kodak, Rochester, NY). These have increased peak meet the needs of the Japanese high-definition TV standard, these QE to the range of 55%. chips offered a set of practical advantages that biologists found very appealing: Micro-lens array chips — As an interline transfer chip, it needed no mechanical shutter • Sony has pioneered a process in which a micro-lens is mounted and could be run so as to produce a continuous stream of above each pixel of a front-illuminated, interline-transfer images. CCD. The lenses focus most of the impinging light onto a part — The high readout speed (up to 20Mhz) allowed real-time im- of the CCD where reflection losses are least, pushing the QE aging compared with the 5–10s/frame readout then common. to 65% in the green, less in the red (because of losses to the — The 6.45 ¥ 6.45 mm pixels were small enough to sample the overflow drain) and purple. image produced with high-NA 40¥ and 60¥ objectives. Back-illuminated chips — The 1k ¥ 1.3k raster size was both sufficient for most biological microscopy and significantly higher than that of the • Made by thinning the silicon and then turning it over so that other scientific chips then available. the light approaches the pixel from what would have been the — Mass production allowed the development of a micro-lens back side. This avoids scattering in the transfer electrodes and

array that increased the QEeff to an acceptable level for a front- increases the QE to about 90% in the green and >70% over the illuminated, interline chip and did so at a price biologists visible range. could afford. • More expensive because of the extra fabrication. Slightly less resolution and more fixed-pattern noise, caused As a result, the majority of CCDs sold for use in biological • by imperfections in the thinning operation, and the presence microscopy today use this chip or its higher-QE cousin, the of two sets of surface states. ICX285. Although mass production made quality CCDs available to many who formerly could not have obtained one, it is impor- Color Chips tant to remember that the read noise of ±8–24 electrons/pixel One-chip color sensors employ a pattern of colored filters, one (depending on read speed) is substantially higher than ±2–3 elec- • over each pixel. Light stopped by any such filter cannot be trons/pixel that characterized the best, slow-scan, scientific CCDs. detected and is therefore lost. The QE of such sensors is there- Although, as noted below, the difference is only important if the fore at least 3¥ lower than for an otherwise comparable mono- dimmest pixel records fewer than ~50 electrons, and this seldom chrome chip. occurs in widefield fluorescence microscopy, the disk-scanning 3-chip color sensors use dichroic mirrors to separate the confocal fluorescence microscopes now available do provide an • “white” light into three color bands, each of which is directed image in which this difference is significant (Chapter 10). to a separate monochrome CCD sensor. While this would seem to ensure that “all photons were counted somewhere,” because 1. Quantum Efficiency (QE): such systems seldom employ microlenses, their effective QE QE is the ratio of photons striking the chip to electrons kicked into is not much better than the 1-chip color sensors and alignment the conduction band in the sensor. It should be at least 40% and of the signal light is important.17

17 While the QE is not much better, the resolution of the 3-chip camera is the 16 There is no multiplicative noise because any spike above the FET noise ﬂoor same as that of each chip, without the interpolation needed to disentangle the counts as one electron, no matter how much it has been ampliﬁed. 3 colored images from the output of a 1-chip color sensor. 928 Appendix 3 • J.B. Pawley

• Color can be detected by making sequential exposures of a TABLE A3.2. Dynamic Range and Pixel Size monochrome chip through colored glass or LCD filters. This 12-bit camera 14-bit camera produces the same QE losses as the patterned filter but has w/small pixels w/large pixels the advantage that it can be removed when higher sensitivity is needed. This design is not suited for imaging moving Pixel Size 6.7 ¥ 6.7mm 24 ¥ 24 objects. Full Well 27,000 345,000 Least significant bit = 6.5 electrons 21 electrons Implied noise level ±13 electrons ±42 electrons 2. Readout Noise: This spec is a measure of the size of the pixels and the quality of the circuitry used for measuring the charge packet in each pixel. It is measured in “±RMS electrons of noise” (i.e., 67% of a series on the chip determines the total specimen-to-chip magnifi- of “dark” readings will be ± this much). cation needed! • A good scientific CCD camera should have a noise level of Two examples: <±5 electrons at a readout speed of 1M pixels/second. • The readout noise increases with the square root of the readout a. 1.4 NA 100¥ objective and a 1¥ phototube. speed (see Table A3.2). • The Abbe Criterion resolution @ 400nm is about • NO Free Lunch! A chip that has ±5e RMS of noise when 0.22mm. Magnified by a total magnification of 100¥, readout at 100k pixels/sec (or 10 seconds to read out a 1024 this becomes 22mm at the CCD. ¥ 1024 chip), should produce ±50e RMS of noise if read out • A CCD having 8 ¥ 8mm pixels samples such an image at 10M pixels/sec (or 0.1 sec to read the same chip). adequately (~2.8 pixels/resolution element). b. 1.3 NA 40¥ objective and a 1¥ phototube. • The Abbe Criterion resolution @ 400nm is now What Is “Good Enough”? 0.25mm. Magnified 40¥ this becomes, 10mm. Very low readout noise is only essential when viewing very dim • A CCD having 8 ¥ 8mm pixels is inadequate to sample specimens: luminescence, or low level fluorescence. Read noise is this lower-mag, high-resolution image. only a limitation when it is more than the statistical noise on the photon signal in the dimmest pixel (i.e., >sqrt of the number of If you must use this objective, you need either a higher mag detected photons = sqrt # electrons). phototube (2.5¥) or a chip with 3 ¥ 3mm pixels or (as CCD Consider the signal levels that you plan to use. Will the darkest pixels are seldom this small), some combination. important part of your image have zero signal or do you expect • Saturation signal level: The maximum amount of signal that some background signal from diffuse staining or out of focus light? can be stored in a pixel is fixed by its area. The proportion is If the dimmest pixel in your image represents ~100 electrons, then 600 electrons/square mm, so a 10mm ¥ 10mm pixel can store the Poisson or statistical noise on this background signal will be a maximum of 60,000 electrons before they start to bleed into ±10 electrons. “Adding” an additional ±10 electrons of readout neighboring pixels. In practice, as fluorescent micrographs of noise will not make much difference to a measurement of this living cells seldom produce signals this large, large pixels are background signal and it will be even less significant when added usually unnecessary. to the even greater Poisson noise present in pixels where the stained parts of the image are recorded. However, the saturation level also represents the top end of This is especially true because RMS noise signals add as the another spec, the dynamic range. This is usually quoted as “sqrt of the sum of the squares” (i.e., the total noise from ±10 elec- 12-bit (4000:1) or 14-bit (16,000:1) etc., and represents the trons of readout noise and ±10 electrons of Poisson noise is only ratio between the full-well saturation level and the readout sqrt (100 + 100) =±14 electrons). noise. Therefore, a camera with relatively high readout noise On the other hand, if you are really trying to keep those cells can still look good in terms of dynamic range if it has large alive and you find that 2,000 electrons in the bright areas is enough, pixels and hence a high full-well capacity. Conversely, a 12-bit the dark areas may now be only 50 electrons. As the sqrt of 50 is camera with small pixels can have less actual noise-per-pixel about ±7, an additional ±10 electrons of readout noise may no intensity measurement than a 14-bit camera with large pixels. longer be acceptable, but only if you have to make measurements In this case, the noise level of the 14-bit camera is >3¥ that in the dark areas on your image. In this case, the obvious choice of the 12-bit camera. Your signal/pixel would have to be is a slower, quieter CCD or an EM-CCD. 3¥ larger in order to be “seen” when using this particular While in widefield fluoresecence, the background stain level 14-camera. is seldom so low that the sqrt of the signal recorded is lower than 19 the read noise, the disk-scanner does provide such an image 4. Array Size: The argument for small (Chapter 10). As one of the main advantages of disk-scanning is • Assuming 0.1mm pixels (referred to the object plane), a 512 that one can scan an entire image plane very rapidly, the fact that ¥ 512 pixel chip will image an area of the specimen that is one can read out the EM-CCD very rapidly without increasing the about 51 ¥ 51 microns. If this is enough to cover the objects read noise makes it the ideal detector for this type of scanner (or, you need to see, this small chip has a lot of advantages over indeed for high-speed line scanning confocal microscopes). chips that are 1024 ¥ 1024, or larger. • Lower cost 3. Pixel Size: • Nyquist sampling: The size of a pixel on the CCD is, in itself, not very important BUT one must satisfy the Nyquist crite- 18 Of two times smaller than the “resolution,” as defined by Rayleigh, or Abbe. rion: The pixels on the chip must be ~4+ smaller than the 19 The array size refers to the number of lines and pixels in the sensor, not to smallest features focused onto it18 (see Chapter 4): Pixel size its total area. More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3 929

• 4¥ fewer pixels to read out, meaning either: if the signal level is so low that 1s/frame is required to accumu- —4¥ slower readout clock, giving 2¥ lower readout noise. late enough signal to be worth reading out, then reducing the read — Same clock speed and noise level but 4¥ faster frame time. time much below 0.1s loses some of its appeal. (Easier to scan specimen to find the interesting part! Time Faster readout speeds are particularly important for moving is money!) specimens, especially when doing widefield/deconvolution or • 4¥ less storage space needed to record data. when following rapid intracellular processes, such as vesicle tracking or ion fluxes. The argument for big: 6. Shutter Stability: • Manufacturing improvements are reducing readout noise Though not strictly a CCD spec, electronic (LCD) or mechanical levels at all readout speeds, and CCDs with more pixels often shutters are often built into modern CCD cameras.21 The latter have also have smaller pixels which can lead to lower read noise. If the disadvantages of producing vibration and having a limited life- your labels are bright, having a larger chip allows you to see time but the advantage that they transmit all of the light when they more cells in one image (as long as they are confluent!). are open (even an “open” LCD can absorb >50% of the light, other Assuming that Nyquist is met in both cases, a large print of an electronic shutters may be better). image recorded from a larger sensor always looks sharper than There seems little point in having a camera capable of record- one from a smaller array. ing (say) 40,000 electrons/pixel with an accuracy of ±200e (or • Binning: Binning refers to the process of summing the charge 0.5%!) if the shutter opening time is only accurate, or even repro- from neighboring pixels before it is read out. This increases ducible, to ±10%. If one shutters the light source instead of the the size of each charge packet read (making it look brighter) camera, similar limitations apply. and reduces the number of pixels. For example: 2 ¥ 2 binning allows the owner of a 1024 ¥ 1024 chip to obtain the 7. User-friendliness: speed/noise performance similar to the smaller chip (512 ¥ State-of-the-art cameras often seem to have been designed to make 512) and to do so in a reversible manner. However, the optical sure that no one unwilling to become a devotee of “CCD Opera- magnification may need to be increased to preserve Nyquist tion” can possibly use them efficiently! Start off by asking to see sampling. an image on the screen, updated and flat-fielded at the frame-scan Before deciding that you need a larger chip, compare what you rate and showing as “white” on the display screen, a recorded would get if the same money were spent on another intensity that is only ~5% of the full-well signal. This is where you scope/CCD/graduate student! should do most living-cell work. Then ask the salesman to help you to save time-series of this image. Increase the display con- Bottom line: trast until you see the noise level of the image, both before and after “flat-fielding.” Put a cursor on one pixel in the top frame of • If more pixels means smaller pixels, they will each catch fewer the stack and plot its intensity over the series. photons unless the magnification is reduced proportionally. More pixels at the same frame20 rate mean somewhat higher 8. “The Clincher” (Well, at least sometimes...): read noise because the pixel clock must go faster. Ask him/her what the intensity number stored in the computer for some specific pixel means, in terms of the number of photons that 5. Readout Speed: were recorded at that location, while the shutter was open. To Although readout speed has been discussed above, we haven’t answer, the salesperson will have to know the QE, the fill factor mentioned that some good CCD cameras have variable speed read- and the conversion factor between the number of electrons in a outs and the new EM-CCDs impose no high read speed penalty pixel and the number stored in the computer memory (sometimes (Table A3.3). called the gain-setting). To help them out, any “real” scientific It is convenient to be able to read out the chip faster when CCD camera has the latter number written, by hand, in the front searching and focusing as long as one can then slow things down of the certification document (usually a number between 3 and 6). to obtain a lower read noise in the image that is finally recorded. If the salesman doesn’t understand the importance of this funda- However, the read speed is only one limitation on the frame rate: mental number, what hope is there for you? (Hint: It is important because the Poisson noise is the sqrt of the number of electrons in TABLE A3.3. CCD Specifications the well, not the sqrt of some arbitrarily proportional number stored in your computer.) Array size Pixel Clock Rate Noise level* Frame time Frame rate/s 640 ¥ 480 13MHz 200e/pixel** 0.033 30 B. Things That Are (Almost!) Irrelevant When (video rate) Choosing a Charge-Coupled Device for 512 ¥ 512 100kHz 5e/pixel 2.5sec 0.4 1MHz 15e/pixel 0.25sec 4 Live-Cell Microscopy 5MHz 35e/pixel 0.05sec 20 1024 ¥ 1024 100kHz 5e/pixel 10sec 0.1 1. Dynamic Range: 1MHz 15e/pixel 1sec 1 This is the ratio of the “noise level” to the “full-well” (or 5MHz 35e/pixel 0.2sec 5 maximum) signal. Although 16-bit may sound a lot better than 12- bit, you need to think before you are impressed. *Assumes conventional FET circuits. **The readout noise is relatively higher at video rate because the higher speed often precludes the use of various techniques, The noise level should not be more than 5 electrons/measure- such as correlated double sampling, that reduce readout noise. ment. Period!

20 The readout speed of a 2 ¥ 2 binned 1024 ¥ 1024 is a bit slower than an 21 Often the same advantage can be gained by shuttering the light source. This actual 512 ¥ 512 because twice as many vertical clock cycles are needed, may become more common as pulsed laser or light-emitting-diode light and one still needs to read out pixel by pixel in the horizontal direction. sources are introduced (see Chapters 5 and 6). 930 Appendix 3 • J.B. Pawley

Twelve bits is 4,000 levels. If the first level represents 5 elec- 3. “Imaging Range” “Sensitivity” (or anything trons (in fact, it should represent half the noise or 2.5e), then the measured in LUX): 4,000th represents 20,000 electrons or (assuming a QE of 50%), Stick to something you (and I?) understand: Photons/pixel or elec- about 40,000 photons/pixel/measurement. trons/pixel. The other conversions are not straightforward. How often do you expect to be able to collect this much signal from an area of a living cell only 100 ¥ 100nm in size? You should 4. “Neat Results”: be able to get a good, 8-bit image using only 6% of the dynamic 22 Unless you know how well stained the specimen is, you cannot range of a 12-bit CCD (Fig. A3.8). evaluate an image of it in a quantitative manner. (Though you As the “full-well” signal is only proportional to the area of the may not want to admit this!) By all means, view your own speci- pixel on the chip (area in sq. mm ¥ 600), the dynamic range is only mens, but viewing “test specimens” that are not expected to fade really impressive if it is high AND a chip has small pixels. Then and have a known structure (fluorescent beads in some stable it means that the readout noise is low. A test for actual dynamic mounting medium?) facilitates A/B comparison. If you do use your range is described below. own test specimens to compare cameras, be sure to view them on Bottom line: For disk-scanning confocal microscopy, a large the same scope, and with the same conditions of pixel size and dynamic range is only important if it reflects a low readout noise readout time etc. level. Better still... Easier to just check the readout noise!

2. High Maximum Signal (high, full-well C. A Test You Can Do Yourself!!! number, because of large pixels): Set up each camera that you want to evaluate on a tripod, add a On living cells, you will probably never have enough light to reach C-mount lens, and an ND 3 or ND 4 filter. Hook up a monitor or a full-well limit of even 20,000 electrons. Even if you do, there computer and view some scene in your laboratory under ordinary are better ways to use it (more lower-dose images to show time illumination (avoid light from windows which may vary from day course?). to day). Close the lens aperture down until you can no longer discern the image (see Fig. 4.20). This is the “noise-equivalent light level”: the signal level at which the electron signal (i.e., photons/pixel ¥ QE) just equals the total noise level. Your measure is the aperture at which the image disappears.23 Because it is sensitive to both QE and readout noise level, this is a very useful measure of what we all think of as the “sensitivity.” Of course, the signal level depends If the read noise is ±8 electrons, or 2 not only on the light intensity but also on the exposure time and gray levels, one can obtain a useful,“8-bit” (256 levels) image by the pixel area, so make sure to keep the former constant and make using only 6% of its dynamic range. allowances for the latter. If you do not have even these meager facilities (a C-mount lens, an ND filter, a tripod and some time), take an image of nothing. Look at “no light” for one second, and for 100 seconds. Ask to see a short line profile that plots intensity vs. position along a line short enough that one can see the intensity of each individual pixel. The difference in the average intensity between the short and long exposure is a measure of the leakage.24 With a little calibration from the published full-well specs (a spec less open to “interpretation” than “noise”), you can even get a direct measure of the read noise level from these dark images. (It should be the standard deviation of the values as long as they are counted in electrons, not “magic computer units” and as long as fixed-pattern noise is not a factor.) And just trying to work it all out will give you some idea if the salesman knows anything...

D. Intensified Charge-Coupled Devices FIGURE A3.8. Not using the full dynamic range of a CCD. As most scientific CCDs have more dynamic range than one “needs” in live-cell fluorescence Intensified CCDs (ICCDs) are just that: the mating of an “image microscopy, the excitation dose to the specimen can be reduced if one sets up intensifier” to a CCD. The idea is that the photon gain of the inten- the CCD control program to display an 8-bit image using only the bottom 1,024 sifier (can be 200–2000¥) will increase the signal from even a levels of a 12-bit image. Such an image is more than adequate for many functions in live-cell biological microscopy (particularly when other factors such as dye-loading etc., may cause larger errors) and will require only 6% as much signal as would a “full-well” image. 23 If the lens doesn’t have a calibrated aperture ring, you can open the aperture all the way and reach the “threshold” exposure level by reducing the exposure time and adding ND filters. Remember to also correct for pixel area. Larger pixels intercept more photons. 22 Remember, given optical and geometrical losses, you can collect no more 24 With a good EM-CCD, this measurement can be done using a short than about 3–10% of the photons produced, and, each fluoroscein molecule exposure and high EM gain, then counting the number of amplified dark will only produce perhaps 30,000 excitations before “dying.” charge/CIC spikes across a typical line of the raster. More Than You Ever Really Wanted to Know About Charge-Coupled Devices • Appendix 3 931 single photoelectron above the read noise of the CCD. This occurs, • Photocathode resistivity can produce “dose-rate” effects: non- and can be particularly useful where fast readout is needed such linearities in which the recorded intensity of the brightest areas as when measuring ion transients. Finally, pulsing the voltage on may depend on (and affect) the brightness of nearby features. the intensifier section makes it possible to shutter (“gate”) the Because I expect that EM-CCDs such as those mentioned camera on the ns time scale, making the ICCD useful for making above will soon supplant ICCDs except where fast gating is fluorescence lifetime measurements (Chapter 27, this volume). needed, I have not gone into more detail here. For more info, go However, ICCDs do not have the photometric accuracy of to: http://www.stanfordphotonics.com/ normal CCDs for a number of reasons: • The relationship between number stored in memory and the number of photons detected is generally unknown and ACKNOWLEDGEMENTS variable. • The intensifier photocathode has low QE25 (compared to that The author would like to thank Dr. J. Janesick, formerly of the Jet of a back-illuminated CCD). Propulsion Lab (California Institute of Technology, Pasadena, • The “resolution” is generally only dimly related to CCD array CA), for many conversations about CCD operation and for the size because of blooming in the intensifier. To check this, original sketches for Figures A3.1, A3.4, and A3.5 and to Colin reduce light intensity until you can see the individual flashes Coates, (Andor Technologies, Belfast, UK) for his helpful com- produced by single photoelectrons. See how many lines wide ments on the manuscript and for Figure A3.6. they are. (They should be one line wide.) • They have additional noise sources: phosphor noise, ions in intensifier section create flashes, high multiplicative noise in REFERENCES the intensifier section greatly decreases QEeff, etc. Inoue and Spring, 1997, Video Microscopy, Second Edition, Plenum, New York, 1-741, particularly Chapters 5–9. Pawley, J.B., 1994, The sources of noise in three-dimensional microscopical data 25 And the GaAsP photocathode with better QE, have to be cooled, making the sets, Three Dimensional Confocal Microscopy: Volume Investigation of assembly very expensive. Biological Specimens, (J. Stevens, ed.), Academic Press. New York, 47-94.

Index

I suppose it is inevitable that indexes are compromises: If one includes every mention of every entry, the index becomes as long as the book. There is also the time dimension: As one cannot start writing the index until the book has been paginated, every day spent on the index directly delays the publication date. For the Second Edition, I prepared the index somewhat in parallel with the page proofs and it took most of a semester. For this Third Edition, a professional indexer was used to compile the initial index. We then expanded the level of cross-referencing through a series of digital searches. The ﬁnal result may show its mixed parentage. As you use this index, please consider the following. I confess that many entries contain far fewer referents when they appear as sub- or sub-sub-heads than when they appear as capitalized headings. In addition, some See alsomarkers use acronyms and it is also true that these can get confused with the real title of the entry. In compensation, have tried to put in bold type those page numbers on which I one would ﬁnd the more comprehensive discussions of the topic we have added a period at the end of the major heads to distinguish them from sub-heads. My apologies for any errors. My thanks to Helen Noeldner for her calm and competent assistance during this long and laborious process. Please use the Feed- back page at http://www.springer.com/387-25921-X to bring errors to our attention so that they can be corrected in future printings. Remember that this Handbook has always been a community project. Good hunting!

JP, 2/21/06

Numbers dose vs. resolution, 616 4D imaging. See Four dimensional imaging. 2D imaging, blind deconvolution approach, layout, 614 4Pi microscopy, 561–570. 476–477. living mouse, 615, 617 4Pi-PSF, 570 2D-time vs. 3D-time, embryo, 762–764. mouse femur, 616 axial resolution, 563 2D pixel display space, 291. operating principle, 614 I5M, 561, 569–570 2DCHO dataset, 818. tumor-bearing mouse image, 617 OTF, 569–570 2DHeLa dataset, 818. optical coherence tomography (OCT), living mammalian cell imaging, 564–565 2-photon, (2PE). See Two-photon excitation. 609–610 Golgi apparatus, image, 566 3D Constructor, 282. human retina 609 lobe-suppression techniques, 561 3D imaging, alternative approaches, schematic, 610 interference of excitation and detection, 475–476, 607–624. See also, Xenopus laevis embryo, 610 561 Confocal topics; Multidimensional objectives on a tandem scanner, 154, confocal detection, 561 microscopy topics. 304 two-photon excitation (2PE), 561 episcopic fluorescence image capture optical projection tomography (OPT), MMM-4Pi microscopy, 554, 556, (EFIC), 607–608 610–613 563–564 light sheet microscopy (SPIM), 613 lamprey larva, 612 basics, 565 magnetic resonance microscopy (MRM), mouse embryo, 612 scheme, 563 618–624 plants, 774–775 optical transfer function (OTF), 562, 563 amplitude modulation of RF carrier, setup, 611 outlook, 568–569 620 real-time stereo imaging using LLCD point spread function (PSF), 562–563 applications, 623–624 related methods, 607–625 signal-to-noise ratio, 561 basic principles, 618–619 selective plane illumination microscopy space invariance of PSF, 457, 490, 564 botanical imaging, 624 (SPIM), 613 theoretical background, 562–563 developmental biology, 624 Medaka heart, 614 type C, with Leica TCS, 4Pi, 565–568 Fourier transform, image formation, surface imaging microscopy (SIM), imaging of living cells, 568 620 607–608 lateral scanning, 567 future developments, 624 3D Scanning Light Macrography, 672. mitochondrial network, image, 568 hardware configuration, 621, 622 3D for LSM, 282. optical transfer function (OTF), 567 histology, 623, 624 3D methods compared, 448–451, 644–647. resolution, 567 image contrast, 622–623 table, 647 sketch, 566 image formation, 619–621 3D multi-channel time-lapse imaging thermal fluctuations minimized, 567 Larmor frequency, 620 (4D/5D). See also, Time-lapse z-response, 563 phenotyping, 623 imaging. 5D image space, display, 291–294. schematic, 619 table, 384. 2D pixel display space, 291 strengths/limitations, 622 3D3T3 high-content screening dataset, 820, animations, 292–293 micro-computerized tomography 821. color display space, 291 (Micro-CT), 614–618 3DHeLa high-content screening dataset, efficient use, 292 contrast/dose, 614–615 820, 821. image/view display options overview, CT scanning systems, 615–618 3PE. See Three-photon excitation. table, 293

933 934 Index

5D image space, display (cont.) blind deconvolution to remove, RESOLFT/STED, 573 multiple channel color display, 292 480–481 self-absorption, 490 optimal use, 293–294 cause of signal loss, 330, 389, 395, spectra, 217, 267, 338–339, 345, 355, pseudo-color, 173–175, 190, 291 413, 457, 542, 661 390, 415–416, 421, 538–539, stereoscopic display, 293 chapter, 404–413 681–682, 706 true color, 291 correction for refractive index in UV, 195 mismatch, 192, 287, 411–412, 542 Absorption coefficient, complex specimen, A corrections for, 145, 411–412, 654–655 164. Abbe, Ernst, 1, 5. corrector optics, 192, 395, 398, 411, Absorption contrast, 164–167, 195, 427. Abbe refractometer, 377. 477, 640, 655, 657 equations, 164, 539 Abbe resolution criterion, 36, 37, 60, 61, deconvolution, 463, 466, 468, 469, 471, heating, 539, 685 65–68, 574, 575, 631–636, 928. See 480, 498–499, 784 Accuracy. also, Rayleigh criterion. generated by specimen, 192, 418, biological vs. statistical, 24, 36–37, 68, breaking the Abbe limit, 573 454–455, 654, 658, 747, 772, 775 312 calculation, 65–66 of GRIN lens, 108 position, 39–41 individual point features separated by, for IR wavelengths, 160 Acetoxymethyl ester indicators, 726. 68 measurement using small pinholes, deposits formaldehyde, 738 pixel size, 62, 65, 634–635, 784, 928 145, 407 derivatization, 738 Abbe sine condition, 151, 239. monochromatic, 147–151 formula/reaction, 359, 738 Abbreviations, list, 125. multi-photon excitation, 542, 407–410 loading method (AM ester), 358–359, Aberrations, 109, 146–156, 241, 411–412, PSF, 148, 407, 455, 471, 481, 492, 657 361, 726, 738–739, 744 471, 480–481, 542, 629, 640–641, secondary, 247, 249 painting brain slices with, 726–737 654, 655, 657–659, 747. See also, in thick embryo imaging, 747 Achrogate beam-splitter/scan mirror, 50, Chromatic aberrations; Refractive Zernicke coefficients, 247, 248 212, 231–232, 916. index mismatch; Spherical wave-front, measuring performance, 145 operation, 50, 232, 916 aberration. Ablation, 2-photon, 107, 764–765. Zeiss LSM5 line scanner, 212, 231–232, astigmatism, 145, 151–152, 245–247, Absorber, saturable-crystal, 107, 111, 112. 916 249, 483, to cover gap in titanium:sapphire lasers, Achromat, 152, 153, 244. axial, 242, 505, 542, 630 112 chromatic correction of, 153 chromatic, 152–156, 160, 177–178, 209, indium-gallium arsenide, InGaAs, 111 flatness of field and astigmatism, 152 242–243, 641, 659 Absorption, 25, 163, 309–312, 338–339, longitudinal chromatic correction, 153 in 2-photon disk scanning, 542, 550, 341, 514–518, 542, 550, 613, 704. measurement, 244 554 2-photon, 405, 535–536, 541, 545, 550, Acousto-optical beam splitters (AOBS), 45, of AODs, 56 552, 705, 719, 764, 884 55–57, 88, 102, 211, 218, 395. axial chromatic registration, 287, 658 caged compounds, 543, 544 to select wavelength and intensity, 88, chromatic registration, 657–658 CARS, 595–596, 599 102 of collector lenses, 657–658 contrast, 162–165, 211, 595, 610, 613, to separate illumination and emission, 45, intentional, for height measurement, 770, 779 218 224 cross-section, 189, 426 Acousto-optical components, 43, 54–57. magnification error, 155, 287, 331, 493, energy levels, 514, 517, 682, 697, 705, tellurium oxide crystal, 55 542, 641, 657, 883, 904 792 thermal stability, 56, 57, 219 measurement of, 243–244, 654, 659 excited state, 544, 692 Acousto-optical deflectors (AOD), 25, 33, multi-photon microscopy, 542 fiber optics, 501, 502 54–56, 88, 447, 519, 543, 664, 762, of optical fibers, 504, 507 filters, 552 908. signal loss, in confocal, 156, 178, 542, of fluorescent dyes, table, 345 as beam-splitters, to reduce loss, 33 641 fluorescent excitation, 45, 88 to gate light source, 25. See also, AOM standards, table, 157 FRET, 184 group velocity dispersion due to, 88, coma, 145, 151–152, 245–246, 249, 483, and heating, 21, 218, 252, 539, 685 540, 646 630 of incident light, 163, 177, 427 multi-photon excitation, 88, 540, 543, detecting, 241 by ink, 73 646 monochromatic, 147–152, 542. See also, and laser operation, 82, 108, 110, 116 multi-tracking, 664performance, 55 Spherical aberration light lost by, 25, 166, 414–418, 457, 654 problem descanning fluorescent light, 56, optical, avoiding with thin disk lasers, lighting models, 283, 285, 309–312 447 109 molar extinction, 80–81, 343, 353, 357, Acousto-optical modulators (AOM), 11, of refractive systems, 146–156 793 55–57, 88, 231, 519, 540, 543. signal loss, 156, 178, 542, 641 nonlinear, 188, 416, 427, 680, 704, FRAP experiments, for controlling laser, spherical, 15, 34, 147–149, 151, 160, 192, 709–710 56 208, 241, 244, 247, 330, 395, of optical materials, 158 group velocity dispersion, 88 404–413, 454–455, 463, 466, 480, and photodamage, 22, 685–686, 690, 750 Acousto-optical tunable filters (AOTF), 43, 542, 629, 640, 654–655, 657, 658, in photodetectors, 253 55–56, 88, 102, 219, 237, 346, 543, 728, 772, 774. See also, Spherical photon, 550, 749 651, 660, 673, 806, 908. aberration; Mismatch, refractive quantum dots, 221, 343, 357–358, 696, for selecting CW laser lines, 88, 102 index 759, 801 blanking, 54, 55, 237, 389, 543, 628, 651 Index 935

leakage, 660 AlexaFluor dyes, 81, 103, 184–185, 190, Anisotropic sampling, 287–288. to regulate light intensity, 43 192, 236, 330, 342–344, 353–357, when resampling, 833–835 to spectrally filter light, 55 360, 363, 393, 395, 416, 533, 540, Anisotropic specimens, 163, 286, 320, 329, thermal sensitivity, 56–57, 219 694, 726, 731, 749, 794, 799, 804, 420, 623, 675, 678, 690, 710, 793. Acridine Orange, 23, 344, 531, 665–667, 810, 814, 854, 878, 880, 905. Anisotropy analysis, chimeric proteins, 794. 691, 774, 874. fluorescence excitation, 355 Anisotropy of fluorescence, 742, 794. bleaching, 693–694 living cells rapid assessment, table, Anisotropy of interference filters, 49. Acronyms, list, 125. 360 Annular aperture, 4, 9, 20, 211, 889. Actin filament, 7, 236, 372, 378, 383, 692, structure, 356 3D pattern of point-source from lens, 3+ 696, 714, 719, 748–749, 753, 756, Alexandrite (Cr in BeAl2O4), tunable laser, 4–20 759–760, 773, 781, 804, 819, 109. in specimen-scanning confocal 824–825, 854, 856. Alga. microscope, 9 widefield source suitability, 142 autofluorescence, 357 Anti-bleaching agents, 36, 340, 363, 368, Active laser medium, defined, 81. autofluorescent image, 173, 175, 192, 375, 499, 694. Active mode-locked, pulsed laser, 111. 194–195, 438–439, 528, 585, 785, Antibody stains, 292, 339, 342–343, 348, Actual focal position (AFP) defined, 405. 870, 881–885 357–360, 375, 528, 576–578, 582, Actuator, galvanometer, 52. biofilm, 870, 881–885 610, 612, 664, 696, 731, 748, 760, Acute neocortical slice protocol, 723. cell chamber for, 429 789, 802–804, 812, 852–855, Adams, Ansel, zone system, 71–72. in laser cooling water, 116 877–880. Adaptive optics, 892. Aliasing, 38–39, 271, 291, 293, 448, 588, artifacts, 664 ADC. See Analog-to-digital converter. 590–592, 640, 830, 833–834, biofilms, 877–880 Adipocyte cells, CARS imaging, 604. 836–839, 903. FRET, 790–791 Adjacent fields, automated confocal and Nyquist criterion, 38–39, 448 high-content screening, 812–815, 818 imaging, 810. temporal, 39, 41, 391, 836–837, 839 in situ, 612 ADU, analog digital units, 74–77, 630, 925. Alignment, 25, 85, 134–135, 157, 505, penetration, 387 Advanced Visual System. See AVS. 629–631, 651. preparation, 369, 371–372, 375–377, 878 Aequorea victoria, biofilms, 348, 356, 736, of laser systems, to reduce instability, and TEM, 852–855 794, 873–874, 877. 85 Antifade agent, 36, 340, 363, 368, 375, 499, variants, table, 873, 874 of optical coherence tomography, 610 694. See also, Antioxidants. Aequorin, Ca2+ reporter,736–737, 739, 741, of optical system, thermal stress, 85 Antiflex optics, to reduce reflections, 158, 802. importance, 25, 630 171, 507, 513. developmental cellular application, 736 and PSF, 646 Antioxidants, living cell imaging, 341–342, ion binding triggers light emission, 737 of source, 134–135, 629–631 363, 389, 390, 729, 794. Ca++ signal detection, 737 Alkali vapor lasers, diode-pumped, Anti-reflection (AR) coatings, 1, 8–9, 25, AFP. See Actual focal position. 103–105. 49, 117, 139, 145, 151, 158–159, AIC. See Akaike Information Criterion. Allium cepa. See also, Onion epithelium. 212, 505–506, 901. Airy aperture, optimum for NA, 28. Alpha blending, 302, 304. color effect, 139

Airy disks, 4, 24, 65, 131, 145–146, 151, Alumina (Al2O3) ceramic tubes for lasers, of optical fibers, 506 156, 210, 443, 444–449, 454–456, 102. AOBS. See Acousto-optic beam-splitter. 463–465, 474, 485, 492–493, 562, Amira, 282–283, 286, 296, 302, 308, 312, AOTF. See Acousto-optic tuning filter. 567, 630, 655–657. 775–778. APD. See Avalanche photodiode. Abbe criterion resolution, 65–66, 225 Amoeba pseudopod, detail, 168. Apochromat, 15, 147–148, 151, 153–155, defined, 146, 444 Amplifier rods, maintenance, 116. 158, 240–245, 409–410, 454–455, diameter in image plane, 210, 225 Analog digitization, for photon counting, 29, 655, 659, 771. four-lobed, from astigmatism, 151 33–37, 41, 65, 74, 78, 251, 254, chromatic correction, 153 image, 38, 146, 225 258–261, 263–264, 404, 460, 495, compared with fluorite objective, 154 intensity ratios, 28, 145–146 522, 525–526, 542, 634, 766. longitudinal chromatic correction, 153 inverse, 11 Analog-digital unit (ADU), to calibrate Apodization, high-NA objective lenses, 240, and line spacing, 24 CCDs, 74, 77, 630, 925. 243, 249–250, 272, 567, 889. radius and pixel size, 4, 24, 38, 39, 60, Analog-to-digital converter (ADC), 31–34, Applied Precision Instruments (API), 131, 65–67, 227, 485 64–66, 70, 72, 74–75, 258–259, 261, 137, 282, 388, 651. vs. NA and wavelength, 1, 4, 146 263, 286, 521, 630–632, 924–925. APSS up-converting dye, saturation, 165. Airy figure image, 38, 75, 79, 146, 147, Analyze (software), 281–282, 288, 290, AR. See Anti-reflection. 225, 479, 486–487, 562. 301–304, 312, 651. A. thaliana, 169, 173, 174, 175, 193, 196, FWHM as optimal pinhole/slit size, 28, Analyzer, in pol-microscopy, 25, 157, 229. 202, 416, 420–421, 423, 425, 426, 36, 225, 232, 443, 454, 463–465, Analyzer, spectrum, 901–902. 427, 431, 771, 772, 773, 775, 778, 564, 567–568, 630–631, 633, Anemonea majano, sulcata, 874. 779, 780. 655–657 Angular deflection, distortion, 211. attenuation spectra, 416 and resolution, 65–67 Aniline Blue stain, 430–432, 435, 438, 774. birefringent structures in cells, 420–421. size, and Nyquist criterion, 38, 39, 60 Animations, 281, 283–285, 289–290, See also, Anisotropic specimens Airy unit, 28, 36, 41, 210, 222, 227, 232, 292–293, 295, 299, 308, 312, 764, bleaching, 203 274, 443–451, 632, 775, 779. 829, 835–839, 841–844. double imaging, 169 Akaike Information Criterion (AIC), 825. Anisotropic crystals, 114. fluorescence spectra, 421, 423, 425 936 Index

A. thaliana (cont.) Atto Bioscience CARV confocal scanning electron micrographs, GFP protein fusion, 773 microscope, 215, 229, 230, 907. 851–852, 855 limitations for imaging, 772 Autofluorescence, 44, 81, 90, 173, 175, 195, TEM implementation, 858–859 mesophyll protoplasts, 196, 426 202, 339–340, 360–361, 369–370, neurobiology example, 320 optical sectioning, 772, 775 387, 414, 416, 421–434, 442–445, quantitative morphometry, 331 protoplasts, 195–196, 203, 416, 421, 447–449, 451, 509–510, 528, 530, rationale, 316 425–427, 429–430 438–439, 693 545, 607, 612, 663, 667–670, 678, registration synthesis, 328–331 root tip fluorescence spectra, 173–175 682, 690, 698, 706, 710–711, 713, defined, 328 seedling, autofluorescent image, 202 729, 742–743, 745, 764–765, landmark-based, 328–329 three-dimensional reconstruction, 190, 769–773, 779, 781–782, 785, 798, multi-view deconvolution, 291, 330, 193, 771, 775, 777–778, 781 815, 874, 876, 881–885. 675–677 two-channel confocal images, 169, 175, of alga chloroplast, 168, 172–176, 202, segmentation methods, 321–322 193, 196, 203, 427, 431, 772 429–435, 556, 785 bottom-up, 321 two-photon excitation, advantages, 779, A. thaliana seedling, 202, 303, 307, hybrid, bottom-up/top-down, 322 780 772 integrated, 322 two-photon fluorescence image, 427, 780 bleaching, 202, 698, 729 intensity threshold-based, 321 two-photon fluorescence spectra, 425, 426 cell wall, 303, 431, 438, 770 region-based, 321–322 Arc lamps, 132, 136–138. emission spectra in plants, 176, 421–423 top-down, 322 current/stability of plasma, 138–139 extracellular matrix, 311 segmentation testing methods, 333–334 monitoring during exposure, 137 fixation, as a cause, 358, 369–370 manual editing, 333–334 radiance, 137–138 fluorescent probes, 339–340, 360–361 specimen preparation, 319–321 sensitivity to environmental variation, 136 harmonic signals. See Harmonic signals imaging artifacts, 320 shape of discharge, 132 lamprey larvae, 612 stereology, 316 shift of wavelength with temperature, 137 multi-photon microscopy (MPM) See time series in vivo images, 319 stability of, vs. filament lamps, 137 also, harmonic signals, 545 tube-like object segmentation example, Area of interest. See also, Region of optical materials, 45, 158 324–328 interest. plants, 190, 193–195, 421–428, 770–772 mean/median template response, 328 identifying, 201–202 plots, 176, 421–423 skeletonization methods, 324–325 Argon-ion laser, 85–86, 90–102, 107, removal using spectral unmixing, 192, vectorization methods, 324, 326, 109–110, 112, 119, 124, 203, 338, 382, 664–667 327 341, 346, 353, 355, 375, 540–541, examples, 665–666 types, 318–319 655, 657. removal on basis of fluorescence lifetime, Automated fluorescence imaging, 814. CW, 90–103, 107, 109–110, 112, 119, 345–346, 348, 349, 528 endpoint translocation assays, 814 124 UV excitation, 347 Automated interpretation of subcellular emission stability, 86, 102 Automated 3D image analysis methods, patterns, 818–828. See also, references, 124 316–335. See also, Automated Automated 3D image analysis Argon-krypton mixed-gas laser, 90, 92, 93, interpretation of subcellular methods 2D dataset analysis. 102, 108, 119, 203, 343, 375, 748, patterns. automated 2D analysis methods, 818 798, 811. biological objects, 319 2D subcellular location features, Artificial contrast, vibration and ambient blob segmentation example, 322–324 819–820 light, 201–204. gradient-weighted distance transform, 2DHeLa dataset images, 819 Artificial lighting, image display, 306–312 323 CHO cell dataset, 818, table, 820 Astigmatism, 145, 151–152, 245, 247, 249, model-based object merging, 323–325 Haralick features, 818–820 483, 505, 542, 630. watershed algorithm, 322–325, 777, HeLa cells 2DHeLa dataset, 818 of AOD, 914 822 Zernike moments, 818–820 and flatness of field, 152 combined blob/tube segmentation, automated 3D analysis methods, 824 and intensity distribution, 152, 246, 630 328–330 classification results, 824 laser optics, 89, 106–107, 505 data collection guidelines, 319–320 feature normalization, 824 measuring subresolution pinholes, 145 defined, 316, 328 feature selection, 824 at off-axis points, 151, 245, 247, 249 future directions, 334 automated classification of location ATP-binding cassette, 362. hypothesis testing, 318 patterns, 824–825 ATP-buffer, 802–803, 812. illustrations, 317 classification accuracy, 826 ATP-caged, 544. image preprocessing, 320–321 confusion matrix for 3DHeLa images ATP-gated cation channels, 359. background subtraction, 320 using SLF10, table, 824 Attenuation of light. morphological filters, 320 confusion matrix for 3DHeLa images by specimen, 164, 287, 298, 304, 320, signal attenuation-correction, 320–321 using SLF17, table, 825 321, 414–418, 428, 439, 538, 558, vs. manual, 316–317 features in SLF17, table, 825 706, 779, 782 montage synthesis, 282, 293, 312, measured classification accuracy, table, plots, 415, 706 328–332, 748, 753, 851–852, 855, 825 of laser beams, 85, 87, 354, 415, 904 858–859 clustering of location patterns with modeling, 309, 311, 320–321, 330 defined, 329–330 clustering consistency, table, 826 of PSF, 456, 462–463, 466, 494 examples, 330–332, 780–781 exclusion of outliers, 825 x-ray, 614–615 neuron, 330 methods, 826 Index 937

optimal clustering determination, focus shift, 243, 407–410 cheek cells, 22, 23 825–826 as function of pinhole diameter, 656 diatom, 145, 438, 638–640, 881 optimal consensus tree, 827 magnification, 215 latex bead, 182, 196, 197, 653 clustering of location patterns, 825–826 measurement, 194, 656–657, 659 transparent ciliate protozoa, 141 downsampled images, different gray multi-photon, 750 LLLCD objectives/3D color-coded BSL scales, 824–825 multiview, 678 as a noise signal, 663 future directions, 827–828 near focal plane, slit-/point-scan confocal optical coherence tomography, 609 high-resolution 3D datasets, 820–822 microscopes, 225–228 practical confocal microscopy, 631 3D3T3, 820 SHG, 704 from specimen, 202 3DHeLa, 820 SPIM, 614, 674, 751 unmixing, 192, 382, 664–667 color images from 3DHeLa, 821 STED, 571–577 Back-thinned CCD, 31, 77, 222, 232, 234, image acquisition requirements, tandem-scanning confocal microscope, 6, 754. 821–822 225 QE plot, 29 images from 3D3T3, 821 tomography, 610–611 Bacteria. See Biofilms. image database systems, 827 using mirror, 656–657 Ballistic microprojectile delivery, 360, 726, image processing/analysis, 822–823 803. 3D SLF, 822–823 B Ballistic photons, 418, 427, 538. edge features, 823 Back-focal plane (BFP), 34, 50–51, 58, Ballistic scans, 40, 41. feature calculation process, 822 61–62, 84, 126–128, 166, 208–210, Balloon model segmentation methods, 776. morphological features, 823 225, 239, 268, 487, 509, 627, 629, Bandpass, optical filters, 43–44, 46, 48, 49, segmentation of multi-cell images, 822 708. 51, 76, 87, 132, 141, 173, 204, 341, texture features, 823 Background light, from transmission 528, 708, 798. protein subcellular location, 818 illuminator, 201–202. for CARS, 598–599 statistical comparison of patterns, Background noise, 260–262, 275. coupling short and long-pass filters, 46 826–827 Background signal, 12, 26, 28, 37, 68–69, excitation and emission, 48, 141, 217, AutoMontage software, 282, 293, 304. 71–72, 88, 90, 112, 115, 158, 162, 341, 708, 757, 798 Avalanche photodiode (APD), 77, 233, 168, 172–173, 175, 184, 188, laser, 106–107 252–255, 404, 527, 542, 558, 567, 201–202, 221–225, 227, 232, 235, liquid crystal, 425 698. 248, 251, 257, 266–275, 278–279, to select range of wavelengths, 43–44 array, for multi-beam sensing, 558 283, 287,-288, 290, 301–302, 305, spectral detector, 203–204, 662–663, noise currents, 256 312, 321, 326, 339–340, 343, 345, 666–667 pulse pileup, 253, 527 348, 360–362, 375, 421, 423, Bandwidth, 32, 64, 69. unsuitability for non-descanned detection, 428–429, 432–433, 442–451, 462, 3dB point, definition, 59, 65 542 465, 472–477, 486, 493, 497, 506, of AOBS, 57 vacuum ADP, 254–255 509–510, 518–519, 535, 541, 543, electronic/optical, digitization, 32, 34, 70, Average intensity, 66, 110, 516, 556, 668, 553, 559, 582, 584–585, 595, 238 684, 695, 747, 763–764, 816, 838, 598–600, 602, 604, 621, 633, 656, head amplifier, 251 930. 663–370, 676, 694, 697, 698, 707, limiting, to improve reconstruction, 69 equation, 302, 309, 668 713, 727, 733–734, 736, 747, Nyquist reconstruction, output, 64, 69, 70, AVS (Advanced Visual System), 282–283, 755–757, 760, 798, 801, 803, 809, 238 286, 300, 311–311, 862, 863. 813, 815, 818, 822, 830, 836, 839, Bead, fluorescence emission, 181, 182, 196. Axial chromatic aberration, 155, 658–659. 851. fluorescent, 454, 477, 493, 499, 527, 652, Axial chromatic registration, 154, 658. Background subtraction, 284, 301, 320, 473, 653, 656, 659, 784, 900, 904, 930 Axial contrast. See z-contrast. 510. image, 656 Axial edge response, 409–410, 654. Back-illuminated CCD, 31, 77, 222, 232, table, 653 calculations for glycerol, table, 409 234, 754. glass, in water, 181, 198–199 calculations for water, table, 409 Back-propagation neural network (BPNN), latex, fluorescence image, 196, 407, Axial illumination, 60–61, 134. 818. 455–457, 463, 471, 656 Axial laser modes, 82, 110. Backscattered light (BSL), 22–23, 57, in water, confocal serial sections, 182 Axial minimum, 3D diffraction pattern, 4, 83–84, 130, 141, 145, 165, 169–170, Beam blanking, 54, 55, 237, 389, 543, 628, 147. 180–182, 191, 196, 202, 212, 221, 651. Axial rays, spherical aberration, 148. 228, 240, 376, 378, 416, 430, 436, Beam collimation, 728. Axial resolution, 3–4, 6, 172, 182, 209, 211, 442, 631, 879. for fiber delivery, 506 225–228, 230, 240–241, 243–244, access to, antiflex optics, 6, 57, 141, 212, Beam delivery, with fiber optic coupling, 320, 370, 395, 407–411, 413, 229, 507, 513, 609, 631, 704, 707, 85–88, 107, 216, 503, 506–508. 444–446, 489, 493, 499, 511, 513, 854, 879, 990 Beam deviation, unintentional, 15–16. 551–553, 559, 561–568, 571–577, biofilm, image, 880 Beam expander, 8, 84, 124, 208, 212–214, 610–611, 613, 649, 651, 654, contrast, effect of specimen absorption, 231, 650, 682, 708, 728, 907. 656–657, 659, 674, 704, 747, 165 advantages, 213 750–751, 822. effect of coherence on, 130–131, 170 Beam pointing, lasers, 85, 103, 107, 201, 4Pi microscopy, 561–568 images made using, 22–23, 154, 436–438, 250. coding, display, 305 513, 638, 855, 880 active cavity stabilization, 87 defined, 3–4, 240, 444–446 Amoeba pseudopod, 168–170, 191 Beam quality, of diode lasers, 107. 938 Index

Beam shift, vignetting due to, 211. pupil engineering, 896 time-lapse confocal imaging, 885–886 Beam-splitter, 33, 46–48, 50–51. See review articles, 889 transmitted laser light image, 880 Dichroic mirrors. technical interests, 891–892 Bioimagers, kinetics, endpoint analysis, Achrogate, 50, 212, 231–232, 916 theory, 890–891 816–817. AOBS, 56–57 thickness, 896 Biolistic transfection, 360, 724–726, 803. broadband, 346 turbidity, 896–897 Biological accuracy, vs. statistical accuracy, dichroic, 25, 33, 35, 43–51, 56–57, variants on main theme, 897–899 24, 36–37, 68, 73, 312. 83–84, 88, 139, 132, 135, 143, 151, Binding equation, for fluorescent indicators, Biological reliability, of measurements, 24, 162, 203–204, 207–208, 211–214, 740. 36–37, 68, 73, 312. 217–218, 229, 231–232, 266, 339, Biocytin, 730, 731. Biological specimens, 6, 11, 12–13. See 341, 346, 375, 386, 424, 469, EM imaging of brain cells labeled, 731 also, Plant cell imaging, Biofilms, 503–504, 552, 563–564, 599, protocol, 730 Specimen preparation, and entries 630–632, 647, 650, 657–658, 664, Biofilms, 287, 688, 529, 530, 624, 779, under specific equipment and 667, 691, 707–708, 747, 771–772, 870–887. cell/tissue type. 810, 846, 879, 910, 907 2-photon imaging, 530, 882–885 backscattered light images, 22–23, 25, table, 799 dual-channel imaging, 884 167–168, 170, 880 fiber-optic, 503–504 limitations of CLSM and 2-photon, 884 CARS imaging, 603–604 forty-five degree, performance, 47 single-photon/2 photon comparison, adipocyte cells, 604 fused-biconic coupler, 503–504 883 epithelial cells, 603 long-pass cut-off, 43, 46, 51, 175, 204, thick environmental biofilms image, erythrocyte ghosts, 603 564, 801, 875 885 distortions caused refractive index multi-photon, 540–541 autofluorescence, 545 inhomogeneity, 40–41, 181, 182, polarizing, 13, 50, 57, 85, 87, 100, 217, backscattered light, 880 198–199, 419 513, 631 fluorescent proteins for, table, 874 tandem scanning systems for, 6, 11 spectral problems, 50–51 future directions, 887 Yokogawa CU-10, 12–13 triple dichroic, 33, 46, 48, 217–218, 658, GFP variants for, table, 873 Biophotonic crystals, 188, 428. 783 imaging extracellular polymeric Bio-Rad, 25, 33, 35–36, 70, 113, 214, 260, losses due to, 33 substances (EPS), 879–882 630, 638–640, 657, 748–752, 757, performance, 46–48 lectin-binding analysis, figures, 881, 759–762, 858, 889. Beam scanning, along optical axis, 215, 882 1024ES, 710–711, 714, 718–719 555. lifetime imaging, 530 data storage, 585 Beam-scanning confocal microscope. See magnetic resonance microscopy, 624 using white light source, 113 Confocal entries; Flying spot making bacteria fluorescent, 873–874 MRC 1024, photon counting, 33 ultraviolet (UV) microscope. pH imaging, 530, 739–745 photon efficiency, 25, 32, 261, chromatic correction, 177 sample mounting, 870–873 748–752 Beam-scanning systems, 6, 7, 16, 132, 146, flow chamber system setup, 872–873 MRC-600 scanner, full-integration 151, 156, 166, 177, 214–215, 218, perfusion chambers, 870–872 digitizer, 70 381, 554, 562, 564, 567, 568, 599. pump selection, 871 PMT, 260–261 coma in, 151 upright vs. inverted microscopes, 870, Radiance-2100, 23, 185 off-axis aberrations affecting, 156 872 resolution, 657 Before-bleach/after-bleach ratio, FRET, 794. water-immersible lenses 149. 161, 209, Biosensors, fluorescent, 33–8348, 799, 805. Benchtop fiber-optic scanning confocal 411, 429, 568, 613, 727, 737, 870, See also, Dyes, Fluorophores, and microscopes, 507–508. 872. Chapters 16 and 17. Bertrand lens, 61, 157, 412, 643. stains for, 874–879, 875 future, 805 Beryllium oxide (BeO), for laser tubes, 102. Acridine Orange, 23, 344, 531, mitotic clock measurements, 799 Beta barium borate (BBO), non-linear 665–667, 691, 774, 874 Birefringence, 6, 15, 54, 83, 103, 109, 113, crystal for frequency doubling, 100, antibodies, 877–878 116, 162–164, 188, 189, 414, 109, 114–115, 125. biofilm community on tooth, 879 420–421, 431, 434, 436, 438, 479, BFP. See Back-focal plane. DAPI, 874. See also, DAPI 503, 710–711, 714, 717, 894. Bibliography, annotated, 889–899. effect of antibiotic treatment, 877 acousto-optics, 54, 55 adaptive optics, 892 embedding for FISH, 876–877 collagen fibers, 164, 188, 717 books on 3D light microscopy, 889 FISH with fluorescent protein, contrast, 15, 162–164, 188, 414–428, differential phase contrast, 892 875–876, 878 431–438, 710–711, 714, 717, 719, display methods, 892–883 imaging bacteria, backscattered light, 894 fiber-optic confocal microscopes, 883 879 deconvolution, 479–480 general interests, 891 live/dead stain, Streptococcus gordonii, defined, 163, 188 historical interests, 889–890 876 in fiber-optics, 503 index mismatch, 893–894 nucleic acid, 874–875 harmonic generation from, 428, 431–438 multiplex, 894 preparing labeled primary antibodies, images of Cymbopetalum baillonii, 189 non-linear, 894 878 in laser components, 85, 103, 109, 113, point spread function, 895–896 SYTO, 874–875 116 polarization, 894–895 temporal experiments, 885–886 quarter-waveplate, 6 profilometry, 895 multi-cellular biofilm structures, 886 table, 715 Index 939

Birefringent crystals, 188, 420–421. Blind deconvolution, 190, 468–487. See Borohydride, to reduce glutaraldehyde optical effects of acoustic fields on, 54, also, Deconvolution. autofluorescence, 374, 770. 55 2D approach, 476–477 Botanical specimens, 414–439, 624, Black-body radiation, 44, 135–136. 3D approach, 475–476 784–785. See also, Plant cell from incandescent lamps, 44, 126, advantages/limitations, 468–472 imaging, and Chapters 21 and 44. 135–136 algorithms, 472–474 birefringent structures, 420–421. See also, spectrum, 136 of A. thaliana seedling image, 190 Birefringence Bleaching, 10, 12–13, 20, 24, 44, 63–64, 90, confocal stack, 470 deconvolution, 784–785 142, 186–187, 194, 202–203, 210, data collection model, 472 effect of fixation on, 195, 428 218, 220, 222, 340, 382–387, 442, data corrections, 477 Equisetum, 774 539–540, 690–702, 797, 905, 907. defined, 469 fluorescence properties, 421–428 2-photon excitation, 539–540, 680–689, DIC schematic, 475 emission spectra, 421–423 905 DIC stack example, 470 microspectroscopy, 421–426 acceleration, 341 different approaches, 475–477 fluorescence resonance energy transfer, of acceptor in FRET, 184–187 deblurring algorithm, 476 425. anti-bleaching agents, 36. See also, Anti- Gold’s ratio method, 476 See FRET, 425 bleaching agents inverse filter algorithm, 476 harmonic generation properties, 428, bleach patterns, 3D, 538, 628, 693 iterative constrained algorithms, 711–715 beam blanking, to reduce, 53–54 475–476 light attenuation in plant tissue, 414–418 before/after ratio, for donor/acceptor pair, Jansson-van Cittert algorithm, 476 absorption spectrum, 415 794 nearest-neighbor algorithm, 476 A. thaliana example, 416 chapter, 690–702 no-neighbor algorithm, 476–477 maize stem attenuation spectra, 417, combining fluorescence with other, processing times/memory table, 476 418 383–386 Richardson-Lucy, 497, 568 M. quadrifolia attenuation spectra, 416 in dye lasers, 103 TIRF microscopy, 477 M. quadrifolia optical sections, 419 dynamics, 202–203 differential interference contrast (DIC), Mie scattering, 162–163, 167, 417–418 fluorescence correlation spectroscopy, 473–475 nonlinear absorption in, 416–417 383, 801 examples, 469, 470, 481, 482, 483 Rayleigh scattering, 162–163, 167, 417, fluorescence lifetime, 382–383 flowcharts, 473, 474 703 fluorescence recovery after future directions, 483 light-specimen interaction, 425–428 photobleaching, 51, 54, 56, 80, 90, Gerchberg-Saxton approach, 472 living plant cell, 429–439 187, 210, 218, 224, 229, 237, 362, hourglass widefield PSF, 474 calcofluor staining procedure, 424, 438 382, 684, 390, 691, 759, 801, 805, light source/optics alignment, 478 callus, 429 850 maximum likelihood estimation (MLE), cell walls, 168–169, 188–189, 303, FRET, 186, 382, 794–798, 800 472–477, 669–670 306, 416–417, 420–421, 428–431, fluorescence speckle microscopy, 383 new developments, 478–480 435–136, 438, 439, 710–711, in four-dimensional imaging, 222 live imaging, 480 713–715, 769–776, 779–781 improvement, recent, 36 polarized light microscopy, 479 chamber slides, use, 429 laser trapping, 383 subpixel imaging, 478–479 culture chamber, 429 linear unmixing, 192, 382, 664–667 optical sectioning schematic, 469 cuticle, 434–437, 715, 717, 779 of living cells, 212, 220, 382, 797. See OTF frequency band, 474 fungi, 438–439, 624, 782, 870 also, FRAP, FLIP simulated example, 481, 482 hairs, 431, 434–436, 772 intensity dependence, 341, 363 speed, 482–483 meristem, 168, 420, 430, 770, 776–778, mechanism, 222–223 spherical aberration correction, 480–481, 783 of non-specific fluorescence, 27, 44, 74 471 microsporogenesis, 431–432 optical tweezers, 383, 385 spinning-disk confocal example, 481, mineral deposits, 163, 420, 436–438, performance limitations, 221, 224, 232, 482, 482 703 381, 448–450, 556, 693. See Chapter transmitted light, bright-field (TLB), 472, pollen germination, 420, 433–434, 781, 39 477 783 photoactivation, 187, 224, 383, 385, 541, two photon example, 481, 483 pollen grains, 202, 305, 313, 420, 544–545, 693, 759 widefield simulated example, 481, 469 431–433, 553, 558, 781, 783 photo-uncaging, 383. See also, Photo- WWF stack example, 469 protoplasts, 195–196, 203, 416, 421, uncaging and signal per pixel, Blind spots, due to sampling with large 423–427, 429–431, 438–439, 693 63–64 pixels, 38. root, 172, 174, 303, 307, 421, 429, spectral unmixing, 192, 382, 664–667 Blue Sky Research, ChromaLase 488, 107. 430–431, 438, 464–465, 556, table, 384–385 Boar sperm cells, 557. 772–773, 775, 777, 779–783 techniques, 125 BODIPY dye, 142, 342–343, 353–356, 389, starch granules, 202, 420–421, 428, temperature as a variable, 696–698 692, 749, 760–762. 432–433, 435, 703, 710–712, 715, time-lapse fluorescence, 382 BODIPY TR, methyl ester dyes, 760–762. 719 Bleedthrough fluorescence, 185, 203, 664, Bolus injection protocol, 360, 726, 728, stem, 168, 172, 180, 417–419, 421, 904. 731. 424, 429, 556, 707, 710–711, multi-tracking, reduces bleed-through, Bone, reflectance, 167. 713–714 664 Books on 3D LM, listing, 889. storage structures, 435–436 940 Index

Botanical specimens (cont.) photoactivation, 383 Ca2+ indicators, 346–347, 738, 742–743. suspension-cultured cells, 189, slice loading, 726 See also, Ca2+ sparks, 737–738, 742. 429–430 linear unmixing, 192, 382, 664–667 discovery, 737, 738 tapetum, 433–434, 779 making brain slices, 393, 722–724 Caenorhabditis elegans. see C. elegans. waxes, 420, 428, 434–435, 714–715 acute slices, 722–723 Caged compounds, 759–760. point spread function in, 784 autofluorescence, 383 multi-photon excitation, 543–544 refractive index heterogeneity, 192, cultured slices, 724 Calcein AM dye, 355, 360, 362–363, 418–420 mouse visual cortex, 723 426–427, 430, 685, 804, 812. maize stem, 419 primary visual cortex, 724 Calcium imaging, 529, 545, 584, 736–737, Bovine embryo, 750. protocols, 731 812. Boyde, Alan, 2, 6, 141, 154, 224. See also, thalamocortical slice, 724 calibration, 742–743 Stereoscopic images. photodamage, 729 data compression, 584 BPNN. See Backpropagation neural pulse broadening, 728 intensity image, 529 network. reducing excitation light, 390–391 introduction, 736 Bragg grating, tuning diode, 107. resolution, 729 multi-photon excitation, 545 Brain slices, 392–398, 722–734. 686. second harmonic imaging (SHG), ratiometric, 189 beam collimation, 728 729–730 signal-to-noise ratio, 737 choice of objectives, 395, 727–728 silicon-intensified target (SIT) camera single-cell kinetic, 812 future directions, 929 use, 730 TIRF for measuring, 180 image processing for, 732–734 slice chamber, 394 very fast imaging, 237 algorithms, 733 protocol, 727 Calcium ion dyes, 183, 189, 237, 736, 737, alignment, center of mass in, 732–733 speckle microscopy, 383 741–743. See also, fura-2, Fluo-3 alignment, based on image overlap, useful techniques, table, 384–385 and Indo-1. 732 time-lapse, 382 Fluo-3 and Fura Red indicator system for automatic detection of neurons, two-photon imaging, 727 determining, 183 733–734 calcium imaging, 729 Fluo-3 indicator system for determining, drift/vibration compensation, 396, 732 z-sectioning, 729 737 image de-noising using wavelets, 734 Breakdown. fura-2 reactions, 741–742 image processing/analysis, 330–331, electrical, in PMTs, 263, 660 Indo-1 and Fura-2 indicator system for 395–396, 730–732 optical, high power density, 198, 680, Calcofluor, 424, 438. biocytin protocol, 730 682, 685, 687, 703, 705 staining procedure, 438 classified using cluster analysis, Brewster surfaces, 83. Calibration, 34, 75–76, 742–745. 731–732 Brewster windows, 83, 102–103, 115. Ca2+ sparks, 742 correlated electron microscopy, 731 Bright-field microscopy, 6, 127, 130, 201, of CCD to measure ISF, 75–76 montaging, 331 224, 229, 448, 468, 649, 728. confocal microscopy, 742 neuron reconstruction, 330–331, 730 CCD for, 127, 483 errors in, 744 protocol for PCA/CA, 731–732 deconvolution, 468, 472–473 of ion concentrations, 742–745 spectral imaging, 382 depth of field, 4 ion interference, 745 two-photon/neurolucida system, 316 low coherence light for, 130, 134–135, of effective pinhole size, 34 image production, 729 139–140 in vitro, 742 2-photon excitation, 727 optical projection tomography, 610–612 Calistics, 726. deep imaging, 395 Brightness, source, 21, 26, 126–127, Callus, 429. living neurons, 725 129–130, 141–142, 215. Cambridge Technology, galvanometers, maintaining focus, 395, 732 and exposure time, 141–142 54. microglia, 397–398 gray levels, 71–73 cAMP indicators, 347. neuronal ensembles, 726 as limitation of disk-scanners, 21, 215 Canna, 422, 710. objective lenses, choice of, 727–728 of non-laser light sources, 126–127 fluorescence spectra, 422 second harmonic imaging, 729–730 of sun, 127, 135 as function of excitation intensity, 165 in vivo observations, 387 Brillouin background, in glass fibers, 88. nonlinear absorption, 710 preparation, 387 Brillouin effect, reduction, 110. Carbon arc lamps, 136. labeling cells, 394–396, 724–727 Brownian motion, microtubules, 11. CARS. See Coherent anti-stokes Raman biolistic transfection, 724–725 BSL. See Backscattered light. scattering. bolus injection, 726 Buffering, fluorescent ion measurement, CARS correlation spectroscopy (CS-CARS), calistics, 726 740. 602. choice of dyes, 729 Bulk labeling, in living embryos, 761. Raman spectra, 602 diolistics, 726 CARV disk-scanning confocal microscope, dye injection/patch clamp, 726 C 215, 226, 229, 230, 907–908. genetic manipulation, 725–726 C. elegans, 746, 748, 766, 856, 857–858. diagram, 230, 907 GFP transgenic mice, 726 cryopreparation, 857–858 CAT. See Computed axial tomography. Helios Gene Gun, 724 FRET imaging, 766 Cathode-ray tube (CRT), 5–6, 53, 67, live-dead staining, 393 as model system, 746, 748 72–73, 291, 293, 588–589. painting with AM-ester indicators, TEM images, 856, 857 gamma, compensation, 73 726–737 Ca2+ imaging, see Calcium imaging. Cavities, of dielectric coatings, 46, 47. Index 941

Cavity-dumped lasers, 111, 114. labeling, 775 specifications, 927, table, 233, 929 for FLIM imaging, 114 viability, 780 user-friendliness, 929 CCD. See Charge-coupled devices. Cell-by-cell analysis, 817. gain-register, 76–78, 460–461 CD. See Compact disks. Cell-cell signaling, 778. intensified, 930–931. See also, Intensified cDNA-GFP fusion, in plants, 773. Cellular structures, optical effects, 22–23. CCD Cedara, 281–282, 288, 302, 308. Center-of-mass alignment protocol, 733. monitoring during exposure, 137 Cell adhesion imaging with TIRF, 90. Center pivot/off-axis pivot mirrors, 1, 214. multi-focal multi-photon microscopy, 552, Cell autofluorescence, 742. Cerium, doping of quartz lamp envelope, 558 Cell chambers, 11, 22, 191, 219, 370–371, 116. noise sources, 256, 924–925 386–387, 394, 429–430, 564, CFP and YFP molecules, in FRET pair, charge amplifier, 925 610–611. 798–800. clock-induced charge (CIC), 234, 926 for 4Pi confocal, 564 Chambers for living cell imaging, 388–389. fixed pattern noise, 924–925 for biofilms, 870–873, 875, 877, 880, 885 commercial suppliers, table, 388–389 multiplicative noise, 77, 234, 257, 262 brain slice, 394, 723, 727, 729 Charge amplifiers, 923–924. noise vs. pixel dwell time, 922 for epithelial cells, 370–371, 377, 386 defined, 923 table, 256 finder chamber, 683 destructive readout, 923 operation, 254, 918–927 flow chamber, 870–873, 875, 877, 880, FET amplifier performance, 923 blooming, 923 885 non-destructive (skipper), 923 charge amplifiers, 923–924 for high-content screening, 810 Charge-coupled device (CCD), 26–28, charge coupling, 918–920 for optical projection tomography, 30–31, 39, 61–62, 65, 70, 74–78, 88, charge loss, 921 610–611 127, 137, 142, 215, 233, 254, dark charge, 921–922 perfusion, 394 458–459, 460–461, 482, 552, 558, destructive readout amplifiers, 924 for plant cells, 191, 429–430 644, 754–755, 784, 918–931. See edge effects, 921 simple, 22, 394 also, Electron-multiplier electron multiplier, 926–927 for SPIM, 613, 625, 673 CCD. FET amplifier performance, 253, 922, table of required functions, 380 bit depth, 75 924 table of suppliers, 388–389 camera, 918–931 frame transfer readout, 920 test chamber/dye, 654, 661 advances in, for speed, 754–755 full-frame readout, 920 Cell cycle, 790, 791. bright-field imaging, 127 gain register amplifier, 925–926 Cell damage, 2-photonmicroscopy, 680–688 for disk scanner systems, 78, 205, 215, incomplete charge transfer, 923 See also, Bleaching; Photodamage. 220, 233–235, 349, 459, 754–755 interline transfer readout, 920 absorption spectra of cellular absorbers, pixel size, 62, 65, 634–635, 784 leakage, 921–922 681 specifications, table, 929 non-destructive (skipper) amplifiers, intracellular chromosome dissection, 688 time for sampling, 70 923–924 mitochondria, 686 choosing, possible problems, 920 nanosurgery, 219, 686–687 color, 927 quantum efficiency vs. wavelength, one-photon vs. multi-photon, 680–689 computer-assisted pulse shaper, 88 922 by optical breakdown, 198, 680, 682, 685, confocal imaging, 458–459 quantum efficiency, 920–921 687, 703, 705 cooled, advantages and limitations, 30–31 readout methods, 920 photochemical, 682–685 quantum efficiency, 26–28 signal level representing zero photons, absorbers/targets, 682 spatial quantization of signal, 39 925 beam power sensor, 683 digital camera, 75 storage array, 920 impact on reproduction, 686, 685 digital vs. video camera, 61–62 performance, table, 256, 459, 923 laser exposure parameters, 682–683 electron multiplier-CCD, 30–31, 76–77, piezoelectric dithering, increases NIR-induced DNA strand breaks, 233–235, 262, 459–461, 482, resolution, 70 683–684 925–926 pixel size, 62, 65, 634–635, 784, 928 NIR-induced ROS formation, 683 multiplicative noise, 77, 234, 257, 262, quantum efficiency and noise, 29, 644, photodynamic-induced, 684 926 920, 922 spectral characteristics, table, 682 result, 205, 234, 755 measuring, 74–76, 926 photothermal, 685 table, 233, 459 sensors size, parallel data collection, 142 reproductive effect, short NIR pulses, evaluating, 927–931 snapshot camera, 65 682, 686 array size, 928–929 specifications, described, 927–930 ultrastructure modifications, 685–686 “the clincher,” 929 testing, 930 Cell microarray (CMA), 815–816. comparison, CCD/EM-CCD, table, Cheek cells, backscattered light image, Cell motility, 757. 233, 459 22–23. Cell nuclei, optical effects, 23. dynamic range vs. pixel size, table, 928 Chemical environment probe, 517. Cell pellet, three dimensional, 815. maximum signal, 930 Chimeric fusion proteins, 794, 801–802. Cell surface targeting assays, 813. quantum efficiency, 927–928 anisotropy analysis, 794 Cell walls of plants, 168–169, 188–189, readout noise, 928 cloning for FRET, 801–802 303, 306, 416–417, 420–421, readout speed, 928–929 overexpression, 802 428–431, 435–436, 438, 439, self test, 930 Chinese hamster ovary cell, 197, 556. 684+, 710–711, 713–715, 719, 769–776, sensitivity, 930 818. 779–782. shutter stability, 929 Chirp, pre-compensation, 88, 111, 602, 907. 942 Index

Chlorophylls, autofluorescence, A. thaliana, CNS, (central nervous system), 392–393, Colloidal gold labels, 167, 241, 846–859. 175, 194, 203, 425–426, 528, 711, 395. See also, Chapters 19 and 41. contrast, 167 714, 779, 782, 881. Codecs, image processing, 831, 836, electron microscope markers, 846–857 bleaching, 203 840–841. correlative, 850, 852, 855 FLIM, 528 Coefficient of variation, 660, 661. SEM, 850 Cholera toxin transport, 790–791, 796–797, Cohen’s k statistic, 826. TEM 802. Coherence length, 7–8, 84. FluoroNanoGold, 854 FRET, 796–797, 802–803 defined, 7–8, 84, 130–131 GFP related, 854–855, 857–858 Chromatic aberrations, 134, 152–156, 178, reducing, for laser light, 84 measuring resolution, 241 242–245, 657–658, 659. Coherence surface, 84. quenches fluorescence, 854 apparatus for measuring, 243 Coherence volume, 84. Rayleigh scattering, 167 axial chromatic registration, 243–345, Coherent anti-stokes Raman scattering Colocalization, 517, 650, 667–670, 794, 658, 657–659 (CARS), 90, 204, 550, 595–605. 813, 881. of incandescent and arc lamps, 134 advantages, 204, 596 FRET, FRET, 519 intentional, for color/height encoding, 154 correlation spectroscopy, 602–603 erroneous, 581 lateral chromatic registration, 657–658 defined, 595 Color display, 291, 292. fluorescent latex bead labeled, 178 energy diagram, 596 display space, 291 linear longitudinal chromatic dispersion, epi-detected, 597–599 multiple channel display, 292 154, 659, 664 forward/backward detected, 597–599 palette, 291 measuring, 242–245 Hertzian dipole radiation pattern, 598 pseudo, 173–175, 190, 291 Chromatic corrections, 157, 177. history, 595–596 resolution, 291 excitation/emission wavelength, 177 imaging of biological samples, 603–604 true, 291 tube length, table, 157 adipocyte cells, 604 Color centers, in optics, avoidance, 116. Chromatic magnification difference. See artificial myelin, 204 Color filters, 43–52. See also, Filters. Lateral chromatic aberration. epithelial cells, 603 long-pass, 43–46, 175, 203–204, 212 Chromatin, 385, 390, 684, 693–695, 812. erythrocyte ghosts, 603 short-pass, 45, 46 Chromophores, 338–348, 543–544, intensity distribution, 597 bandpass, 44, 45 803–804. See also, Dyes; mapping intracellular water, 90 Color print images, 592. Fluorophors; Fluorescent probes etc. microscope schematic, 599 Color reassignment, 173–175, 190, 291. cellular introduction methods multiplex CARS microspectroscopy, Coma, 145, 151, 245, 247, 249, 483, electroporation, 359–360, 803 601–602 630. microinjection, 360–361, 388, 739, non-resonant background suppression, distortion away from optical axis, 151 748, 755, 795, 803–804 600–601 observation using point objects, 145, table, 344–345, 803 energy diagram for multiplex CARS, 246 transfection reagents, 358, 360, 362, 601 Commelina communis, images, 712. 556, 682, 790–791, 795, 803 epi-detection, 600 Commercial confocal light microscopes, multi-photon excitation, 543–544 phase control of excitation pulses, 600 906–917. CIC, clock-induced charge, EM-CCDs, 234, picosecond vs. femtosecond pulses, BD-CARV II, 230, 907 926. 600 La Vision-BioTec TriM-Scope, 907 Circular exit pinhole, 9. polarization-sensitive detection, 600 Leica, TCS SP2 AOBS, 910 Circular laser beam, corrective optics, 106. time-resolved CARS detection, 600 Leica MP RS Multi-photon, 910 Classification, pattern. See Automated optimal laser sources, 599–600 Nikon C1si, 911 interpretation of subcellular patterns. pumped optical parametric oscillator Olympus DSU, 913 Clathrin-GFP dynamics, 236. (OPO) Olympus Fluoview-1000, 912 Clearing agents. See also, Mounting media. systems, 600 optical parameters of current, table, optical projection tomography, (OPT) perspectives on, 604–605 908–909 610, 624 unique features under tight-focusing, Visitech VT Infinity, 914 plant material, 166, 417–420, 439, 596–597 Visitech VT-eye, 914 774–775 Coherent illumination, 1, 83–84. Yokogawa CSU 22, 231, 915 Clock, role in digitizing and reconstructing properties of laser light, 83–84 Zeiss LSM 510 META optical, 916–917 analog signal, 64. and resolution, 1 Zeiss LSM-5-LIVE Fast Slit Scanner Clock-induced charge, in EM-CCDs, 234, Collagen fibers, 164, 188, 313, 361, 393, schematic, 232, 916 926. 514, 703–704, 715. Compact disks (CD) for data storage, 499, Closterium, 192–194. autofluorescence, 545 586–587, 588, 731. chloroplast autofluorescence, 192–195 birefringence, 164, 188, 717 Compact flash cards, 588. signal variation with depth, 194 gels, 393 Components, of confocal fluorescence CLSM. See Confocal laser-scanning polarization microscopy, 164, 188 microscopes, 43–58, 207–208. microscopy. second harmonic image (SHG), 703–704, acousto-optical devices, 54–57 Cluster analysis (CA), 731–732, 826. 715 chapter, 43–58 neurons classified using, 731–732 Collector optics, elliptical and parabolic, electroptical modulators, (Pockels cells), protocol with PCA and, 731–732 129. 25, 54, 57, 87, 116, 543, 701, subcellular patterns, 826 Colliding-pulse, mode-locked laser (CPM ), 903–904 CMA. See Cell microarray. 540. filters/beam-splitters, 44–51 Index 943

mechanical scanners (galvanometers), fluorescence lifetime imaging, chapter, alignment of optics, 629–630 51–54 518 back-focal plane (BFP), 210, 509, 629, polarizing elements, 58 laser power required, 81 633 Computed axial tomography (CAT), laser requirements for, 89 focus, 629 610–611. vs. multi-photon laser-scanning low signal, 631 Compression, data see, Data compression. microscopy, 750–751. See Chapters mirror test specimen, 630 Condenser lens, size, 129. 22, 23, 24 no signal, 631, 660 magnification, 128–129 photobleaching, 690, 697 simultaneous BSL/fluorescence, 631 Configuration of pixels in image plane, 62. vs. selective plane illumination high-content screening systems, table, 811 ConfMat. See Confusion matrix based microscopy, (SPIM), 678 illumination sources, 126–144, 650–651 method. stage-or object-scanning, 13–15 See also, Lasers; Non-laser sources Confocal disk-scanning microscope. See TEM mode, 118 acousto-optic tuning filter (AOTF), also, Disk-scanning confocal zoom magnification and number of pixels, 651. microscopy. 38 laser sources, chapter, 80–125 Confocal fluorescence microscope, 73, 207, Confocal microscopy, 90, 141, 265, laser stability, 651 404–413. See also, Confocal 381–399, 444–447, 453–467, power measurement, 650–651 microscopy; Confocal laser-scanning 650–670, 742, 770, 774, 779, 810, living cells, 381–399. See also, Living microscopy. 811, 815, 870–887. See also, cells basic optical layout, 207 preceding major head and Chapters Minsky first confocal design, 2, 4–6, 11, limitations due few photons, 73, 459 35 and 36. 141, 216, 890 refractive index mismatch, 404–413 See art of imaging by, 650 monitoring instrument performance, also, Refractive index automated, platforms used for, 810 650–663 Confocal imaging, 4–5, 232, 235–236, 737, balancing multiple parameters for, 650 illumination source, 650–651 738, 746–766, 809–817. See also, of biofilms, 870–887 optical performance, 652–660 next major head and calibration of, 742 photon efficiency, 14–15, 24 Chapters 35 and 36. cell microarray and, 815 scan raster/focus positioning, 651–652 4Pi. See 4Pi microscopy colocalization, 667–670 signal detection, 660–663 automated effect of MLE and threshold, table, 669 with non-laser light, 141 for cytomics chapter, 809–817 fluorogram analysis, 669 objective lens, 652–660. See Chapter 7 of microarray slide, 816 image collection, 667–668 optical performance, 652–660. See also platforms used for, 810 nerve fiber, 669 Chapters 7, 11 real-time, 810 quantifying, 668 axial chromatic registration, 658–659 temperature control, 810 setting thresholds, 668 axial resolution vs. pinhole, 656–657. types of assays for, 811, 813–814 spatial deconvolution in studies, See also, Axial resolution workstations, 814 668–670 contrast transfer function, 656. See of biofilms, Chapter 50 vs. deconvolution, 644–647, 453–467. See CTF deconvolution, 753. See Deconvolution also, Chapters 22, 23, 24 coverslip thickness and RI, table, 654 by disk-scanning confocals, 232 CCD/confocal imaging combination, field illumination, 658 fast, 235–236 458–459 flatness of field, 659 of fluo-3 loaded cardiac myocyte, 737 deconvolving confocal data, 461–464, Focal Check™ beads, 657–659 fluorescent indicators for, 738 466, 488–500 lateral chromatic registration, 657–658 high-resolution datasets, cell fluorescence excitation, 459 lateral resolution, 655 arrangements, 776 fluorescent light detection, 459–460 refractive index, 654. See Chapter 20 of living cells, 813 gain register CCDs, 460–461 resolution test slides, 656 of living embryos, chapter, 746–766 image sections, figures, 455, 456, 462 self-lensing artifacts, 659 methods compared, 459, 644–647. See imaging as convolution, 453–457 spherical aberration, correction, 654, Chapters, 22, 23, and 24 integration of fluorescence intensity, 655 of plants, 773. See also, Chapters 21and 459 subresolution beads, 655–656 43 limits to linearity, 457 x-y and z resolution using beads, 656 vs. non-confocal, 746 model specimens, 461 optimizing multi-labeling, 663–667 time-lapse. See Time-lapse imaging noise, 459–463 bleed-through between channels, 663 Confocal laser-scanning microscopy out-of-focus light, 461 control samples, establishing limits, (CLSM), 9–15, 32, 38, 81, 89, 118, point spread function, 453–457 663 222–224, 408, 518, 678, 690, 697, practical differences, 458, 463–466 measuring autofluorescence, 663 750–751, 754, 884–885. See also, resolution, 459–463 multi-tracking, reduces bleed-through, next major head same specimen comparison, 465 664 advantages and limitations, 11–12, sensitivity, 459–463 positively labeled sample, 664 222–223, 644–647, 884–885 shift invariance, 457, 490, 564 reflected light contribution, 663 alternatives to, 644–647, 754 single point imaged, 454 secondary conjugate contribution, 664 comparisons, 644–647 summary of pros/cons, table, 459 photon efficiency, 24, 26, 28, 30 33–34, disk-scanning and scanned slit, table, 224 temporal resolution, 458 36 digitizer employing full integration for, 32 focus positioning, 651–652 polarizing elements, 57 edge response, 408 getting a good confocal image, 629–631 scan raster, 651–652 944 Index

Confocal microscopy (cont.) defined, 162 different requirement of LM/EM, malfunctioning system, 653 flare, 649 846–850 phototoxicity from uneven scan speed, formation of, chapter, 162–206 early 4D microscopy, 846 651 fluorescence. See Dyes, and Fluorophores fluorescence/TEM to analyze sources of fluorescent beads, table, 653 as function of feature size, 16, cytoskeleton, 854 well-calibrated system, 652 61–62, 37, 634 fluorescent micrographs, 851 x and y galvanometers, 651–652 intrinsic, 633 FluoroNanoGold for cryosections, 854 z-drive mechanism, 652 measuring, 16, 59 GFP, 854. See also, Green fluorescent z-positioning calibration, 654 polarization. See Polarization microscopy protein z-positioning stability, 652 second harmonic generation. See SHG HVEM stereo-pair, 848–849 separating signal by spectral regions for, and statistics, 633 immuno-stained bovine aorta, 852 664 third harmonic generation. See THG LVSEM of FRAPed microtubules, 849, sequential collection reduces bleed- Contrast medium, and laser power, 80–81. 850 trough, 664 Contrast method, defines signal required, phalloidin as correlative marker, 235–236, signal detection for, 660–663 126. 344, 376, 378, 694, 696, 756, 804, coefficient of variation, 660–661 Contrast transfer function (CTF), 16, 35, 854–856 instrument dark noise, 660 37–39, 59–62, 656, 747. phase-contrast imaging, 851 PMT linearity, 661–662 in confocal vs. non-confocal microscopy, postembedding, 855 signal-to-noise ratio, 660 16. quantum dot labeling, 853 spectral accuracy, 662 See Chapter 11 same cell structure LM/SEM, 850–852 spectral detector systems, 662 as function of grating period, 16 same cell structure LM/TEM, 852–856 spectral resolution, 662–663 of microscope optical system, 35 SEM images at 5kV and 20kV, 847, 848 wavelength response, 663 relationship with objective BFP, 61 TEM cross-section of C. elegans, 856 signal level, 444–445 and spatial frequencies, 16, 37 TEM longitudinal section of C. elegans, signal-to-noise ratio, 444–447 and stages of imaging, 62 857 spectral analysis, of plants, 770 Control, of non-laser light sources, 138–139. tetracysteine tag labeling, 221, 348, 357, spectral unmixing, 192, 382, 664–667 Convalaria majalis, 425, 556. 853 limitations to, 667 fluorescence microscopy of rhizome, 425 tiled montage TEM images, 858 overlapping fluorophores separation, multi-focal multi-photon imaging, 556 time-series DIC images, 847 664–667 Conversion techniques, 259–260. Correlative LM/EM. See Correlational light removing autofluorescence, 667 analog-to-digital, 259 microscopy/electron microscopy. stage-scanning, 9 digital-to-analog, 259–260 Coumarin dye, 114, 339, 344–345, 353, 355, staining plant cells, 774 Convolution, a primer, 485–487. 654–655, 661, 693. vs. structured-illumination methods, 265 3D blurring function, 486 Counting statistics, 20, 30. See Poisson vs. two-photon excitation, 779 Fourier transforms, 487 statistics. Confusion matrix based method (ConfMat), geometrical optics, 487 Cover glass. See Coverslip. 826. out-of-focus light, 486–487 Coverslip, and spherical aberration, 15, Constant output power laser stabilization, Cooling water, checking/maintaining, 147–150, 201. See also, Spherical 86. 116–117. aberration. Continuous wave (CW) laser, 87, 88, Cork microstructure, 770. CPM laser. See Colliding pulse mode-locked 90–118. Correction collar, (spherical aberration), 15, laser. beam intensity stabilization, 86–87 145–149, 158, 160–161, 178, Crane fly spermatocyte, metaphase spindle, diode (semiconductor), 105–110 241–242, 247, 377, 407, 410–412, 15. output power/cooling, 108 471, 654–655, 657. Creep, in piezoelectric scanners, 57. pumped solid-state, table, 94, 95 adjustment, 377, 407, 471, 499, 654–655 Cr:Fosterite, femtosecond pulsed laser, 109, dye lasers, 86, 103, 112, 114, 124, dry objectives, 410 114, 415, 541, 706–709, 712–714. 540–541 multimedia, 640 Critical angle, for reflection of incident light fiber up-conversion, 109–110 Correctors, 70, 147. surface of refracting medium, 167, gas lasers spherical aberration, 15, 151, 147, 192, 502. Argon-ion, 90, 102 411–412 Critical illumination of the specimen, Krypton-ion, 102 Intelligent imaging innovations, 78–79, 128–129. HeNe, 102 151, 192, 395, 411, 654 Crosslinking fixatives, 369. HeCd, 103 to stored data, second Nyquist constraint, Crosstalk. cesium and rubidium vapor, 103–105 70 between fluorescence channels, 203, 424, table, 92–93 Corrective optics, for diode lasers, 107–108. 882 titanium-sapphire, 109 Correlational light microscopy/electron between disk pinholes, 227 Contrast, 7, 11, 16, 37, 39, 49, 59–62, 68, microscopy, 731, 434, 436–437, between excitation foci, 553–556, 159, 162–204, 248, 421, 473, 488, 846–860. 558–559, 564 542, 599–600, 607, 622, 656, 657, BSL image, 855 CRT. See Cathode-ray tube. 675. See also, Rose criterion and brain slices, 731then CLSM, 856–857 Crystal Fiber A/S, HC-800-01 bandgap CTF. cryopreparation of C. elegans, 857–858 fiber, 88. absorption, equations, 164 DIC image tracking, 849 CSU. See Confocal scanning unit. chapter, 162–206 DIC image/UV fluorescence image, 850 CTF. See Contrast transfer function. Index 945

Curtains, laser, safety, 118, 904. file formats for, 580–588 modern microscopes design aims, Cuticles, plant, 434–437, 715, 717, 779. fractal compression, 581–582 862–865 insect, 166 GIF (graphics interchange format), projects, 865–866 maize, 436 580 BioImage, 865–866 CW. See Lasers, continuous wave. JPEG (Joint Photographic Experts Biomedical Image Library (BIL), 866 Cyan fluorescent protein (Cyan), 221–222. Group), 581–584 Scientific Image DataBase (SIDB), 866 Cyanine dyes, 339, 342, 344, 353–355, MPEG, 836–839, 840–841 recent developments, 861–862 362–363, 374, 443, 540, 587, 760, PNG (portable network graphic), 581, MPEG-7 format, 862 854, 874. 584 relational database management Cytomics, 810, 811. QuickTime, 829, 831, 836–837, systems (RDBMS), 862 automated confocal imaging, 810 840–844 TIFF format, 861 automated confocal imaging, table, 811 TIFF (tagged image file format), 580 software for, 868–869 Cytoskeletal structures, 24, 188, 190, wavelet compression, 581–584, 819 ACDSee, 868 328–329, 368, 370, 372, 378, 383, movies, 836–842 Aequitas, 868, 869 461–462, 577, 703, 715, 719, artifacts, 839 Cumulus, 868 773–774, 813, 846–848, 852, 854. compression ratios, 842–843 Imatch, 868, 869 LM-TEM analysis, 846, 854 entrope, 841 iView, 868, 869 stabilizing buffer, 852 MPEG formats, 840–841 Portfolio, 868 Cytosolic markers, 757. Up-sampling, 838 price, 868 Cytotoxicity, reducing, 36–37. See also, pixel intensity histograms, 584 Research Assistant, 868 Bleaching; Phototoxicity. testing, 830, 835 ThumbsPlus, 868, 869 time required, table, 581for WWW use, system requirements, 864 D 816 DBR. See Distributed Bragg reflector. DAC. See Digital-to-analog converter. useful websites, 844–845 DCT. See Discrete cosine transform. Damage threshold, LED sources, 139. Data projectors, 590. Deblurring algorithm, 476. DAPI, 140, 344–345, 355, 358, 376, 431. Data storage, 106. See also, Mass storage. Deconvolution, 7, 26–28, 39, 40, 66, plants, 431 Data storage systems, 287, 395. 580, 594, 189–190, 222–223, 278, 456–458, use of, 376 764. 464, 468, 488–500, 542, 564, 736, Dark current, 29, 76, 234. chapter, 580–594 746, 751–753, 778, 784–785, 828, fixed-pattern noise due to, 76 characteristics of 3D microscopical data, 864, 900, 929. See also, Blind of photomultiplier tube, 29, 660 287 deconvolution. reducing, 234 databases, 861–869. See Databases of 2-photon images, 498 Dark noise, defined, 232. random access and 3D Gaussian filtering, 70, 281, 285, Darkfield microscopy, 5, 7, 172, 474, 672. CDR, CDRW, 586–587 323, 392, 395, 667. See also, depth of field, 4 DVD, 587 Gaussian Data, 11–12, 33, 64, 76, 237. Magnetic disks, 586 4Pi lobe removal, 562, 565 conversion from ADU to electron data, 76 semiconductor, FLASH memory, 588 advantages and limitations, 458, 475 degradation by multiplicative noise and for remote presentation, 842 algorithms, 472–476, 490, 495–497, 751, digitization, 33 role for STED, 577 778 reconstructing, 64 Databases, 2D/3D biology images, 827, comparison, 497–498 speed of acquisition, 11–12 861–869. iterative constrained Tikhonov-Miller, storage of volume of data, 237 benefits, 863–864 497 Data collection guidelines, 319–320. fast, simple machine configuration, 863 Jansson-van Cittert, 496 Data collection model, blind deconvolution, improved analysis and access, 863 nearest neighbor, 495–496 472. performance, 863 non-linear constrained iterative, Data compression, 288–289, 292–293, 295, remote monitoring, 863 496–497 319, 499, 580–585, 762, 764, 819, repeatability of experiments, 863 Richardson-Lucy, 497, 568 835–836. submissions to other databases, 863 Weiner filtering, 496 algorithms, 580 criteria/requirements, 866–867 background history, 488–490 discrete cosine transform (DCT), 581 digital rights management, 867 blurring process contributions, 488 Huffman encoding, 580 metadata structure, 867 equation showing restoration possible, Lempel-Ziv-Welch (LZW), 580 query by content, 866–867 489 run-length encoding (RLE), 580 user interface, 866 image formation, 489–490 archiving systems, 580 data/metadata management, 861–862 schematic diagram of convolution, 489 gzip, 580 future prospects, 867 blind, 189–190, 431, 463, 469, 472–473, PKzip, 580 image database model, 864–865 478, 486, 492, 496–497, 646 WinZip, 580 image information management, 862 chapter, 468–487 calcium imaging, 584 image management software, table, 865, maximum likelihood estimation, color images, 581 868 472–477, 483, 669–670 different techniques, table, 581 instrument database model, 864 blurring process contributions, 488 Dinophysis image, 585 laboratory information management confocal data, 39, 40, 453–467, 488–500, effects on confocal image, 583 systems (LIMS), 862 753, 778. See also, Confocal examples, 583–585, 592, 834–837 microscopy data/metadata life cycle, 863 microscopy, vs. deconvolution. 946 Index

Deconvolution (cont.) purpose, 468 of confocal fluorescence microscope, 208 of simulated confocal data, 40 requirements and limitations, 489–494 efficiency in, 43 CARS data cannot be deconvolved, 397, diagram demonstrating convolution, fast-scanning confocal instruments, 237 399 489 intermediate optical system, 207–209 chapter, 453–467 linearity, 490 of microscope optics, 145 colocalization, 668–670 comparison of missing cone problem, 494 MMM, 552, 555 methods, 66, 453, 467, 475–477, noise, 495 practical requirements, 210–211 497–499, 644–648 optical transfer function, 490–491 of transmitted confocal microscope, 166 convolution primer, 485–478 point spread function, 489, 492–494 Detection efficiency, 34, 35, 210–211. convolution and imaging, 490–491 shift invariance, 457, 490, 564 measurement, 34–35 Fourier transform of PSF, 489, 490 sampling frequency, 635 practical requirements, 210–211 linearity, 490 spherical aberration, 471, 480–481 Detection method, multi-photon, 541, 542. optical transfer function, 490–491 stain sparsity, 28 descanned, 542 shift invariance, 457, 490, 564 structured illumination, comparison, Detectors, 9, 11, 25, 28, 251–264. See also, and data compression, 584–585 265–279 Photomultiplier tube; Charge- test results, 401, 461, 464–466, subpixel refinement, 478–479 coupled device, etc. 481–482, 483 temporal/spatial, 392, 458, 753 area detectors. See Image detectors defined, 189–190, 468 transmitted light imaging, 472, 475, assessment of devices, 260–262 display of data, 301, 830, 835–836 478 CCD, 254 examples, 40, 190, 392, 411, 462, 466, of wavelength spectra, 382, 663–667, noise vs. pixel dwell time, 922 471, 488–498, 510 771–772 comparison, table, 255–256 4Pi, 468, 565 limitations, 667 conversion techniques, 259–260 botanical specimens, 784–785 Deconvolution lite, 68–70. descanned, 208, 212, 537, 540–542, 774, brightfield, 411, 475, 478 Deflector, acousto-optical. See Acousto- 904 cardiac t-system, 498 optical deflector. direct effects, 252 confocal, 470 Defocusing, size and intensity distribution, errors, 211–212 DIC, 470 146. evaluation, 211–217 polarized, 479 Degree of modulation, 268–270. future developments, 262–264 of simulated confocal data, 40 locally calculated, 268–270 history, 262–264 STED, 574–576 absolute magnitude computation, image dissector, 254–255 flatfielding the data, 477 268–269 image intensifier, 13, 232–233, 519–520, black reference, 76 equations, 269 524, 555–556 white-reference, 76 homodyne detection scheme, 268–269 gated, 233, 519–522, 524, 555–556 fluorescence lifetime imaging, 521 max/min measured intensity difference, intensified. See Intensified CCD four dimensional deconvolution, 391–392, 268 MCP-PMT. See Microchannel plate 752 scaled subtraction approach, 269–270 microchannel plate, 232–233, 255, 262 Fourier transform of PSF, 489, 490 square-law detection, 268–269 MCP-CCD, 262 future directions, 483, 766 synthetic pinholes, 268 gated, 519, 523–524, 527, 532 and image formation, 490–492 Delamination, and interference fringes, noise internal, 256–259 linearity and shift-invariance, 457, 564 168–170. internal detection, 256 live imaging, 480, 564, 751–754 Delivery, dye, 355, 357–360, 810. noise currents, table, 256 missing cone problem, 494 Deltavision, 132, 282. photoemissive devices, 256–257 model specimens, 461, 464–466, Demagnification, and numerical aperture, photon flux, 257–258 481–482, 483 127. pixel value represented, 258–259 multi-photon, 488–500, 542 Depth discrimination, in LSCM. See Axial non-descanned, 185, 201, 218, 381, 447, multi-view montaging, 330, 677 resolution. 456, 507, 542, 552, 559, 643, 646, ion imaging, 736 Depth of field, 4, 9, 13. 727, 750, 779, 904, 909–910 noise, 495, 635 extended-focus images, 9 phase-sensitive, 518–520, 619 and Nyquist reconstruction, 59, 65, 67, fluorescence microscopy, 4 photoconductivity effects, 252, 253 68, 222–223, 635 phase-dependent imaging, 13 photoemissive, 254 suppressing Poisson noise, 39 Depth-weighting, projection images, 304, photography. See Photographic systems optical sectioning, 752 306. photomultiplier tube, 9, 11. See also, out-of-focus light, 26–28, 431, 487, 644 exponential, 304 PMT and pinhole, 26, 487 linear or recursive, 304 photovoltaic effect, 252–253 point-spread function (PSF), 223, 241, Derived contrast (synthetic contrast), photon interactions in, 252–256 247, 453, 463, 471, 489–492, 635, 188–201. point detectors, 260–261 655 Descanned detection, 166, 208, 212, 537, quantal nature of light, 251–252 approximations, 493 540–542, 754, 904. quantum efficiency (QE) vs. wavelength, measuring PSF, 492–494 Design of confocal microscopes, 43, 145, 25 and Poisson noise reduction, 320 166, 207–211, 237. See also, for second harmonic detection, table, 707 pre-filtering, 281, 497, 581 Commercial confocal light silicon-intensified target (SIT) vidicon, problem with specimen heterogeneity, 22, microscopes. 730 648 4Pi, 563, 566 spectral, 203–204, 662–663, 666–667 Index 947

TCPSC, 518, 520–523, 526 Digital-to-analog converter (DAC), 64, Dispersion, optical, 56, 88, 152, 154, 242, time-gated, 522 259–260. 411, 542–543, 609, 683. thermal effects, 252 operation, 64 in acousto-optical devices, 3, 15, 55–56, work functions, table, 252–253 Digitization, 25, 31–32, 36, 38–39, 59, 88 vacuum avalanche photodiode, 254, 255 62–63, 66, 72, 75, 79, 259, 261, 286, CARS signal generation, 728 Developmental biology, 545, 624. 460, 495, 639, 911. compensation, 566–567 multi-photon microscopy (MPM), 545 aliasing. See Aliasing defined, 152 Dextran labeling, 173–174, 292, 512, 757. blind spots, 38 in fiber lasers, ultra-fast pulses, 88, 110, DFB. See Distributed feedback. and Nyquist criterion, 38–39 113 4¢,6-diamidino-2-phenylindole, 140, precision, 25 by filter blank material, 211 344–345, 355, 358, 376, 431. See and pixels, 62–63 generates third harmonic signal, 704–705 DAPI. of voltage output of photomultiplier rube, group delay dispersion, 537–538, 543 plants, 431 31–32 group velocity dispersion, 88, 111, 210, use of, 376 DiI derivatives, 760. 537, 609, 903 Diatom, 438, 638–640, 881. Dimethylsulfoxide (DMSO), 697, 726–727, in optical coherence tomography, (OCT), as standard for measuring objectives, 145 760, 875. 609 test specimen, 638–640 handling, 739 in optical fibers, 502, 504, 507 DIC. See Differential interference contrast. DIN standard, microscopes, 156. and temperature, 15, 411 Dichroic filters, 212. Dinophysis image, 585. for multi-channel detection, 51 intensity loss, 212 Diode injection lasers, 105–108. using to correct for chromatic aberration, transmission, 212 Diode lasers, 86, 87, 107, 112, 116. 153 Dichroic mirrors (beam-splitters), 44, 45, distributed feedback, 107 Display software. See Presentation software. 50–51, 129, 211, 217–218. emission stability, 86 Displays, 580, 588–590, 594, 892. coating for collection mirrors, 129 intensity, 87 cathode ray tube (CRT), 5–6, 53, 67, double and triple, 217–218 maintenance, 116 72–73, 291, 293, 588–589 effect of deflection angle, 211 modulated, 112 data projectors, 590 separating emission/excitation, 44–45, noise sources, 86 digital light processor (DLP), 590 50–51 physical dimensions, 106 halftoning vs. dithering, 589 Die, of light-emitting diode, 133, 134. violet and deep blue, 107 international television standards, 589 Dielectric butterfly, galvo feedback, 54. visible and red, 107 liquid crystal (LCD), 589–590 Differential interference contrast (DIC) wavelength stabilization, 87 supertwisted nematic (STN), 589 imaging, 10, 14, 76, 127, 146, 171, Diode-pumped alkali lasers (DPAL), thin-film transistor (TFT), 589 453, 468, 473–475, 846. 103–105. monitors, 588–589 blind deconvolution, 473–475 Diode-pumped lamp (DPL), 108–109. Distortion, 39–41, 152. converting phase shifts to amplitude, 171 Diode-pumped solid-state lasers (DPSS), and resolution, practical, 39–41 narrow bandpass filter use, 76 108–109, 111, 112. Distributed Bragg reflector (DBR) diode Nomarski DIC contrast, 2, 268, 746, 892 kits, companies offering, 109 laser, 107. photon flux reduction, 127 passively mode-locked, 111 Distributed feedback (DFB) diode laser, schematic for, 475 ultrafast, 112 107, 113. three dimensional, 470 Diolistics, ballistic gene transfer, 726. ultrafast, 113 Wollaston prism, 156, 468, 473, 475 Dipping objective, 149. 161, 209, 411, 429, Dithering vs. halftoning display, 589. Diffraction, 61, 65. 568, 613, 727, 737, 870, 872. DLP. See Digital light processor. contrast transfer function, 16, 35, 37–39, Direct permeability, 358–359. DMSO, 697, 726–727, 760, 875. 59–62, 656, 747 Discrete cosine transform (DCT), 581. handling, 739 and sharpness of recorded data, 65 Disk-scanning confocal microscopy, DNA damage, 390, 517, 539, 680, 682–684, Diffraction limit, 210–211. See also, 215–216, 224, 225, 228–229, 812. Rayleigh criterion. 234–235 754, 755. DNA probes, 273, 317, 339, 343, 354, 358, defined, 210 advantages and limitations, 223–224 360, 362, 369, 393, 396, 459, 520, point-spread function, 146 for backscattered light imaging, 228–229 531–532, 539–540, 691–695, 774, practical requirements for, 210–211 chapter, 221–238 779, 782, 812, 818–825, 828, 874. Digital light processor (DLP), projectors, commercial instruments, 907, 913, 915 DAPI, 140, 344–345, 355, 358, 376, 590. comparing single- vs. multi-beam, 224 431 Digital memory system, 64. table, 226 DRAQ5, 343 Digital microscopy, optics/statistics/ and electron multiplier CCDs, 78, 205, Hoechst, DNA dye, 136, 339, 344, 360, digitizing, 79. 215, 220, 233–235, 349, 459, 362, 520, 565–566, 683, 782 Nyquist sampling, 146 754–755 DNA sequencing, constructs for, 801–802. Digital printers, 591–593. embryo, 754 DNA transfer, 724, 756, 760, 773, 790, Digital processing, in disk-scanning high-speed image acquisition, 216, 802–804. confocal, 12. 222–224, 754 Dominant-negative effects, 755. Digital projectors, 590. image contrast in, 168–171 Donor/acceptor pair (FRET), 790, 792–794, Digital rights management (DRM), 830, microscopes, table, 224 796–797. See also, FRET. 844. optical sectioning, 235 before bleach/after bleach ratio, 794 Digital video disks (DVD), 587–588. types, 228–232 equations, 790, 792–794 948 Index

Donor/acceptor pair (FRET) (cont.) diI derivatives, 355, 362, 389, 726, 760 photoactivatable, 187, 210, 224, 383, 385, fluorescence, 796–797 donor acceptor pair, 794. See also, FRET 541, 544–545, 693, 729, 759–760, fluorophores, 794 DNA probes, 343–344, 531–532, 912 separation in nm, table, 793 818–825, See also, DNA probes Kaede, 187, 383, 385 Double-image, diagram and example, 169. DRAQ5, 343 Kindling, 574, 760 Double-label, 375. dyes vs. probes, 353, for embryos, 748, PA-GFP, 187, 383, 385, 752, 759–760 Down-conversion, parametric, 114. 761 photodestruction, 340–341. See also, DPAL. See Diode-pumped alkali lasers. exciting efficiently, 44 Bleaching DPL. See Diode-pumped lamp. fade-resistant, 36 See also, Antifade; and Chapter 39 DPSS. See Diode-pumped solid-state lasers. Bleaching photophysical problems, 338–340 Drift, 386–387, 652, 655, 732. for fatty acid, 347. See also, FM4–64, absorption spectra, 339 CCD read amplifier, 76 below autofluorescence, 339–340 compensation for, 392–393, 732–733, Feulgen-stained DNA, 166, 200, 298, contaminating background, 339–340 886 433, 437 optimal intensity, 340 focus, 16, 40, 115, 190, 219, 386, 489, Fluo-3 and Fura Red, for calcium, 180, Rayleigh/Raman scattering, 339 567, 652, 720, 729, 886 183, 345, 434 singlet state saturation, 338–339. See compensating, 396, 732 Fluo-3 for calcium, 737 saturation, below lasers, 85–86, 115 fluorescein, 353, 355. See Fluorescein; triplet state saturation, 339 DRM. See Digital rights management. FITC phycobiliproteins, 338, 341, 343, Drosophila, 273, 675–676, 747–748, fluorescence lifetime, 517, 527–528 355–357, 693 751–752, 754, 756, 759, 804, 810. FluoroNanoGold, 854 for plants, 774–775. See Chapters 21 and living embryo, 675–676, 752 FM4–64, FM1–43, lipophilic dyes, 236, 44 salivary chromosomes, 273 355, 359–360, 389, 556, 755, two-photon, 782 SPIM, image, 675–676 760–761 propidium iodide, 344, 355, 360, 426, Duty cycle, laser, defined, 110. fura-2, 103, 189, 234, 257, 345, 346, 348, 651, 693–695, 773, 778–779, 782, DVD, 587–588. 358–359, 361, 529, 531, 726–727, 812, 875, 877 Dye lasers, 86, 103, 112, 114, 124, 540–541 730, 733, 741–743, 810, 812, 846, quantum yield, 172, 180, 184, 338–845, in cancer treatment, 112 850 347, 353–354, 360, 363, 383, 421, colliding-pulse mode-locked, 112 Fura Red, 180, 183, 345, 454 543–544, 574, 661, 683, 690–692, with intra-cavity absorbers, 112 future developments, 348–349 710, 737, 792, 794–795 noise and drift, 86 genetically expressed, 348 ratio methods, 346–348, 742–743 references, 124 Glutathione, 342, 358, 545, 694, 779, rhodamine, 353, 355. See also, as wavelength shifters, 103 782 Rhodamine Dye-filling, studying micro-cavities, hazards in using, 116, 118 excitation, 109 173–174. for ion concentration, 346–347 saturation , 21–22, 41, 142, 222, 265, Dyes, 22–23, 36, 44, 90–102, 109, 116, 118, ion-sensitive probes, table, 531 276, 338–340, 448, 643, 647, 899 165, 173, 183, 212, 222, 342–346, kinetics, 741–742 Schiff-reagent, 262, 369, 770, 774–775, 353–358, 360, 430, 461, 462, 527, lanthanum chelates, 345–346 778 528, 575, 726, 736–738, 740–745, laser/filter configuration, table, 799 selection criteria for, 353–358 748, 749, 755, 759–760, 774, 775, lineage tracers, 461 signal optimization strategies for, 782, 804. See also, Green fluorescent lipid dyes, 236, 355, 359–360, 389, 556, 341–342 protein (GFP); Rhodamine dyes; 755, 760–761 SNARF, 345–346, 531, 739, 744–745 Fluorescein. living cells, rapid assessment, table, 360 specimen damage, 340–341 affect on living cells, 391, 748 loading, uniformity, 749. See also, spectral properties, 212, 342, 344–345 AlexaFluor, 353–355 Loading spectral unmixing, 192, 382, 664–667 Aniline Blue, 430–432, 435, 438, 774 LysoTracker Red DND-99, 360 for STED, table, 575 APSS and Canna yellow, non-linearity, membrane labels, 344–345 Dynamic Image Analysis System (DIAS), 165 membrane potential, 205, 346 396–397, 783–784. bandwidth of emission, 44 microinjection, 360–361, 388, 739, 748, living cells of rodent brain, 396 BODIPY TR, methyl ester, 760 755, 795, 803–804 of plant cells, 783–784 BOPIDY, 142, 342–343, 353–355, 389, MitoTracker Red , 142, 170, 353, 358, Dynamic range, 929–930. 692, 749, 760–762 360, 430–431, 692, 750 Calcein AM, 355, 360, 362–363, multi-photon excitation, 543–544 E 426–427, 430, 685, 804, 812 nano-crystals, 343, 345. See also, e2v Technologies, EM-CCDs, 76–77, calcium dyes, 346–347 Quantum dots 233–234, 237, 262, 460, 925–926. cAMP, 347 Nile Red, 435, 528, 575, 774, 782 E-CARS. See Epi-detected CARS. characteristics of probes/specimen, table, organic, 342–343, 353–356 ECL. See Emitter-coupled logic. 344–345, 354–355 oxygen sensor, 347 Edge detector (software), 309, 322, 327, coumarin, 114, 339, 344–345, 353, 355, patch clamp loading, 360, 726, 734, 396, 823–826. 654–655, 661, 693 738–740 Edge effect, self-shadowing, 172. cyanine, 339, 342, 344, 354–355, pH indicator, 346, 739–745. See also, Edge-emitting diode laser, 89, 106. 362–363, 374, 443, 540, 587, 760, pH corrective optics for, 89 854, 874 imaging cross-section through, 106 Index 949

Efficiency, laser, 102, 105–106. See also, labeled proteins, 756 European Molecular Biology Laboratory Quantum efficiency (QE); Photon photobleaching, 759 (EMBL), 53, 212. efficiency. transcriptional reporters, 756 compact confocal camera, 212 of diode injection lasers, 105–106 EM-CCD. See Electron-multiplier CCD. Evanescent waves, 90, 177, 180, 245, 503, wall-plug, of argon-ion lasers, 102 Emission filter. See Filters. 801. EFIC. See Episcopic fluorescence image Emission spectra, of arc sources, 136, 176. defined, 90, 180 capture. Emission spectra, fluorophores, 1- vs. 2- optical fibers, 503 EGS. See Ethylene glycol-bis-succinimidyl. photon excitation, 421. resolution measurement, 245 E-h. See Electron-hole. Emitter-coupled logic (ECL), 259. Excess light. See Stray light. Electro-magnetic interference, in electro- EMT. See Electron microscopy tomography. Excimer lasers, 112, 116. optical modulators, 57. Endomicroscopy, 511, 513, 514. maintenance, 116 Electron microscopy, 167. distal tip for, 514 for tissue ablation, 112 brain slices, 730–731 fiber-optics, 513 Excitation efficiency, multi-focal multi- chapter, 846–860 human cervix image, 513 photon microscopy, 552. cryo-techniques, 854 human gastrointestinal track image, 514 Excitation filter, requirements, 44. See also, fixation, 167, 368–369 miniaturized scanning confocal, 511 Filters. immuno-stained, 371–372, 852 Endoplasmic reticulum, 374, 770, 819. Excitation source, laser. See Lasers; Non- micrographs, 479, 847–853, 855–858 and DiOC6, 390 laser sources. tomography (EMT), 610–611 FLIP, 382 Excitation wavelength change, contrast, 173. Electron-multiplier CCD (EM-CCD), 30–31, FRET, 795 Explants, for imaging living embryo, 74–75, 78, 142, 233–235, 262, genetic fluorescent probes, 771, 783 748–749. 466–467, 482, 647, 678, 737, and harmonic signal generation, 703 Exposure time, 62, 65, 71–76, 81, 127, 137, 753–754, 784, 923–926. in ion-imaging, 738 141–142, 176, 212, 219, 224, 226, advantages and disadvantages, 30–31, and phototoxicity, 685 231–236, 267, 270, 276, 346, 363, 220, 228, 233–235, 237, 459–460, table, 363 392–393, 423, 427, 459–460, 477, 647, 737, 909, 923–926 Endpoint data analysis, 816–817. 495, 556, 613, 627–628, 651, 655, CIC, clock-induced charge, 234, 926 Endpoint translocation/redistribution assays, 681–686, 692–697, 708, 746–747, and disk-scanners, 76, 205, 215, 220, 814. 753–755, 760–764, 783–784, 816, 270 Energy diagram, lasers, 102, 105, 106. 822, 850–851, 873. frame-transfer, 262, 234 argon-ion laser, 102 for CCDs and EM-CCDs, 127, 137, gain-register amplifier, 76–77, 258, 753, helium-cadmium laser, 105 141–142, 231–236, 267 925 helium-neon laser, 105 disk scanners, 231–235 interline-transfer, 233–234 semiconductor laser, 106 laser, safety, 117–118, 839, 900, 903–904 mean-variance curves, 78 titanium:sapphire four-level vibronic reducing, 753–755 multiplicative noise, 77 laser, 109 and source brightness, 141–142 noise currents, 256 Energy, of single photon, 35, 127. total, comparison of methods, 442, 449 parameters, vs. normal CCD, table, 233 Energy transfer rate, for FRET, 790, 792. UV, 116 QE(effective), 78, 927 Entrance aperture. See Back-focal plane. x-ray, 614–616 readout amplifier, 76–77, 258, 753–754, EOM. See Electro-optical modulators. External laser optics, maintenance, 117. 925 Epi-detected CARS (E-CARS), 597–599. External photoeffect. See Photoemissive results, 235, 237, 755 erythrocyte ghosts, 603 effect. Electron-beam-scanning television, 6–7. Epi-fluorescence microscopy. See External Pockels cell, 25, 54, 87, 116, 543, Electron-hole (e-h) pairs and photon Fluorescence microscopy, 44, 166, 701, 903–904. counting, 29. 172–173, 195, 202, 235. External-beam prism method, laser control, Electronic bandwidth, 64–65. See also, Epi-illuminating confocal microscope, 9, 90. Bandwidth. 166. See also, Confocal laser Extracellular polymeric substances (EPS), Electronic noise, defined, 232. scanning microscopy; Confocal 183, 311, 358, 376, 703–704, 717, Electronik Laser Systems GmbH, VersaDisc, microscopy. 760, 783, 870, 879–880. See also, 109. Episcopic fluorescence image capture Collagen. Electrons, interaction with light, 129–130. (EFIC), 607–608. bleaching, 693 Electro-optical modulators (EOM), 25, 54, mouse embryo image, 608 damage, 685 57, 87, 116, 543, 701, 903–904. Epithelial cells, 14–15, 603. dye, 361 Electroporation, 359–360, 795, 803. CARS image, 603 lectin-binding in biofilms, 870, 879–880 for chromophores, 803 oral, optical sections, surface ridges, matrix, 760 Ellis, Gordon, 2, 3, 7, 8, 13, 14, 84, 129, 14–15 negative contrast, 173 131, 478, 507. EPS. See Extracellular polymeric in optical projection tomography, 612 Embryo imaging. See Living embryo substances. plants, 438, 783 imaging. Erythrocyte ghosts, CARS imaging, 603. preparation, 376 Embryos, 761–766. Ester-loading technique. See Acetoxymethyl Extrinsic noise, reduction, 21. bulk labeling, with dyes, 761 esters loading method. depiction, in time and space, 762–764 Ethylene glycol-bis-succinimidyl (EGS), F dyes, for multi-wavelength analysis, 756 369. Fabry-Perot interferometer, optical cavity, FRET, 764–766 Euphorbia pulcherrima, spectrum, 710. 81–82. 950 Index

Fast Fourier transform, 487. step-index vs. gradient index, 502 conventional, 45 to identify interference fringes, 202 step-index optical fibers, 501–502 hard vs. soft coatings, 45–49 Fast line scanner, 231–232. transmission losses in silica glass, 502 intensity loss, 212 Fatty acid indicator, 347. Fiber-optic confocal microscopy, 501–515, interference, 45–51 FBG. See Fiber Bragg Grating. 893. conventional and hard coatings, 46 FBR. See Fiber Bragg Reflector. benchtop scanning microscopes, 507–508 multi-channel detection, 51 FBTC. See Fused biconical taper couplers. clinical endomicroscopy, 513 ND filters, 43, 89 F-CARS. See Forward-detected CARS. distal tip, 514 notch and edge, 50 FCS. See Fluorescence correlation human cervix image, 513 tuning with angular dependence, 50 spectroscopy. human gastrointestinal track image, to select image contrast features, 162 Feedback, 136, 139. 514 short-pass, interference type, 46 for control of light-emitting diode, 139 image transfer bundles, 504–505 transmission vs. laser line, 212 to increase source stability, 136 managing insertion losses, 506 types, 46 Femtosecond pulsed lasers. See Ultrafast miniaturized scanning confocal, 508–512 wavelength selective, 43–51 lasers. bundle imagers for in vivo studies, 509 FiRender, 281–282. Feulgen-staining, DNA, 166, 200, 298, 433, with coherent imaging bundles, First or front intensity, projection rule, 302, 437 508–509 304. Fianium-New Optics, Ltd., FemtoMaster- imaging heads, 508–512 FITC. See Fluorescein isothiocyanate. 1060 objective lens systems, 509 Fixation, specimen, 368, 378, 428, 852, 854, fiber laser, 113–114. optical efficiency, 509 856. Fiber Bragg Grating (FBG), laser optical schema, 508 antibody screening with glutaraldehyde stabilization, 87. resolution, 509 fix, 377 Fiber Bragg Reflector (FBR), stabilizes rigid endoscope, 511 artifacts, 195, 369–373, 428, 624, 815, laser, 87. vibrating lens and fiber, 510–511 854, 857 Fiber lasers, 85, 101, 109–110, 113–114, in vivo imaging in animals, 510–514 autofluorescence, 358, 663 124. Fiber-optic interferometer, 240–241, 504, borohydride to reduce autofluorescence, defined, 109–110 609. 374, 770 temperature sensitivity, 85 diagram, 241 chapter 368–378 tutorial reference, 124 for measuring point spread function, characteristics, 368–370 ultrafast, 101, 113–114 240–241 chemical fixatives, 369 Fiber optics. See Chapter 26. Fiber-optic light scrambler, 8, 13, 131–132, crosslinking fixatives, 369 beam-splitters, 503–504 143. freeze substitution, 369, 769, 854–856 Bow-tie, pol-preserving fiber, 503 Fibroblasts, 292, 361, 691, 798, 803, 852. microwave fixation, 369 cable, for delivering ultrafast pulses, 88 Field diaphragm, 34–35, 127–128, 139, 461, protein coagulation, 369 laser output, 106 627, 648–649. cryo-fixation, 854 pigtail, 106 Field effect transistor (FET) CCD amplifier, dehydration, 166, 368, 417–418, 481, Fiber optics used in microscopy, 501–507. 30–31, 77, 922–927, 929. 611, 623–624, 815, 849, 854–855 evanescent waves in optical fibers, 503 noise vs. pixel dwell time, 922 effect on plants, 428 fiber image transfer bundles, 504–505 Filament-based lamps, 34, 44, 126–132, for electron microscopy, 167, 368–369, fiber-optic beam-splitters, 503–504 135–138, 346, 507, 648, 663. 372, 479, 731, 851–860 fused biconical taper couplers, 503–504 fiber optic, 507 ethylene glycol-bis-succinimidyl, 369 glass made from gas, 501 image, 100 W halogen bulb, 135 evaluation, 371–374 gradient-index optical fibers, 501–502 size, 126–127 cell height to measure shrinkage, key functions of fibers, 505–507 spectrum, 44, 136 371–373 delivering light, 505–506 stability, 34, 137 MDCK cell example, 372, 373 detection aperture, 506 File formats, multi-dimensional images, formaldehyde, 369–370, 373 diffuse illumination, 507 288–289. general notes, 374–378 for femtosecond laser pulses, 507 Fill factor. geometrical distortion, 372–373, 815 large-area detection, 507 of CCD, 920–921, 927, 929 GFP, 854, See also, Green fluorescent large-core fibers, as source/detection disk-scanning microscopes, 224–228, 233, protein apertures, 507 552 arsenical derivatives, 348 same fiber for source and detection, Filtering, digital, 281, 810. See also, glutaraldehyde, 369, 370 506 Deconvolution. high-content screening, 815 single-mode fiber launch, 505 Gaussian, 41, 65. See also, Gaussian immunofluorescence staining, 371, 372, SMPP optical arrangement, 216 filters 852 managing insertion losses, 506 multi-dimensional microscopy display, improper mounting, 376 angle polishing of fiber tips, 506 281 microwave, 377–378 anti-reflection coating of fiber tips, 506 nonlinear, deconvolution, 190 mounting methods, 370–374 index matching of fiber tips, 506 sets, for automated confocal imaging, 810 critical evaluation, 371–374 microstructure fibers, 504 smoothing, effect on contrast, 59 media refractive index, table, 377 modes in optical fibers, 502 to reduce “noise” features, 70 technique, 371 polarization effects in optical fibers, 503 Filters, optical, 43–51, 70, 89, 162, 190, optical properties of plants, 428 polarization-maintaining fibers, 503 212, 753. See also, Heat filters. pH shift/formaldehyde, 370–371, 373 Index 951

plants. See also, Botanical specimens, laser requirements, 81 methods, 518–527 Plant cells, 428, 769–770, table, 385 comparison, 523–527 773–774 Fluorescence emission, botanical specimens, frequency domain, 518–520 refractive index of mounting media, table, 425–428. time domain, 520–523 377 1- vs. 2-photon excitation, 421 multi-focal multi-photon microscopy, optical effects, 428 Fluorescence imaging, deconvolution vs. 555–556 refractive index of tissue/organs, table, confocal, 459–460, 644–648. quantitative fluorescence, 517–518 377 Fluorescence in situ hybridization (FISH), quantum efficiency, 516 shrinkage, 369–373, 624, 815, 854 316–317, 319, 323, 331, 333–334, spectroscopy, 516 staining, 370–371 343, 875–878. table, 385 tissue preparation, 376 biofilms stains, 875–878 time domain detection methods, 520–523 Fixed wavelength lasers, table, 119–120. with fluorescent protein, 878 point-scanning, 522 Fixed-pattern noise, 74–76, 278, 924, 927, Fluorescence ion measurement, 736–738, streak camera, 520 931. 740–745. See also, Calcium imaging, TCSPC FLIM, 522–523 Flare, out-of-focus light, 6, 132, 157–158, pH, etc. time-gated FLIM, 523 172, 395, 456, 465–466, 469, 471, calcium imaging, 736–737 use of intensified CCDs for, 233 481, 649, 731. concentration calibration, 742–745 Fluorescence loss in photobleaching (FLIP), Flatness of field, 145, 151, 154, 418, 457, indicator choice, 738 187, 382, 384, 801. 639. interpretation, 740–741 FRET, affected by, 801 measurement/ small pinholes, 145, 457, pH imaging, 346, 739–745 table, 384 639 water-immersion objectives, 737 Fluorescence microscopy, 4, 9. 13, 43–44, objectives, to improve, 151–152 Fluorescence lifetime imaging microscopy 154, 166, 172–173, 195, 202, 235, Flat-fielding CCD data, 76, 477. (FLIM), 108, 111, 114, 139, 204, 251, 448–451, 809–810 See also, black reference, 76 233, 382–383, 385, 516–533, Widefield (WF) fluorescence white-reference, 76 799–801. microscopy. Flexible scanning, 51–52. advantages, 766, 800 chromatic correction, 154 FLIM. See Fluorescence lifetime imaging alternatives to, 766 compared to disk-scanning microscopes, microscopy. analysis, 251 235 FLIP. See Fluorescence loss in applications, 516–518, 527–532 vs. confocal imaging, 13 photobleaching. calcium imaging, 529 depth of field, 4 Flip mirrors, to control laser, 58. chemical environment probe, 517 filters for selecting wavelengths for, Floppy disks, 586. FRET, 517–518 43–44 Fluorescein,48, 80–81, 88, 203, 261, ion concentration, 517, 528–530 folded optical path, 166 353–355, 375, 443, 582, 697, 781, multi-labeling with dyes, 517, 527–528 increase contrast with less intensity, 794, 930. pH imaging, 529–530 172–173 arsenical derivatives, 348 probes, 517 signal-to-noise ratio comparative, calculating laser power needed, 80–81, table, 530–532 448–451 443 comparison of methods, 523–527 bleaching-limited performance, derivatization, diagram, 354 acquisition time, 525–526 448–450 double-labeling, 375 bleaching, 524 configurations of microscope, 448, 449 filters for, 48 cost, 526 disk-scanning microscope, 449 photobleaching quantum yield, 363 detector properties, 526–527 line illumination microscope, 449 rhodamine and, FRET between, 794 multi-exponential lifetime, 523–524 saturation-limited performance, 450 Fluorescein isothiocyanate, 88, 198, 203, photon economy, 524–525 scanning speed effects, 450–451 261, 263, 335, 375, 394, 397–398, pile-up effect on detection efficiency, S/N ratios, table, 450 511–512, 527–528, 582–583, 526 wide field (WF) microscope, 450 693–694, 781, 794, 799, 884, 885. shortest lifetime, 523 spectral problems, 44 See also, Fluorescein. table, 526 Fluorescence, quenched by colloidal gold, 2-photon, 781 decay process of excited molecule, table, 854. biofilms, 884–885 518 Fluorescence recovery after photobleaching dextran, 292, 512 frequency domain methods, 518–520 (FRAP), 51, 54, 56, 90, 187, 210, filter sets, 48–49 disk-scanning implementations, 520 218, 224, 229, 237, 362, 382, 384, FRET, 794, 799 phase fluorometry method, 518–519 390, 691, 759, 801, 805, 850. lifetime, 527–528, 532 point-scanning implementations, 520 in biofilms, 874 photobleaching quantum yield, 363 widefield, spinning-disk, 519–520 damage to cellular structure, 341, toxicity, 391, 693–694 frequency-domain, 108 859–851 Fluorescence anisotropy measurements, 742. reducing repetition rate, 111 damage to microtubules, 341, 850–851 Fluorescence contrast, 172–173. FRET, 799–801 efficiency of illumination light path, 210 Fluorescence correlation spectroscopy history, 516 related to TEM of same specimen, (FCS), 5, 363, 383, 385, 602, 801, Jablonski diagram, 516, 517, 697, 792 850–851 803, 805, 917. with light-emitting diode sources, 139 setups for, 218, 907 and CARS, 602 limitations, 800 table, 384 FRET, 801 living cell images, 204 using CARV2 disk-scanner, 229, 907 952 Index

Fluorescence resonance energy transfer Fluorescent constructs for FRET, 801–802. dye classes, table, 355 (FRET), 26–28, 34, 184–187, 204, cloning of fluorescent chimeras, 801–802 dye vs. probes, 353 218, 221–222, 382, 384, 425, expression and over-expression, 802 fluorescein, 353, 355. See also, 517–518, 556, 650, 691, 741–742, functional activity of expressed, 802 Fluorescein fluorescent proteins, 764–766, 788–806, 796–797. Fluorescent dyes. See Dyes; Fluorescent 355–357 based on protein-protein interactions, 800 indicators; Fluorescent probes. GFP, 355–357. See, Green fluorescent based sensors, 798–799 Fluorescent efficiency, 34. protein indicators of intracellular botanical specimens, 425 Fluorescent emission, incoherence, 130. sate, 346–348 C. elegans, 766 Fluorescent indicators, 346–348, 736–743. Ca2+ indicators, 346–347 chapter, 778–806 See also, Fluorescent probes, and protein cloning and expression of fluorescent particular ions. multi-photon excitation, 357–358 constructs for, 801–804 binding equation, 740–741 phycobiliproteins, 355–357 donor/acceptor pair, 790, 792–794 buffering, 740 probes/specimen characteristics, table, donor, 796–797 calcium imaging, 736–737 354 efficiency, 792 calibration, 742–743 quantum dots, 357 experimental preparation, 795 indicators, 738 rhodamine, 342–345. See also, FCS and, 801 cellular introduction, 738–739. See also, Rhodamine FLIM and, 799–801 Loading excitation, 737, 344–345 between fluorescein and rhodamine, 794 cellular trapping, 738 for fluorescence lifetime imaging, 517, fluorescence lifetime imaging, 517–518 choice, 738 530–532 fluorescent proteins, 794–795 concentration, 741–742 genetically encoded, for plant imaging, FRAP and, 801. See also, Fluorescence dialysis, 740 769, 771, 773, 783. See also, recovery after photobleaching future free diffusion, 741 Transcriptional reporters; perspectives, 805 genetically expressed intracellular, 348 Transfection agents for high-content induced by cholera toxin transport, 797 green fluorescent protein, 348 screening, 810 intramolecular, 765 ion indicators, 348 high specificity/high sensitivity, 806 kinetics, 741–742 ligand-binding modules, 348 living cell imaging, 387–389 in living cells, 195–186, 204 handling, 739–740 rapid assessment by, table, 360 chapter, 788–806 inaccurate measurements, 740–741 loading methods, 358–360. See also, in living embryos, 764–766 intracellular parameters imaged, 346–348 Loading MMM, 797–798 Ca2+, 346–347 acetoxymethyl esters, 359 nanobioscopy of protein-protein cAMP, 347 ATP-gated cation channels, 359 interactions fatty acid, 347 ballistic microprojectile delivery, 360, acceptor bleach for, 797–798 ion concentrations, 346–347 724–725, 802–803 donor fluorescence for, 796–797 membrane potentials, 346 direct permeability, 358–359 measurement methods for, 795 other ratioing forms, 347–348 electroporation, 359–360, 795, 803 sensitized emission of acceptor, oxygen, 347 microinjection, 360–361, 388, 739, 795–796 pH, 346, 739–745 748, 755, 795, 803–804 photobleaching, 691 wavelength ratioing, 346 osmotic permeabilization, 359 practical measurements, 792 positive pressure, 740 peptide-mediated uptake, 359 probes, 221–222 selectivity, 743 transient permeabilization, 359 quantum dots, 801 Fluorescent intensity (IF), TIRF, 180. whole-cell patch pipet delivery, 360, setups, 218 Fluorescent labels, 342–346, 530–532, 761, 726–727, 734, 738–740 small molecules, 794–795 775. See also, Dyes; Fluorescent photoactivatable, 210, 224, 383, 385, 541, spatial orientation factor, 792–793 probes; Chapters 16–17, and by 544–545, 693, 759–760, 912 spectrofluorimetry, 793 name of dye. Kaede, 187, 383, 385 spectroscopic properties used for, 795 Fluorescent probes. 353–364, 387–389, 517, Kindling, 574, 760 standards for, 34 530–532, 736–737, 739–740, 755, PA-GFP, 187, 383, 385, 752, 759–760 table, 384 769, 771, 773, 783, 806, 810, 811. photobleaching, 362–363. See also, theory, 790–794 See also, Dyes, Fluorescence Bleaching TIRF and, 801 indicators and by name of dye, phototoxicity, 363–364 See also, total, measured with widefield, 26–28 Chapters 16, 17. Phototoxicity factors influencing, in transgenic animals, 765 automatic living cell assays, 811 table, 363 wavelength depiction, 793 bound, 737 specimen interactions, 361–362 Fluorescence saturation, singlet-state, 21–22, care, 739–740 cytotoxicity, 362 41, 142, 265, 276, 339, 448, 643, characteristics, table, 344–345, 354 localization, 361–362 647, 899. development, 736 metabolism, 361–362 Fluorescence speckle microscopy (FSM), dye criteria for, 353–358 perturbation, 362 13, 383, 385, 889. AlexaFluor dyes, 353–355 target abundance/autofluorescence, table, 385 BOPIDY dyes, 353–355, 749, 760–762 360–361 Fluorescent biosensor, 799, 805. coumarin dyes, 353, 355 tissues, 360 future, 805 cyanine dyes, 353, 374, 587, 760, 854, Fluorescent proteins, 187, 355–357, 739, mitotic clock measurements, 799 874 794–795. Index 953

+ emission change after photodamage, Forsterite laser (Cr4 in MgSiO4), 109, FRAP. See Fluorescence recovery after 187 114, 415, 541, 706, 707–709, photobleaching. FRET, 794–795 712–713. Free diffusion, of fluorescent indicators, genetically engineered variants, 739 second/third harmonic generation, 114 741. ion binding regions, 739 tunable, 109 Free-ion concentration, 742. Fluorescent lights, stray signal, 201, 632, Forward-detected CARS(F-CARS), Freeze thawing, 731, 739. 904. 597–599, 603. Frequency, 52, 65, 82. Fluorescent staining, 371, 393, 438, 774. erythrocyte ghosts, 603 laser vs. pumping power, 82 See also, Dyes; Staining. Foundations of confocal LM, chapter, 1–19. of resonant galvanometer, 52 immunofluorescence, 371, 372, 852 Four-dimensional images, 746–749, 752, of sampling clock, 64 living cells, 393 761–764. Frequency doubling. See Second harmonic microglia, 319–320, 393–398 advantageous techniques, 746–747 generation. nuclei of living or dead cells, 393 automatic image analysis, 321 Frequency-resolved optical gating (FROG) Fluorite (CaF2), optical to reduce chromatic deconvolution, 495 for pulse length measurement, 115. aberration, 153. embryogenesis visualization strategies, FRET. See Fluorescence resonance energy FluoroNanoGold, cryosections, 854. 761–764 transfer. Fluorophores, 44, 338–349, 543–544, living cells, 393 Frustrated total internal reflection, defined, 664–667, 748, 794, 799. See also, of living embryos 177. Dyes, Fluorescent labels. cellular viability, 747–748 FSM. See Fluorescence speckle microscopy. Flying spot detector for measuring photon challenges, 762 Full-well of CCD pixel, defined, 75. efficiency, 34–35. dataset display strategies, 393, 763–764 Full-width half maximum (FWHM) Flying spot ultraviolet (UV) microscope, deconvolution, 752 resolution. 6–7. for large thick specimen, 746–747 4Pi, 562, 567 Fly’s-eye lenses, for diode lasers, 107–108. photobleaching during, 747–748 of beams in scanning disk, 554 FM4-64, FM1-43, and other lipophilic photodamage during, 746 of CARS, 597, 599 membrane dyes, 236, 355, 359, 360, required datasets for, 746–747 of confocal performance, 656–657, 389, 556, 775, 760–761. multi-photon, 535 661–662 Focal CheckTM beads, 657–659. structured illumination, 482 of emission wavelength Focal-plane array detection, 2-photon, SPIM, 676 LED, 136 542. Fourier analysis. quantum dots, 343 Focal shift for mismatched RI, 405, 4Pi microscope, 563, 576 of interference filters, 44 407–410, 553. analogy with image reconstruction, 69 laser bandwidth, 93, 95, 100, 101 defined, 405 of blind deconvolution, 472–476, 478 laser pulse length, 109, 112, 507, 537, dependence, 410 and convolution, 485–487 538, 902 for glycerol, table, 409 of image formation, 446, 454, 456–457 micro-surgery precision, 219, 687 for water, table, 409 MRM, 618–620 multi-photon, 682–683, 901–902 Focus, 3–4, 13, 36, 197. of periodic test specimen, 638–639 objective resolution (PSF), 149, 209, 225, for confocal microscope, 36 of short laser pulses, 88, 728 444–445, 456, 492, 509, 552, 571 displacement, by living cell specimen, SPIM multiview processing, 675–677 PMT rise time, 225 22–23 STED, 574 resolution, with spherical aberration, effect of coverslip, 197 of structured-illumination images, 268, 407 extended, 9 270–273 table, 409 in phase-dependent imaging, 13–14 and wavelet processing, 734 SPIM, 675 planes, diagram, 27 Fourier plane. See Back-focal plane, 201, STED, 572, 576–578 position, confocal microscopy, 651–652 245, 509. z-resolution, measured, 194 Focused spot. See Point spread function. Fourier space, 270–271. Fundamental limits, chapter, 20–42. Folded optics, for trans-illuminated confocal Fourier transform, 201, 202, 271, 487, 489, Fungi, 438–439, 624, 782, 870. microscopy, 166. 490–492, 620. Fura-2 [calcium ion] indicator dye, 103, Formaldehyde, 369–370, 373–377, 428, of AC interference in image, 201–202, 189, 234, 257, 345, 346, 348, 738. 651 358–359, 361, 529, 531, 726–727, AM-loading releases formaldehyde, and convolution, 487 730, 733, 741–743, 810, 812, 846, 738 and deconvolution, 487, 490–492 850. fixation protocol, 371 for detecting stray light into detector, Fused bi-conical taper couplers (FBTC), permeabilization agents for, 375 201 503–504. pH shift method, 370–371, 373 identifying interference fringes, 202 Future, 143–144, 160, 192, 219–220, 234. for plants, 428 of microtubule TIRF image, 183 of EM-CCD with interline transfer, 234 stock solutions, 370–371 missing cone problem, 494 of laser-scanning confocal microscopes, Förster distance, defined, 184, 790, 792, MRM image formation, 620 219 793. of point spread function, 489, 490 of non-laser light sources, 143–144 Förster equation, 184, 790, 793. Fractal compression, 581–582. spherical-aberration corrector, 15, 147, Förster resonance energy transfer. See also, Frame rate. See also, Speed 151, 192 Fluorescence resonance energy in confocal microscopy, 11 of tunable objective, 160 transfer. matching, 838–839 FWHM. See Full-width half maximum. 954 Index

G “Gaussian-to-flat-wavefront” converter, immersion objective lenses, 412, 563, 567 Gain, 31, 232. 554 example, 785 of image intensifier, 232 Kerr effect produces self-focusing, 111 mounting media, 371, 373, 375, 377–378, photomultiplier tube, from collisions at laser beam profile, 538–539, 554, 597, 420 first 635–636 RI-mismatch, table, 409, 410 dynode, diagram, 31 noise, 473, 497, 925 Goggles, laser, for eye protection, 118. Gain register, (EM-CCD) 76–78, from optical fiber, 502, 505–506 Gold’s ratio method, 476. 233–234. optical tweezers, 89. See also, Laser Golgi receptor, 374, 389, 556, 564–566, CCD (CCD), 76–78 trapping spatial filter, 89, 729 791. of electron multiplier-CCD, 233–234 Gaussian filters, digital, 39, 41, 65, 70, 89, Golgi stain, 107, 283, 298. Gain setting, 75, 115. 281, 285, 301, 323, 338, 391–392, Gourard shading, 308, 309, 311. defined, 75 399, 497, 499, 510, 650, 667–668, Gouy phase shift, 597. effect of bandwidth on, 115 676, 729, 734, 753, 764, 830. Graded index (GRIN) lenses, 84. GAL4 genes, 773. of 3D data to reduce Poisson noise, 39, in diode lasers, 108 Gallium arsenide (GaAs). 41, 65, 69–70, 269, 281, 285, 323, Gradient index optical fibers, 501–502. diode laser, 107, 111 391–392, 399, 499, 510, 635–636, Gradient-weighted distance transform, InGaAs photodiode, 707–708 650, 667–668, 676, 764, 830 323. LEDs, 133, 138, 143 “Gaussian blob,” 635–636 Graphics interchange format. See GIF. PMT photocathode, 4, 28–29, 232, 252, and Nyquist reconstruction, 65 Grating, periodic. 255, 263, 464, 527, 931 in presentation displays, 830 GVD compensator, 88, 504, 538, 686 Galvanometer, 11, 25, 36, 40, 51–54, 56, 57, results, 285, 676, 733, 835–837 laser tuning, 90, 103, 106–107, 111 63, 211, 215, 223, 231–232, 513, Gaussian laser pulses, 536–536, 902. minimum spacing, 1, 16, 652 543, 552, 558, 599, 651–652, 753, Gaussian noise, 473, 497, 925. OCT phase-delay, 609 806, 907, 910–911, 914, 931. See Gaussian norm statistical tests, 830, 835, pulse compressor, 113 also, Linear galvanometers. 837. spectral detector, 87, 346, 422, 664, defined, 52–54 GDD. See Group delay dispersion. 772 distortion, 211 Gene gun, 360, 724–724, 730. structured illumination, 266–267, 273 electromechanical properties, 40 Geometric contrast, 180–187. Gray levels, 71–76. errors, 40 Geometric distortion, 6, 23, 36, 39–41, 53, intensity spread function, 74–76 in fiber-optic micro-confocal, 513 152, 211, 215–216, 265, 297, 329, printer, 592 figure, 63 372–373, 448, 480, 590, 641, Green fluorescent protein (GFP), 90, 174, line-scanner, 231–232 653–654, 741, 835. 221–222, 348, 355–357, 429, linear, 52, 53, 223 kinetic, 741 478–479, 556, 568, 571, 612, 614, measurement, 651–656 measurement, 651–656 625, 675–676, 690, 692, 698–699, multi-focal, 554 projector, 590 724–725, 727, 731, 741, 747–752, multi-photon, 543 of specimen preparation, 372–373, 815, 755, 756–763, 766, 769–773, resonant, 25, 52–54, 56–57, 223, 447, 872 781–785, 798–806, 812–815, 820, 510, 539, 543, 552, 558, 910 Gerchberg-Saxton algorithm, deconvolution, 854–859, 862, 873–875, 877–879, specifications for, 214, 543 472. 885. See also, Transfection reagents; ultra-precise, 211 GFP. See Green fluorescent protein. Transcriptional reporters. x-y scanners, 213–215, 223, 651–654, Ghost images, from transmission biofilms labeling, 873 806, 907, 910–911, 914 illuminator, 201–202. or CFP molecules, as FRET pair, 798 Gamma, brightness non-linearity, 72–73, GIF (Graphics interchange format), 580. constructs, in embryos, 756 287, 832–833. Gires-Tournois interferometer (GTI), to EM imaging, brain cells, 731, 854–859 data projector, 590 reduce GVD, 88. FRET, 793–795, 798–803 display, 582–583, 589, 832–833 Glan-Taylor polarizer, 85, 87, 100, 171. image contrast, 174 Gas lasers, 86, 90–105. See also, CW lasers; in single-sided confocal microscope, limitations, 760 Pulsed lasers. 171 membrane localized, 749 continuous wave, 90–105 Glan-Thompson polarizer, attenuator, 85, methods with Correlative LM/EM, 854 maintenance, 116 904. mice, 727 noise sources, 86 Glutaraldehyde, fixative, 369, 369–374, photoactivatable, 187, 383, 385, 752, pressure, 102 377–378, 428, 438, 731, 852. 759–760 Gating, intensified CCD, 25, 233, 262, 522, antibody screening with, 377 photobleaching, 690, 692, 698 555. autofluorescence of, 374, 428, 770 for plant imaging, 424, 429–430, Gaussian beam profile, lasers, 80–81, 83–84, fixation protocol, 370 769–773, 781–785 108–109, 111, 113, 116, 231, 269, stock solutions for, 370 direct visualization, 773 338, 456, 496, 502, 538–539, 554, Glutathione (GSH), 342, 358, 545, 694, 779. genetic fusions, 773, 783 891. visualization, in plant cells, 782 genetic marking, 773 in CARS, 597 Glycerol, immersion/mounting medium, two-photon excitation, 782–783 converted into line, 231, 916 404, 407, 409–410, 435, 563, 654, protein fusions/cytoskeleton, 773–774, fiber optic, 502, 505, 506 698, 785. 801 filling back-focal plane, 210, 509, 629, clearing, 198, 200 tagged proteins, 758 633 diffusion in, 698 TIRF, 90 Index 955

FRET, 794 Heat, 84–85, 89–90, 109, 129, 133. Holographic diffusers, to reduce coherence, Grey levels, 71–76. filtering, dichroic filters, 43–44, 129, 84. printer, 592 132 Holography, holomicrography, 7–8. GRIN. See Graded index. heat sink for LED light source, 133 Hooke, Robert, image of cork, 769–770, Ground state depletion (GSD), 573. from laser cooling, 84–85, 109 785. Group delay dispersion (GDD), 537–538, of optical trap, 89–90 HTS. See High throughput screening. 543. placing system components, 129 Huffman encoding, 580–581. Group velocity dispersion (GVD), 88, 111, Heat filters, to exclude IR light, 43–44, 129, Human endomicroscopy, confocal. 210, 537, 606, 609, 903. 132. cervix, 513 in optical coherence tomography, 609 liquid, 132 gastrointestinal track, 514 pulse broadening due to, 88, 111, 210, Heating. See also, Thermal variables. Human retina, viewed with OCT, 609. 537–538, 543, 606, 609, 728, detectors, 252 Huygens, 3D software, 104, 413, 669, 778. 903 microwave fixation, 377 Huygens-Fresnel wavefront construction, GSD. See Ground state depletion. in magnetic resonance imaging, 621–622 406. GTI. See Gires-Tournois interferometer. multi-focal, multi-photon, 551, 556, 685, HVEM. See High-voltage electron Guinea-pig bladder, calcium sparks, image, 903 microscope. 237. specimen, by the chamber, 387–389, 394, Hybrid mode-locked dye laser, 540–541. GVD. See Group velocity dispersion. 732 Hymenocallis speciosa, fluorescence spectra, Gzip, 580. specimen, by the illumination, 43, 89, 422. 132, 211, 218, 341, 536, 539, 544, Hysteresis. H 556, 621–622, 681, 685, 884, 903 in Piezoelectric scanners, 57, 754 Hairs, plant, 431, 434–436, 772. calculation, 89, 685, 904 temperature cycling of lenses, 249 Halftoning vs. dithering, 589. stability, 652 Halogen lamps, 126–127, 132, 136–139, HeLa cells, 391–392, 693, 799, 812, 814, I 143, 159, 663. 820, 828, 854. I5M, (Incoherent Illumination Image brightness vs. temperature, 136 Helios Gene Gun System, 724. Interference Imaging), 275, 561, filaments, 132 Helium-cadmium (He-Cd) laser, 83, 86, 90, 569–570, 672. image, 135 93, 103, 105, 115. optical transfer function (OTF), 569–570 lifespan, 136 operational lifetime, 115 ICNIRP. See International Commission of power available, 126–127 output variation, 86 Non-Ionizing Radiation Protection. stability plot, 137 transverse electromagnetic mode, 83 ICTM. See Iterative constrained Tikhonov- Haralick features, 818–820. Helium-neon (He-Ne) laser, 82, 84, 88–90, Miller algorithm. Hard coatings, for interference filters, 45, 93, 102–103, 105, 107, 240, 241, IEC. See International Electrotechnical 48. 376, 673, 680, 798, 799, 864, 875. Commission. Hard copy, 580, 590–594. four state, 82, 105 IF. See Fluorescent intensity. photographic systems for, 590–591 Heterectis crispa, 874. Illumination, 44, 210. See also, Structured- printers, 591–593 Hidden-object removal, 304–305. illumination microscopy, and aliasing, 592 High content screening (HCS), 809–817. Chapter 6. color images, 592 for cytomics chapter, 809–817 brightness, table, 140 digital, 591–593 data management/image informatics, errors, 211–212 grey levels, 592 816–817 evaluating, 211–217 ink jet, 593 fluorescence analysis of cells, table, 812 goal in confocal microscopy, 210 laser, 593 multiple fluorescent probes, 810 path, 211–212 posterizing, 591 High resolution spatial discrimination, 813. types of lamps, 44 scaling techniques, 592 High throughput screening (HTS), 809. vignetting caused by beam shift, 211–212 Harmonic signals, 2, 49, 80, 90, 100, 109, High voltage electron microscope (HVEM), Image(s), 9, 11–12, 30–31, 38–39, 59, 145, 113–114, 162–163, 174, 179–180, 846. 192, 210, 219, 280, 286–290. See 188, 243, 361, 414, 428, 535, 545, stereo images of platelets, 848–849 also, Multidimensional microscopy 550, 556, 577, 596–597, 682, Hippocampal brain slices, 268, 316–317, images. 703–704, 708–719, 722, 729, 734, 393, 556–557, 722, 724–725, 727. contrast, 7, 11, 16, 39, 49, 60–62, 68, 894 See also, Second harmonic calcium imaging, 556–557 159, 162, 165, 167, 173–175, 180, generation; Third harmonic culture protocol, 724–725 189–190, 192, 201–204, 248, 421, generation see Structured damage, 341 473, 488, 542, 599–600, 607, 622, illumination. at neurons, 205, 268, 316–317, 393 656, 657, 675 chapter, 703–721 Histology, 623, 624. chapter, 162 contrast, 179–180, 188 Historic overview of biological LM, table, flare, 649 descanned detection, 56 2–3. definition, 280 in lasers, 109, 113, 114, 115 Hoechst, DNA dye, 136, 339, 344, 360, 362, degradation of, measuring, 145 plants, 428 520, 565–566, 683, 782, 812. extended-focus, 9 second and higher, 114 4Pi, image, 565–566 motion between specimen and objective, Haze, from out-of-focus light, 227. FLIM image, 521 39 HBO-50 mercury-arc bulb, 126. high-content screening, 812, 814 multi-dimensional microscopy, 286–290 HCS. See High content screening. Holey optical fiber/non-linear effects, 88. anisotropic sampling, 287 956 Index

Image(s) (cont.) multi-channel time-lapse fluorescence, Indo-1, calcium indicator, 103, 189, 257, calibrating image data, 286–288 382 345, 346, 348, 529, 531, 544, 693, contrast transfer function (CTF), 61. optical tweezers, 383 697, 742–743. See CTF photoactivation, 187, 224, 383, 385, 541, Infinity corrected optics, 155–157, 166, 239, data type/precision in computations, 544–545, 693, 759 405. 288–289 photo-uncaging, 383. See also, Photo- advantages, 156–157, 166, 239, 405 digitization, defined, 62 uncaging physiological fluorescence, Infinity PhotoOptical, InFocus spherical dimensions, 286–288 383 aberration corrector, 15, 151. display devices, non-linearity of, 72–73 spectral, 382 Infinity space, generating, 157. file formats, table, 288–289 table, 384–385 Information, 27, 60, 64, 73–74, 179, 235, processor performance, 289–290 time-lapse fluorescence, 382 241, 243, 268, 270–275, 278, 330, Voxel rendering speed, 290 Imaris, software, 193, 281–282, 284, 334, 353, 369, 382–383, 396, 398, real, disk- and line-scanners, 30–31 287–288, 290–291, 299, 301–303, 443, 448, 459, 468, 475–476, 481, reconstructing, and noise reduction, 308, 311–312, 764, 795. 487, 488–490, 494, 496–499, 506, 38–39. See also, Reconstruction; In vitro fertilization, mitotic apparatus, 188. 512–513, 517, 519, 522–524, Nyquist reconstruction sharpness of In vitro preparations. 543–544, 556, 559, 570, 580–587, vs. signal intensity, 192 2D mixed-cell, assays, 813 596, 643, 650, 732, 715, 769, 774, of source and detector pinholes, 210 antifade agents. See also, Antifade, 342 776, 779, 782, 790, 794, 800. speed of acquisition, 11–12. See also, automated analysis, 318–320 3-dimensional, 321, 378, 396, 747 Speed as sum of point images, 59 backscattered light image, 513 4Pi, 570 thermal distortion, 219. See also, Thermal biofilms, 870, 872, 879, 884 and bleaching, 222, 690–692, 705 variables bleaching, 551, 851 CARS, 597–598, 602 Image analysis. See Automated 3D image brain slices. See Brain slices, 392–393, colocalization, 668 analysis methods; Automated 725 confocal, 461, 462 interpretation of subcellular location cell maintenance, 387 contrast, see Chapter 8 and Contrast pattern. cytoskeleton, 368 crystal orientation, 179, 188 Image dissector, 254–255. fertilization, 188 display of, 280–281, 288–291, 293, in trans-illumination mode, 10 GFP, 357 295–297, 299–301, 304–305, 311 Image enhancement. See Deconvolution, high content screening, 809, 813–816 efficiency, 336, 628, 631 488–499. high speed imaging, 11, 237, 809, 813, of electronic signal, limitations on, 64 Image iconoscope, for television, 6–7. 815–816 genetic, 756, 762–763 Image intensifiers, 13, 232–233, 235, 255, ion imaging, calibration, 742 lost signal, 25–28 460, 477, 519–520, 522, 524, living cell imaging, 387 matching gray levels to, 73–74 555–556, 730, 737, 784, 801, 930. micro-CT, 614, 617 micro-CT, 615 Image Pro Plus, 282, 290. micro-MRI, 618, 621, 623–625 micro MRI, 618 Image processing. See also, Automated 3D multi-photon, 535 and Nyquist sampling. See Nyquist analysis methods, and Multi- optical coherence tomography image, 609 sampling, 38, 39, 634–637 dimensional microscopy display. photodamage, 684 chapter, 59–79 for display, Chapter 14 In vivo (intact animal) imaging, 112, optical projection tomography, 612 for measurement, Chapter 15 368–377, 512, 545, 806. out-of-focus light, 27, 368, 458, 461, 746, Image resolution, 8, 9. See also, Resolution. 2-photon microscopy (MPM), 535, 543, 784 Image substrate, automated confocal, 810. 545 parallel vs. serial acquisition, 223–224 ImageJ, free software, 282, 290, 395, cell preparations, 387 PSF, 245, 247, 250 732–733, 762–764, 795, 858. comparison with fixed material, 368–377 from second harmonic generation signal, Imaging system, optics characterized by FLIM calibration, 517 179 CTF, 61. labeling, 372–373 Shannon theory, 443 Imaging techniques, 382–386, 394–395. miniaturized confocal, 504, 508, 511–513 on source brightness, 137 combining fluorescence with other, micro-CT, 614, 617 spectral, 665–667 383–386 micro MRI, 618, 621, 623–625 SPIM, 614, 675–378 fluorescence correlation spectroscopy, molecular imaging, 806 storage, 106 383 photodamage, 684, 693–694, 698 chapter, 580–594 fluorescence lifetime (FLIM), 382, “stick” lenses, 806 theory, 4, 64, 443 516–532 Incandescent lamps, 34, 126, 133–137, 477, transmission, contrast transfer function, fluorescence loss in photobleaching 499 See also, Halogen lamps. 37, 60 (FLIP), 382 black-body radiation emitted by, 135–136 Index mismatch. See Spherical aberration. fluorescence recovery after spectrum vs. temperature, 137 Infrared (IR) lasers, 89, 383, 385. See also, photobleaching, 382 stability, 137, 477 Ultrashort lasers; Laser tweezers. fluorescence resonance energy transfer, Incidence angle, 49, 50. solid state lasers, 108–109 382 efficiency, 143 Infrared paper, to identify infrared beams for fluorescence speckle microscopy (FSM), interference filters/transmission, 49 safety purposes, 118. 383 reflectivity, diagram, 50 Ink jet printers, 593. laser trapping, 383 Incident light beam, sample interaction, Innova Sabre/frequency-doubling crystal, linear unmixing, 192, 382, 664–667 162–163. 102. Index 957

Insect cuticle, transparency to NIR light, phase-contrast, 9, 171, 368, 372, 453, Ion-concentration imaging, 736–738, 166. 506, 643, 649, 731, 851, 854, 890, 740–745. See also, Calcium imaging, Installation requirements, for laser sources, 892. See also, Phase contrast pH, etc. 85. centering the phase rings, 643. See calcium imaging, 736–737 Instrument dark noise, 660. See also, Noise also, Bertrand lens scanning, 9, 13 concentration calibration, 742–745 Integrated circuit (IC) chip, 9. using fiber optics, 506 indicator choice, 738 Intelligent imaging innovations, (III), 3D RI inhomomogeneity and contrast, 22–23, interpretation, 740–741 imaging system supplier, 78–79, 151, 41 pH imaging, 739–745 192, 395, 411, 654. Interference filters, 45–51, 102, 136, 212. water-immersion objectives, 737 Intensified CCD, 13, 232–233, 460, 477, in argon-ion laser systems, 102 Ion sensitive probes, optical, 348, 737. 519–522, 524, 555, 556, 737, 784, continuously-graded, 137 table, 531–532 930. destructive and constructive reflections, IR. See Infrared; Near infrared. Intensity, light, 26, 37, 43, 58, 59, 61, 45 Irradiance, arc and halogen light sources, 71–72, 86, 87, 133, 136, 163, 165, transmission, 212 130. 180, 189, 192, 208, 217, 222, 228, types, 46–49 table comparing, 130 258, 270, 391, 413, 426, 459, 461, Interference fringes, coverslip surface, 168, ISO standard, microscope dimensions, 156. 487, 536, 538, 571–573, 633, 681, 170. Iso-intensity surface, or arc sources, 304. 693, 705, 810, 901. Amoeba plasma membrane/coverslip, 170 Iterative constrained algorithms, 475–476. of excitation light, 80, 222, 680–682 in close proximity, 168 See also, Deconvolution; Nonlinear laser beam, stability, 86 Interference mirrors, 46. constrained iterative deconvolution losses Interference mode, coherent light, 130. algorithms. detection path, table, 217 Interference, speckle pattern, 8, 13, 84, 90, Iterative constrained Tikhonov-Miller illumination path, table, 217 130–132, 144. algorithm (ICTM), 497. minimum needed, 392 in backscattered light images, 448 on optical response of specimen, 165 fluorescence speckle microscopy (FSM), J in photons/second, 80 13, 383, 385, 889 Jablonski energy diagrams, 516, 517, 697, regulating, 43, 88 Interferometer. 792. singlet-state saturation, See Saturation 4Pi microscopy, 561 Jansson-van Cittert algorithm, 476, 496. and visibility, 37 Fabry-Perot (laser), 81–82 Jitter, defined, for scanners, 54. Intensity control. fiber-optic, for testing objectives, JND. See Just noticeable difference. continuous wave laser, 88 240–241 Joint Photographic Experts Group. See non-laser, 128 Gires-Tournois, 88 JPEG. Intensity distribution, 146–154. Mach-Zender, to measure pupil function, JPEG (Joint Photographic Experts Group), of Airy disk, 65, 146. See also, Airy disks 245 581–584. changes with focus, 147, 407, 455, optical coherence tomography, (OCT), Just noticeable difference (JND), ocular 463, 471 504, 609 response, 72–73. effect of coverslip thickness, 149 Twyman-Green, 239 effect of RI mismatch, 148. See also, Inter-fluorophore distance, measurement, K Spherical aberration 184. See also, Fluorescence Kaede, photoactivatable fluorescent protein, in focal spot, plots, 147–154 resonance energy transfer. emission change after photodamage, nonsymmetrical change with focus, Interfocal crosstalk, 227–228. 187, 383, 385. 148 disk scanners, 227–228, 444, 449 example image, 187 unit image, 147 time multiplexing as solution to, Kalman averaging, 21, 39, 53, 304, 306, with astigmatism, 152 553–554 627, 638, 655, 750, 754, 781. with coma present, 151 Interlocks, laser safety, 118. comparison with deconvolution, in with spherical aberration, 148–150, Intermediate optical systems, LSCMs, reducing 212 chapter, 207–220. intensity, 39 Intensity loss, with spherical aberration in Internal focusing elements, in objective, Kepler, Johannes, 788. detection path, 148–150, 212. 157, 511. Kerr cell, 516. See Spherical aberration. International Commission of Non-Ionizing mode-locking (KLM), 111, 133 Intensity spread function (ISF), 74–78. Radiation Protection (ICNIRP), 117. of titanium:sapphire lasing rod, 113 CCDs and PMTs compared, table, 78 International Electrotechnical Commission Kerr effect, defined, 111, 179. defined, 75 (IEC), 117. self-focusing of pulsed laser light, 111 estimating intensity measurement error, International television standards, 589. Kindling proteins, 574, 760. 76 Internet sources. See Links. Kinetics, 691, 694–698, 741–742, 774, 796, and gray levels, 74–75 lasers, 123, 124 810–812, 816–817. measuring, 75 Intrinsic noise, 21. See also, Poisson noise. bleaching, 691, 694–698 Interference contrast. Inverse filter algorithm, 476, 477. and endpoint data analysis, 816–817 differential interference contrast, (DIC), Ion-binding in Aequorin emits light, 737. fluorescence, 262–263, 348, 383, 385, 10, 14, 76, 127, 146, 171, 453, 468, Ion concentrations, 346–347, 517, 528–530, 571, 578, 741–742. See also, FLIM 473–475, 846, See also, Differential 741. FRET, 796 interference contrast. chapter, 736–745 high content screening, 810–812, deconvolution of, 473–475 determination, 517, 528–530 816–817 958 Index

Kinetics (cont.) excitation wavelength choice, 540–542. saturable Bragg reflector, 111 ion concentration dyes, 741 See also, Acousto-optical devices, ultrafast, DPSS lasers, 112 and STED, 571, 578 filters ultrafast, fiber lasers, 113 Kino, Gordon, confocal design, 6. femtosecond pulsed laser, 44. See also, white-light continuum lasers, 113 KLM. See Kerr lens mode-locking. Ultrafast lasers why are they useful?, 110 Köhler illumination, 34, 127–128, 131, 229, fiber-based lasers, 109–111, 113–118 pumping power requirements, 82 251, 627, 648–649. table, 94 safety, 117–118, 839, 900. See also, coherence of light, 131 ultrafast, 113–114 Safety in disk scanner, 229 up-conversion fiber lasers, 109–110 goggles, 118 field diaphragm, 35, 127–129, 139, 461, fiber light delivery, 107,See also, Fiber- screens and curtains,118, 904 627, 645, 648–649 optics solid state, 103. See also, Solid-state to limit non-uniformity of illumination, GaAs, 107, 111 lasers 127–128 gas, 90, 91–10. See also, lasers by gas. semi-conductor, 105–107 to measure photon efficiency, 34 alkali-vapor, 103 thin-disk lasers, 109 Krypton laser, 102, 119, 346, 355. Ar-ion, 90, 101–102 spectrum of light, 44 comparison with argon-ion laser, 102 Kr-ion, 102 stabilization, 85–87 wavelength, 102 HeNe, 102–103 active, 87 Krypton/argon (Kr/Ar) laser, 90, 92, 93, HeCd, 103 titanium:sapphire laser, 82, 84–86, 88–91, 102, 108, 119, 203–204, 343, 375, heat removal, 84 94, 100–103, 105, 107, 109, 748, 798, 811. hybrid mode-locked dye laser, 540–541 111–112, 114, 123–124, 165, 346, stabilization, 88 important properties for confocal, 80 358, 415, 423–424, 459, 541, 550, KTP. See Potassium titanium oxide light delivery, 87–89 551, 645–647, 688, 706–708, 713, phosphate. fiber-optic, 106 727, 750, 756, 759 mirrors, 88 4Pi, 563–564, 567 L longitudinal modes, 82–83 brain slices, 731 Labeled structures, plants, 757, 761, 775. maintenance, 115–116 CARS, 599 bulk labeling, living embryos, 761 active media replacement, 115 compared to other fast lasers, 82–83, cell walls, 775 cooling components, 116–117 85, 110, 112–113 selective labeling, 757 optical resonator, 116 embryos, 731, 750, 756, 759, 764 Label-free microscopy, noise, 114. metal vapor, 112 maintenance, 116 Lamp housing, 134. microscopical uses and OPO, 114–115 Lamprey. nonlinear: 2- 3-photon, 90 plants, 415, 423–424, 706–708, labeled axons, 235, 236 Raman and CARS, 90 713–714, 717, 781–783 larva, optical projection tomography TIRF, 90 popular models, specs, table, 120 image, 612 tweezers, 89. See Laser trapping STED, 575 Landmark-based registration synthesis multi-photon. See Multi-photon transverse modes, 82–83, 85, 110 method, 328–329. microscopy tweezers, 89. See Laser trapping Lanthanide chelates, 345–346. Nd:glass, 706–708 types, 90 Large mode area photonic crystal fiber Nd:YAG, lasers, 88–89, 91, 95, 97, 103, ultrafast fiber, 113–114, See also, (LMAPCF), 110. 107–109, 111, 113–115, 117, 218, Ultrafast lasers Larmor frequency, MRM imaging, 245, 514, 680, 798 wavelength expansion by sum-and- 618–622. Nd:YLF, lasers, 89, 98, 100, 103, 109, difference mixing, 114 Laser(s), 7–9, 44, 80–83, 88, 90, 94, 112–114, 750, 760–761 optical parametric oscillators, 114–115

112–114, 119–120, 131, 540–543, Nd:YVO4, lasers, 89, 95, 100, 103, second/third harmonic generation, 114 599–600. See also, Fiber lasers; 107–109, 111, 113–114, 541 white light continuum lasers, 88, 109, 113 Mode-locked lasers; Multi-photon NO SMOKING, 116 continuum, 88, 109 ultrafast lasers; Up-conversion fiber performance tables, 91–101 He:Cd, 113. lasers; Ultrafast lasers. phase randomization, 8, 13, 131–132, 143 Laser cavity stabilization, active, 87. Alexandrite, 109 pointing error, 87 Laser cutters, 686–687. amplifier rods, 116 active cavity stabilization, 87 integration, 218–219 attenuation of, 85, 87–88, 354, 415, 904 polarization, 83, 88–89 Laser illumination, conditions for, 8. axial or longitudinal modes, 83 power control, 543 Laser lines, using acousto-optical tunable basic operation, 81–83, 116 pulse broadening/compensation, 88, filters, 56. CARS microscopy requirements, 599–600 901–904 Laser media, maintenance, 115–116. chapter, 80–125, table, 119–120 pulsed, 110–115. See also, Titanium- Laser printers, 593. coherence, spatial and temporal, 83–84 sapphire, Cr:Forsterite, Laser rods, maintenance, 116.

colliding-pulse mode-locked (CPM), 540 Nd:glass,YAG/YLF/YVO4, etc. Laser Safety Ofﬁcer, 117. for confocal, 7, 9–10, 77–78, 280, cavity dumped, 111 Laser sources, 9, 80–125. See also, Lasers. 535–545 Kerr lens mode-locked, 111 Laser speckle, 84, 90, 130–132, 448. continuous-wave, 90–110 modulated diode lasers, 112 removing, 84. See also, Scramblers control of power, 543 pulse-length measurement, 115, source, 130 Cr:Forsterite, 109, 114, 415, 541, 901–903 Laser trapping, 80, 89, 110, 218–219, 383, 706–709, 712–714 purpose, 110 385, 539, 646, 680. Index 959

Laser tubes, operational lifetime, 102, 115. Leonardo da Vinci, early optical studies, Light flux, light-emitting diode temperature, components likely to fail, 115 788–790. 133. Laser tweezers. See Laser trapping. Leukocytes, 347, 387, 520, 815, 854. Light intensity, 71, 163. LaserPix, 282. automatic analysis, 815 Light microscopy history, 1–4. Laser flying-spot microscope, 7. multi-photon, phase-based FLIM, 521 Light paths. See also, Commercial confocal Lasersharp, confocal microscopes, 282, 284, Lifetime. See Fluorescence lifetime imaging light microscopes. 285, 288, 292, 296, 302–306. microscopy. separating excitation/emission, 44–45 LaserVox, 281–282. Ligand-binding modules, 256, 348, 741, Light piping by specimen vs. depth, 182. Lateral chromatic aberration (LCA), 14, 846. Light-sheet illumination, 672–673. 155–156, 239, 242–243, 287, 640, Light detection, general, 28–33, 251–264. Light sheet microscopy, 613. 657–658. See also, Detectors; specific chapter, 672–679 correction in conventional optics, 155 detectors: CCDs, PMTs, etc. optical setup for, 613 measured, 657–658 assessment of devices, 260–262 white-light continuum lasers, 113 Lateral coherence, 8, 84, 267. charge-coupled device (CCD), 254 Light sources, widefield, 132–139, 143. See Lateral resolution, 1–4, 9, 11–13, 28, 207, comparison, table, 233, 255–256, 647 also, Chapters 5 and 6, Arc lamps, 209, 222, 225, 230, 238, 270, 320, conversion techniques, 259–260 LEDs, Lasers; Nonlaser light 409, 453, 511, 513, 542, 552, 554, direct effects, 252 sources; Filaments; Halogen. 563, 568, 651, 654–656, 747. See future developments, 262–264 commercial sources, 143 also, Resolution. history, 262–264 solar, 126–127, 131, 135 4Pi, 568 image dissector, 254–255 stand-alone, 143 CARS, 596–597, 599 microchannel plate, 232–233, 255, 262 table, comparative performance, 140 confocal endoscopy, 511, 513 gated, 519, 523–524, 527, 532 types, 132–139 confocal optics, improvement, 9, 651, MCP-CCD, 262 Light transmission, 11, 139, 160–161, 654–656 noise internal to, 256–259 223–229. of display, 292 internal detection, 256 cummulative loss along optical path, 139 light microscopy, 1–3 noise currents table, 256 of Nipkow disk system, 11, 223–229 optical coherence tomography, 609–610 photoemissive devices, 256–257 specifications for objectives, table, with pinhole and slit disks, 225 photon flux, 257–258 160–161 and spherical aberration, 409 pixel value representation, 258–259 Lighting models, 3D image display, SPIM, 613, 674 photoconductivity, 252, 253 306–312. STED, 573–575, 578 photoemissive, 254 absorption, 309–312 table, 209, 409 photon interactions, 252–256 advanced reflection models, 309 Laterally-modulated excitation microscopy, work functions, table, 252–253 artificial lighting, 309–312 see Stuctured-illumination. photovoltaic effect, 252–253 Gourard shading, 308 LCA. See Lateral chromatic aberration. point detectors, 260–261. See also, PMT gradient reflection models for voxel LCD. See Liquid crystal display. quantal nature of light, 251–252 objects, 309 LCOS. See Liquid-crystal-on-silicon. thermal effects, 252 Phong shading, 308–309 LCS (Leica Microsystems AG), 282, 312, vacuum avalanche photodiode, 254, 255 Phong/Blinn models, 308 910. Light dose, related to pixel/raster size, 64. simulated fluorescence process, 310 Lecithin myelin figures, CARS image, 204. Light, effects, on plant cells, 770. See also, surface shading, 310 LED. See Light-emitting diode. Bleaching, Phototoxicity. transparency, 280, 284, 287, 300, 304, Leica, confocal manufacturer, 51–53, 56–57, Light-emitting diode (LED), 34, 54, 309, 311–312 160, 218, 797, 910. 132–133, 135–139, 143, 237. Lilium longiflorum, image, 783. acousto-optical beam-splitter, 160, 218 aligning, 135 Limitations, confocal microscopy, chapter, objective lens transmission, 160 control by current-stabilized supply, 20–42. RS Scanner, 52–53 138–139 fundamental, 20–42 spectral confocal, TCS SP2, 51, 56–57, definition, 105 table, 41, 647 910 to detect galvanometer rotor position, 54 typical problem, 21, 24 tube length conventions, 157, 239 excitation wavelength for fluorophores, Linear galvanometers, 54. Leica Microsystems AG, 282, 910. 136 Linear longitudinal chromatic dispersion Leica TCS 4Pi, 119–120, 565–568. expected cost reduction, 237 (LLCD), stereoscopic confocal 4Pi microscopy type C, 565–568 fluorescence image, 142 image, 154. imaging of living cells, 568 galvanometer position feedback, 53 Linear unmixing. See Spectral unmixing. lateral scanning, 567, 910 lifespan, 137 Line-scanning confocal microscope, 50, 51, mitochondrial network image, 568 to measure photon efficiency, 34 231–232, 237, 784, 908, 916. optical transfer function (OTF), 567 microscope illumination, 131–139, 141, Linearity, 72, 490. sketch, 566, 910 143 deconvolution for image enhancement, thermal fluctuations minimized, 567 organic, projected development, 143 490 Lempel-Ziv-Welch (LZW), 580–582, 584. radiance, 138 display advantages and disadvantages, 72 Lens aberrations, 13–15. See also, spectra, 133 Links (Internet addresses). Aberrations. stability, 136 2 photon excitation spectra, 546, 727, Lens focal length, change, with wavelength, temperature effects, 137 729, 782 152. wavelength vs. current change, 137 brain slices, 727 960 Index

Links (Internet addresses) (cont.) for 4Pi confocal, 564 of external membranes, 90 CCDs, 76, 234, 927, 931 for biofilms, 870–873, 875, 877, 880, no damage, 114 components, 58 885 test specimen for, 390 confocal Listserve, 390, 901 for brain slices, 394, 723, 727, 729 widefield, 646–647, 751–753 deconvolution, 495 for epithelial cells, 370–371, 377, 386 working distance, 5, 9, 129, 145, 154, dyes, 221, 343–344, 782 finder chamber, 683 157, 198, 249, 511, 568, 598, 634, fluorescent beads, 653 flow chamber, 870–873, 875, 877, 880, 673, 678, 727–728, 747, 774, 779, FRET technique, 185, 803 885 781, 872 high-content screening systems, 811 for high-content screening, 810 table, 158 image management, 865 for optical projection tomography, Living embryo imaging, 749–751, 762–764. lasers, 104, 115, 120, 123–125 610–611 aberrations caused by, 747 live-cell chambers, 388–389, 870 perfusion, 394 apparatus, 748 movies related to book, 235, 392 for plant cells, 191, 429–430 C. elegans, 746, 748 muscles, 237 simple, 22, 394 deconvolution helps confocal, 751–753 non-laser light sources, 138, 143 for SPIM, 613, 625, 673 developmental changes, 746 plants, 769 table of required functions, 380 Drosophila, 273, 675–676, 747–748, safety, 900 table of suppliers, 388–389 751–752, 754, 756, 759, 804, 810 software, 282, 376, 594, 734, 762, 764, test chamber/dye, 654, 661 dyes, 748 776, 777, 820, 824, 827, 831–833, cell-cycle effects, 790 introduction of, 755 844, 845, 864–862, 865–867, 869 chromatin, 385, 390–392, 684, 693–695, embryo size vs. speed acquisition, SPIM, 672 812 753–754 Lipid dyes, 236, 355, 359–360, 389, 556, chromatin dynamics, 390–392 explants, 748–749 755, 760–761. CNS tissue slice preparation, 393 future developments, 766 Lipid receptors, 790. confocal microscopy, 381–399, 746, 813 fluorescent probe Liquid crystal-on-silicon (LCOS), 266. difficulties, 381 four dimensional, 746–747, 749 Liquid crystal display (LCD), 39, 67, 73, future directions, 398–399 cellular viability, 747–748 291, 293, 589–590. considerations, 386–390 challenges, 762 digital projectors, 590 antioxidants, 390 dataset display strategies, 761–764 filters, 928 experimental variables, table, 386 photodamage during, 746–748 non-linearities, 73 fluorescent probes, 387–389 high speed acquisition shutters, 299, 929 maintenance of cells/tissues, 387 disk-scanning confocal microscopy, supertwisted nematic (STN), 589 minimizing photodynamic damage, 754 thin-film transistor (TFT), 589 136, 389 hardware, 754–755 Liquid crystal technology/dynamic photon efficiency, 141–161, 389–390 light scattering, 747 polarization microscopy, 188. See in vitro preparations, see In Vitro optimal acquisition, parameters, 753–754 also, Pol-scope. in vivo preparations, see In Vivo refractile specimens, 747 Lissajous pattern, circular scanning. 554. contrast, 747 superficial optical sections, 748 “tornado” mode, SIM scanner, 52 dyes, 748. See also, Dyes; Fluorophors thick specimens List servers, 125. etc. effective strategies, 748–753, 755–761 Lithium triborate (LBO), as non-linear for rapid assessment, table, 360 inherent trade-offs, 747–748 crystal for multiplying infrared embryos, imaging, 746–766. See also, selective plane illumination (SPIM), output, 109, 115. Living embryo imaging 751 Living cells, 80, 90, 114, 136, 145–161, external membranes, SHC image, 90 “Test drives,” for living embryo imaging, 167, 219, 221–222, 381–399, fluorescent staining, 393 752. 429–439, 480, 564–566, 568, microglia, 393 widefield/deconvolution, 751–752 746–766, 770, 772–773, 788–806, nuclei, living/dead cell, 393 LLCD. See Longitudinal chromatic 811, 813. See also, Brain slices, fluorophore effects, 748 dispersion. Plants cell imaging, and by FRET imaging, chapter, 788–806 LMA-PCF. See Large mode area photonic cell/organism name. future, 221–222 crystal fiber. 2-photon, penetration, 749–751 handling data, 395–396 Loading methods, fluorescent probe, 347, 2D plus time, 753–754, 762–764 imaging techniques, 382–386, 394–395 358–360, 430, 732–734, 738, 739. 3D projection, 763 low-dose imaging, 391–392 acetoxymethyl esters, 359, 360. See also, 4D data, 746–747, 764 microglial cell behavior example, Acetoxymethyl esters 4Pi microscopy, 564–565, 568 392–398 ATP-gated cation channels, 359 acquisition speed, 222, 753–754 no damage from SHG imaging, 114 ballistic microprojectile delivery, 360, algorithms, 763–764 online confocal community, 390 726, 803 assays, 811 photon efficiency, 141–161, 389–390 direct permeability, 358–359 beauty and functionality, 790 phototoxicity, 390–391 electroporation, 359–360, 795, 803 bleaching of, 797. See Bleaching; assays for, 813 ion indicators, 738–739, 742 Photodamage plant, 429–439. See also, Plant cell low level, 430 cell-chamber, 11, 22, 191, 219, 370–371, imaging reflectance imaging, 167 membrane permeant esters, 359–360 386–387, 394, 429–430, 564, second harmonic generation. See also, microinjection, 360, 361, 388, 739, 748, 610–611 SHG 755, 795, 803–804 Index 961

neurons, 722, 726, 730, 732–734 confocal, 52–53, 62–64 multi-dimensional image display, osmotic permeabilization, 359 effect on pixel size, 24, 928 294–296 peptide-mediated uptake, 359 factor, 24, 28 G function, 294 plant cells, 769 and lateral chromatic aberration, 278 image/space view, 296 stabilizing chemicals, 341–342, 362 for line-scanner, 232 orthoscopic view, 294 transient permeabilization, 359 over-sampling, 68–70, 493, 509, 635, 729 reducing geometric dimensions, 294 whole-cell patch pipet, 360 high-content screening, 816 rotations, 294–296 Local projections, display, 305–306, 307. and pinhole size, 28 visualization process, 294 Location proteomics, 818. under-sampling, 68 MAR. See Mark/area ratio. Longitudinal chromatic aberration, 152–155. zoom magnification, 11, 24, 37, 63–34, Marching cubes algorithm, 301–302, 304, Longitudinal coherence length, 7, 8, 84, 66, 70, 79, 317, 389, 493, 627, 776. 130, 131. 634–636, 731 Marconi, CAM-65 electron multiplier CCD Longitudinal linear chromatic dispersion Maintenance. camera, 76. See also, EM-CCD. (LLCD) objectives for 3D color- cell viability, 387 Mark/area ratio (MAR), 279. coded BSL confocal, 154. dye lasers, 114 Marsilea quadrifolia, 416, 419. Long-pass filters, 43–44. lasers, 115–117, 124 attenuation spectra, 416 Low-voltage scanning electron microscope remote logging of, 864 optical section, 419 (LVSEM), 846–847, 849–850, 852. troubleshooting reference, 124 Mass balancing, to reduce scanner vibration, LSM. See Laser-scanning confocal Maize (Zea mays), 167–168, 172, 179, 202, 54. microscopes; Laser-scanning flying- 417–424, 428, 438, 710–711, Mass storage, 580–588, 593–594. spot microscope. 6–7 713–714. data compression for, 288–289, 292–293, Lucoszs formulation, 273. 2-photon, time-lapse microspectroscopy, 295, 319, 499, 580–585. See also, Luminescent nanocrystals, 343, 345. 423 Data compression Luminous intensity vs, color, dye molecule, abnormal vasculature, 437 algorithms, 319, 580 138. anther, 420, 433 archiving systems, 580 LVSEM, 846–847, 849–850, 852. attenuation spectrum, leaf, 418 color images, 581 LysoTracker Red DND-99, 359–360, cross-sections, stem, 172, 707 file formats, 580–588 709–710. emission spectrum, 710, 711, 713 removable storage media, 585–588. See rapid assessment table, 360 fluorescence spectra, 422–424 also, Removable storage media spectra, 710 leaf, random-access devices, 586–588 LZW compression. See Lempel-Ziv-Welch. attenuation spectrum, 418 sequential devices, 585–586 optical section, 172, 179 solid state devices, 588 M reflectance, 167 time required, table, 581 Mach-Zehnder interferometry, 245. surface, 436 Materials, silicon, fused quartz, beryllium, Machine learning. See Automated meristem, 420, 430–432, 707 52. interpretation of subcellular patterns. multi-photon excited signals, 422–424 Mathematical formulas, for confocal Macrography, 3D light scanning, 672. polarization microscopy, 707, 711 microscope performance, table, Magnesium fluoride (MgF2). pollen grain, 202, 433–434 209. for anti-reflection coating, 158 protoplast, 424 Maximum intensity projection, 180, Magnetic disks, 586. root, 432 284–285, 292, 294, 298, 302–304, Magnetic resonance imaging (MRI), 618. second harmonic imaging, 707, 711 307, 313–314, 319, 325–326, Magnetic resonance microscopy (MRM), silica cells, 428, 437, 707 330–331, 585, 755, 763–764, 770, 618–624. spectrum, 422, 423, 710 774, 881, 884. amplitude modulation for RF carrier, 620 starch, 420, 435–436, 707, 711 local, 305 applications, 623–624 stem Maximum likelihood estimation (MLE), botanical imaging, 624 attenuation spectra, 417, 418, 713 472–475, 495, 497–498, 669. developmental biology, 624 optical sections, 419, 714 blind deconvolution, 472–475, 498, 784 histology, 623 storage structures, 420, 435–436, 707, effect on colocalization, table, 669 phenotyping, 623 711 M-CARS. See Multiplex CARS basic principles, 618–619 Manufacturers. See also, Commercial microspectroscopy. Fourier transform/image formation, 620 confocal light microscopes; MCP. See Microchannel plate, 232–233, future development, 624 Appendix 2. 255, 262. hardware configuration, 621–622 listing with web addresses, table, MCP-CCD, 262 image contrast, 622–623 104–105. Gated intensified, 519, 523–524, 527, image formation, 619–621 Mapping conventions, in image processing, 532 Larmor frequency, 620 294–296, 300–304. MCP-PMT. See Microchannel plate Schematic diagram, 618–619 data values, 300–304 photomultiplier. strengths/limitations, 622 choosing data objects, 300–301 MDCK cell, 372–374. Magnification, 24, 35–41, 62, 131, 215, 443. object segmentation, 301–302 actin cytoskeleton, 374 See also, Nyquist sampling; Over- projection rules, 302–304 Golgi apparatus, image, 374 sampling; Undersampling. scan conversion, 301–302 morphologic changes, 374 calibrating, 653, 658 table, 300 stereo image, 373, 374 and CCD pixel-size, 62, 70 visualization, 300 vertical sections, image, 372 962 Index

Measurements, 20, 33–36, 76, 139–141, Metal vapor lasers, 112. GFP, 12. See also, Green fluorescent 159. Metamorph, 281–282, 290, 311, 817. protein achromat performance, 194 Microchannel plate (MCP) image intensifier, in mitosis, 759. See also, Mitotic buffering of, ion measurement, 738, 740 233, 255, 519, 532. apparatus field flatness, 26–28 multiplicative noise, 233 polarization microscopy, 15, 173, 188, geometric distortion, 653–654 photocathodes, 262 420–421 laser pulse length, 109, 112, 115, 507, PMT, 255, 523, 532 photodamage of, 341, 850–851 537, 538, 902–903 Microchannel plate PMT (MCP-PMT), 255. stabilizing buffers, 852 light throughput, 139–141 Micro-computerized tomography (Micro- STED, 576–577 limits on confocal intensity, accuracy, 20 CT), 614–618. stereo image, 752 photon efficiency, 33–36 contrast/dose, 614–615 TIRF, 180, 183 pinhole, effective size, 34 dose vs. resolution, graph, 616 second harmonic generation, for tracking, intensity spread function histogram, layout, 614 90 74–78 mouse images, 615–617 Microwave fixation, 377–378. resolution, 241–245, 657, 658 tumor-bearing, 617 Microwire polarizer (Moxtec Inc.), 85. shrinkage, specimen preparation, operating principle, 614 Mie scattering, 162–163, 167, 417–418. 371–373 Micro-CT. See Micro-computerized clearing with index-matched liquid, 167 spectral transmission of objective, 159 tomography. comparison with Rayleigh scattering, spherical aberration, 145, 407 Microdissection. 163 surface height, using LLCD BSL with multi-photon IR light, 686–687 light attenuation in plant tissue, 417 confocal, 224 with nitrogen lasers, 112 by refractive structures, 162–163 z-resolution, 194 Microelectrodes, for introducing indicator, MII. See Multi-photon intrapulse Mechanical scanners, 51–54. 738. interference, 88. Melles Griot catalog, real lens, performance, Microglial cell behavior, 392–398. Mineral deposits, plant, 163–420, 436–437, 210. Microinjection, 360–361, 388, 739, 748, 703. Membrane permeant esters, 361, 358–359, 755, 795, 803–804. Miniaturized fiber-optic confocal 361, 726, 738–739, 744. of chromophores, 803–804 microscope, 508–512. Membrane potentials, 179, 188, 204–205, Microlens array, 12, 134, 135, 216, 225, bundle imagers for in vivo studies, 509 346, 353, 383, 517, 743, 811–813. 231, 235. clinical endoscope, 514 Memory stick, 588. for 4Pi confocal, 563–565 objective lens system, 509 Mercury arc lamp, 37, 44, 132, 135–138. for CCD, 237 optical efficiency, 509 fluorophores matching excitation, for disk scanners, 6, 12, 216, 224, 226, optical schema, 508 135–136, 139 231, 458 resolution, 509 iso-intensity plots of discharges, 132 for light-emitting diode source, 134–135 rigid endoscope, 511 and pinhole size, 37 for multi-focal, multi-photon, (MMM), single fiber designs, 510 radiance, improvements, 137–138 537, 551–555, 558 vibrating lens and fiber, 510–511 wavelengths, 44 principle, 135 in vivo imaging in animals, 512 Mercury-halide arc source, 136, 138, in Yokogawa disk-scanning confocal, 12, Minolta, CS-100 radiospectrometer, 139. 143–144. 224–226, 231, 235 Minsky, Marvin, 2, 4–6, 11, 141, 216, 890. spectrum, 144 Microscopes, 217, 226. See also, particular Mirror coupling, pulse width and pulse Mercury-iodine (Hg-I) arc lamp, radiance, types. shape, 88. 138. attachment of confocal scanner, 217 Mirrors, 26, 48, 54, 63, 209–210, 214. Mercury-xenon arc lamps, 136–138. specification comparisons, table, 226 galvanometer, 54. See also, spectral lines, 136 Microscopy laboratory URLs, 125. See also, Galvanometers Meristem, 168, 420, 430, 432, 770, Links. internal, testing reflectance losses, 26 776–778, 782. Microspectroscopy, 421–425, 426, 516. laser-line, 48 maize, 168, 432 CARS, 601–602 performance, 54, 63. Merit functions, confocal scanners, 217. fluorescence properties of plants, 421–425 scan angle and magnification, 63 object-dependent, defined, 217 lifetime, 516 size calculation for LSCM, 209 object-independent, defined, 217 of maize, 424 x-y scanning mirror orientations, 214 Mesophyll cells, 169, 193, 195, 417–418, multi-photon setup, 424 Mismatch, 893. 423, 428, 430, 711–712, 714, 779. Microspores, birefringence images, 189, probe shape/pixel, 39, 466 A. thaliana, 193, 196 431–432. caused by chromatic aberration, 243 photodamage, 203 Microsporogenesis, 431–432. refractive index, 377, 404–412, 411, 654, protoplasts, 196, 203, 424, 425–426, Microstructure fibers, 504. 658, 747, 863, 893 430, 439 Microsurgery, 112, 219, 686–687, 764–765. 4Pi, 568 harmonic images, 711–712, 714 Microtubule, 11, 68, 80, 188, 222, 292, 432, causing signal loss, 148–150, 408–409, image, 424 582, 703, 714, 752–753, 759, 773, 654 spectra 790, 852. See also, Cytoskeleton. chapter, 404–412 attenuation, 416, 418 birefringence, 714–715 corrections, 411–412 change with 1- vs. 2-photon, 421, 423 Brownian motion of, 11 embryos, 747 emission, 423 electron microscopy, 848, 850 film vs. CCD, 590 Metal-halide light source, 136, 143–144, fixation, 369, 372–375 harmonic signal generation, 704–705 907, 908. fluorescence correlation spectroscopy, 383 less, at long wavelength, 416 Index 963

measurement, 148–150, 655–656 MRM, 624 MPA. See Multi-photon absorption. of movie frame rate, 839 Monitors, computer display, 588–589. MPE. See Multi-photon excitation. MRM, contrast agent vs. imaging time Monkey cells, 693, 803. MPEG display formats, 836–841. mylar flakes, 198 Monomeric red fluorescent protein (mRFP) MPEM. See Multi-photon microscopes. resolution loss measured, 192–194 constructs, 756, 760, 798. MPLSM. See Multi-photon laser-scanning vector mismatch in CARS, 596–597, with CFP or GFP molecules, as FRET microscopy. 600 pair, 798 MPM. See Multi-photon microscopy. z-distortion, 287 Montage synthesis method, 281, 312, 318, MQW. See Multiple quantum wells. Mitotic apparatus, 15, 173, 373–374, 377, 328–331, 748, 753, 851–852, 855, mRFP. See Monomeric red fluorescent 386, 421, 431, 693, 749, 752, 799. 858–859. protein. See also, Microtubules. defined, 329–330 MRI. See Magnetic resonance imaging. damage, 693 examples, 330–332, 780–781 MRM. See Magnetic resonance microscopy. fixation, 373–374, 377 scanning electron micrographs, 851–852, Multi-channel experiments, 813. FRET, 765 855 filters and dispersive elements, 51 marker, cyclin-B, 790 TEM methods, 858–859 time-lapse fluorescence imaging, 382, Pol-scope, 13, 188, 432, 468, 479–480. Moon, early phase measurements, 788, 789. 384 deconvolved, 479 Morphological filters, 285, 300–301, toxicity, 755 images, 15, 188, 479, 717 316–317, 320–322, 730–734, 817, Multi-dimensional microscopy, display, SHG imaging, 702, 718–719 826. 280–314. See also, Automated 3D in vitro fertilization, 188 high-content screening, 812, 819, 826 image analysis methods. Mitotracker stain, 142, 170, 353, 358, 360, Morphometry, 145, 316, 319, 331, 726, 728. 2D pixel display space, 291 430–431, 692, 750. group properties, 331 efficient use, 292 living cells rapid assessment, table, 360 intensity/spectral measures, 331 animations, 292–293 Mixing, sum or difference, to generate laser interest points, 331 artificial lighting, 306–308 wavelengths, 114. invariants, 331 CLSM images, 286–290 MLE. See Maximum likelihood estimation. location/pose, 331 anisotropic sampling, 287 MMM. See Multi-focal, multi-photon shape measures, 331 calibrating image data, 286 microscopy. size measures, 331 data type/computational precision, MMM-4Pi microscopy, 556. texture measures, 331 288–289 MO (magneto-optical) disks, 586. topological measures, 331 dimensions available, 286–287 Model-based object merging, 323–325. Mosaicing. See Montage synthesis method. file formats for Mode-locked lasers, 87, 101, 111–114, 118, Mounting medium, 166, 198, 342, 370–371, calibration/interpretation, 288–289 124, 358, 520, 646, 728–729, 749, 373–377, 404–413, 418, 454, 457, image data, 286 901–904. 473, 493–794, 499, 564, 631, 642, image size available, 287 active, pulsed laser class, 111 652, 655, 696, 730, 774 See also, image space calibration, 287–288 adjustment of, 901–904 Clearing agents. image/view dimension parameters, for CARS, 599–600 brain slices, 730 table, 288 colliding pulse, 112 chapter, 404–413 processor performance, 289–290 fiber, ytterbium and neodymium, clearing solutions, 166, 417–418, 420, storing image data, 286 113–114 439, 610, 624, 774–775 voxel rendering speed, 290 fiber/diode, ultrafast, 113–114 effect of glass bead, 199 color display space, 291–292 FLIM, 520 plant specimens, 418, 431, 774 commercial systems, tables interference with, by specimen, 171 refractive index, tables, 377 available systems, 282–283 Kerr lens, 111 selection, 198, 631 desirable features, 288–289 modulator, fiber lasers, 111 Mouse, 192, 376, 393, 608, 612, 615+, 723, display options, 293 multi-photon, 535–536, 540–541, 726. geometric transformations, 295 550–551, 563–564, 567, 646, confocal colonoscopy, 509, 512 projection options, 300 728–729, 749 embryo realistic visualization techniques, passive, 111, 113–114 optical projection tomography image, 307 saturable Bragg reflector, 111 612 criteria for choosing visualization, 281 SHG, THG, 706–707 SIM/EFIC image, 608 data values, definition, 222, 280 Mode-locked oscillators. See also, Mode- GFP transgenic, 726 dimensions, 280, 323 locked lasers. hippocampus, 393 degrees of freedom, optical image, 8–9 nanojoule pulse energies, 111 micro-CT image, 614–615, 617 depth-weighting, 304, 306 Moiré effects, 270–271, 755. femur, 616 exponential, 304 ambient fluorescent room lighting, 201 tumor, 617 linear, 304 banding patterns, 755 spectral unmixing image, 192, 382, recursive, 304 disk-scanners and CCDs, 231, 754–755 664–667 display view, definition, 280 structured-illumination methods, 268–271, examples, 665–666 hidden-object removal, 304–305 273 visual cortex brain slices image, 723 local projections, 305–307 Molecular imaging, in vivo, 387, 618, 624, Movement contrast, 190. z-buffering, 304–305 790, 803–806. Movie compression, 836–840. highlighting previously defined structures, FRET, 790, 803–806 Moving-coil actuators, galvanometer, 52. 284 micro-CT, 618 Moxtec Inc., Microwire polarizer, 85. image, definition, 280 964 Index

Multi-dimensional microscopy, display pixel-shift/rotation stereo, 297 bleaching, 218, 338, 539–540, 680–689, (cont.) stereo images example, 298 692–693, 905. See also, Bleaching; image/view display options, table, 293 synchronizing display, 297 Chapter 38 geometric transformations, table, 295 true color, 291 caged compounds, 187, 383, 543–544, intensity calibration, 304 unknown structure identification, 281–284 692, 729, 912 iso-intensity surface, 304 viewing data from, 283 cell viability during imaging, 544–545 laser-scanning microscopy, 280 visualization parameters, table, 285 chromophores for, 543–544 lighting models, 306–312 z-coordinate rules, 304 detection, 538 absorption, 309–312 z-information retained by, 296–300 duty cycle, 644 advanced reflection models, 309 non-orthoscopic views, 299 excitation localization, 538 artificial lighting, 309–312 stereoscopic views, 296–299 excitation spectra, 125 Gourard shading, 308–309 temporal coding, 299–300 FLIM, 576 gradient reflection models/voxel z-depth, 299–300 fluorophores for, 543–544 objects, 309 Multi-fluorescence, systems for utilizing, FRET, 797 Phong shading, 308–309 217+. heating, 539–540 Phong/Blinn models, 308 Multi-focal, multi-photon microscopy history, 535 simulated fluorescence process, 310 (MMM), 221, 276, 550–559, 797. image formation, 535–540 surface shading, 310 4Pi-MMM, 563–564 instrumentation, 540–543, 900–905. See transparency, 309–312 basics, 565 also, lasers for. See also, Ultrafast living cells of rodent brain, 392–398 scheme, 563 lasers mapping data values, 300–304 alternative realizations, 554–555 Alexandrite, 109 choosing data objects, 300–301 background, 550 Cr:Forsterite, 109, 114, 415, 541, object segmentation, 302 beam subdivision approaches, table, 558 706–709, 712–714 projection rules, 302–304 current developments, 558–559 Nd:glass, 706–708 scan conversion, 301–302 experimental realization, 551–555 Nd:YAG, 88–89, 107–109, 514, 680, segmenting data objects, 301 FRET, 797 798 visualization model, 300 imaging applications, 556 Nd:YLF, 89, 112–114, 750, 760–761

mapping into display space, 294–296 boar sperm cells, 557 Nd:YVO4, 89, 95, 107–109, 113–114, G function, 294 Convallaria, 556 541 image/space view, 296 FRET, 556 Ti:Sapph. See Laser, titanium-sapphire orthoscopic view, 294 hippocampal brain slices, 557 laser reducing geometric dimensions, 294 pollen grains, 556 multi-focal, multi-photon microscopy rotations, 294–296 Prionium, 556 alignment, 900–901 visualization process, 294 interfocal crosstalk, 553–554, 556 beam delivery requirements, 541 measurement capabilities See also, time-multiplexing, 553–555 control of laser power, 543 Chapter 15 limitations, 556–558 CPM laser, 540 reconstructed views, 312–313 localization, 538 descanned detection, 166, 208, 212, results, 284–285 Lissajous pattern of scanning foci, 554 428, 537, 540–542 objective vs. subjective visualization, 281 “tornado” mode, SIM scanner, 52 excitation wavelengths, 541 prefiltering, 281 Nipkow-type microlens array, 551–552 focal plane array detection, 542 principle uses, 281–285 optimum degree of parallelization, hybrid mode-locked dye laser, 540–541 projection/compositing rules, 302–304 550–551 lasers/excitation wavelength choice, alpha blend, 302, 304 resolution, 552–553 540–542 average intensity, 302 schematic diagram, 552 non-descanned detection, 185, 201, first or front intensity, 302 time multiplexing, 553–554 218, 381, 447, 456, 507, 542, 552, Kalman average, 304 variants, 555–556 559, 643, 646, 727, 750, 779, 904, maximum intensity, 302 FLIM, 555–556 909, 910 pseudo color, 173–175, 190, 291 MMM-4Pi, 556 non-mechanical scanning, 543 purpose, 281–285, 293–295 SHG, 556 optical aberrations, 542 realism added to view, 306–308 space multiplexing, 555 power requirements, 541, 903, 904 techniques for, table, 307 Multi-length fiber scrambler, 8. See also, pulse spreading due to GDD, 547, 538, reconstructed view generation, 290–312 Scramblers, light. 543 5D image display space, 291–294. See Multi-photon absorption (MPA), 535. resonant scanning, 543 also, 5D image display space Multi-photon excitation (MPE), 356–358, whole-area and external detection, choosing image view, 291–294 535–545, 894. See also, Multi-focal 541–542 subregion loading, 290–291 multi-photon microscopy. optical pulse length, 537–538 reconstruction, definition, 280 absorption, 705–707 group delay dispersion, 537–538, 543 reflection models, 306–308 advantages/disadvantages, 644–647, group velocity dispersion, 88, 111, 210, rendering, definition for, 280 749–751 537, 606, 903 software packages, table, 282–283 autofluorescence, plants, 424, 427 measurement, 115, 901–903 stereoscopic display, 293, 296–299 background from SHG/THG, 361, penetration, 749–750 color space partitioning, 297 708–709, 728 photodamage, 539–540, 680–688, interlaced fields of frame, 297 backscattered light imaging, 429 692–693 Index 965

physical principles, 535–540 Nd:glass, 706–708 Nanosurgery, 219. refractive index mismatch, 404–413 Nd:YAG, 88–89, 107–109, 514, 680, with multi-photon systems, 90 resolution, 539 798 NCI60 CMA, standard encapsulation, 816. SHG and THG background, 361, Nd:YLF, 89, 112–114, 750, 760–761 NCPM. See Non-critical phase matching. 708–709, 728 Nd:YVO4, 89, 95, 107–109, 113–114, ND. See Neutral-density filters. two-photon absorption cross-sections, 125 541 Near infrared (NIR) lasers, 10, 90, 106. See (URL) 543–544 Ti:Sapph. See Laser, titanium-sapphire also, Lasers: titanium-sapphire; Nd:; wavelengths, 538–539 laser Cr:Forsterite. Multi-photon intrapulse interference (MII), uncaging, 545 Near infrared (NIR), 10, 90, 106. 88. Multi-photon-based photo-ablation, 764. diode injection lasers, 106 Multi-photon microscopy (MPM), 10–11, Multi-slit design, for disk-scanning for laser tweezers, 90 56, 172–177, 210, 535–545, 681, confocal, 229. objective lenses designed, 174 682, 685–688, 746–766, 894, Multi-view deconvolution, 330, 675–677. Nearest-neighbor deconvolution algorithm, 900–905. Multiple quantum wells (MQW), diode 476. advantages/disadvantages, 644–647, injection lasers, 106. image enhancement, 495–496 749–751 Multiplex CARS microspectroscopy (M- Negative contrast, for fluorescence alignment, 901–902 CARS), 601, 602. microscopy, 173–174. autofluorescence, 425–427, 545 Multiplicative noise, 28–33, 51, 77–78, 224, Negative feedback, to correct mirror motion, SHG, THG, 361, 708–709, 728 234, 256–258, 262, 275, 443, 460, 53. calcium imaging, 545 633, 661, 667. Neodymium glass laser, 706–708. cell damage during, 544–545, 682, 685 of EM-CCD, 30–31, 77–78, 264, 256, Neodymium-yttrium aluminum garnet 1-photon vs. 2-photon excitation, 681 262 (Nd:YAG) lasers, 88–89, 91, 95, 97, absorption spectra of cellular absorbers, losses in effective QE from, 33, 234, 103, 107–109, 111, 113–115, 117, 681 443 245, 514, 680, 798. intracellular chromosome dissection, from PMT, 29, 51, 77–78, 233, 256–258, infrared range, 108 688 460, 633, 661, 667 pumping non-linear crystal/green light, mitochondria, 686 and quantum efficiency, 33, 234, 443 114–115 nanosurgery, human chromosomes, photon counting, 32–33, 78 Neodymium-yttrium lithium fluoride 686–687 pulse pile-up, 32–33, 35, 78, 521, 523, (Nd:YLF) laser, 89, 98, 100, 103, by optical breakdown, 198, 680, 682, 526–527 109, 112–114, 750, 760–761. 685, 687, 703, 705 table, 256 Neodymium-yttrium orthovanadate

photochemical, 682–685 why it is usually unnoticed in LSCM, (Nd:YVO4) laser, 89, 95, 100, 103, photothermal, 539, 545, 681, 685, 633, 661 107–109, 111, 113–114, 541. 904 Muscle, 737, 739–742. kits utilizing, 113 reproduction affected by ultrashort NIR fatigue, 739–740 Nerve cells, images. pulses, 686 Alexa stained, 330 ultrastructural modifications, 685–686 N backscattered light images, 167 cell viability, 544–545 NA. See Numerical aperture. eye, optic nerve, 481 compared with other 3D methods, Nanobioscopy, protein/protein interactions, Golghi-stained, 298 644–647, 748–751 795–798. Lucifer-yellow, 314 deconvolution, 495–498 acceptor bleach, 797–798 microglia, 396–398 developmental biology, 545, 746–754, donor fluorescence, 796–797 rat-brain neurons, 398 757, 759–760, 764 FRET measurement, 795 transmitted light, 475 dispersion as problem, 56. See also, sensitized acceptor emission, 795–796 Neutral-density filters (ND), 43, 76, 126. GVD; GDD Nanoscale resolution with focused light, in fixed-pattern noise measurements, fluorescence, contrast, 172–177 571–578. See also, Stimulated 76 for living embryo imaging, chapter, emission depletion (STED) to reduce source brightness, 43, 126 746–766 microscopy. NFP. See Nominal focal position. need for efficient illumination light path, breaking the diffraction barrier, 571–573 Nikon, confocal manufacturer, 13, 15, 210 different approaches, 573–574 119–120, 161, 199, 201, 507, optical layout, 540 ground state depletion (GSD), 573 638–640, 657, 750, 910. photobleaching, 545, 680–688, 692–693 STED, 573–574 C1 confocal microscope, 119–120, 507 practical operation, 900–905 outlook, 577 C1si spectral confocal microscope, 908, protein damage/interactions, 765 RESOLFT concept, 571–573 910 resolution, 552 resolution, new limiting equation, 571 CF objectives, 154–156, 217, 669, 779 setup/operation, 540, 900–905 measured, 578 confocal x-z, BSL image, 22 schematic diagram, 540, 901–902 stimulated emission depletion (STED), Plan Apo objective, 13, 15, 638 in vivo (intact animal) imaging, 545 573–578 resolution, measured, 16, 638–640, ultrafast lasers, 88, 90, 109. See also, axial resolution increase, 576 657 Ultrafast lasers compared to confocal microscopy, 576 water-immersion lenses, 15 Alexandrite, 109 dyes, suitable, table, 575 high-content screening, 810 Cr:Forsterite, 109, 114, 415, 541, OTF comparison, 578 tube length conventions, 157, 239 706–709, 712–714 PSF comparison, 578 Nile Red, dye, 435, 528, 575, 774, 782 966 Index

Nipkow disk scanning, 2, 5–6, 11, 12, 41, shot, 442–443. See also, Poisson noise harmonic generation, 704–705 215, 223, 231, 276, 551, 754, single-pixel, 65, 67, 190, 635, 832, emission, 710–711 783–784, 810, 894. See also, 835–836 energy state diagram, 705 Yokogawa; Disk-scanning confocal deconvolving, to reduce, 39–40, 392, multi-photon absorption/fluorescence, microscopy. 498, 667, 784, 835–836 705 commercial systems, 907, 913, 915 reducing, 39, 40, 190, 41, 65, 392, 498 second harmonic generation (SHG), compared to single-beam scanning, 458 sources of, 442–444 704–705 for high-content screening, 810 wavelet transform to reduce, 733–734, setup, 708–709 micro-lens system, 6, 12, 216, 224–226, 819–820 third harmonic generation (THG), 705 231, 234, 237, 551–552 Nomarski DIC contrast, 2, 368, 746, 892. light sources/detectors, 706–708 multi-photon, 537, 551–558, 563–565. See also, Differential interference light attenuation spectra in plants, 706 See also, Multi-focal, multi-photon contrast. photodetector characteristics, 707 microscopy rotation, 754 Nominal focal position (NFP), 405, 408, pulsed-laser, table, 706. See also, for single-sided confocal, 6, 141, 223, 409. Ultrafast lasers 229 calculations for glycerol, 409 in optical fiber, 504–508 source brightness, 141 calculations for water, 409 optically active animal structures, speed of image acquisition, 11, 220, z-responses, diagram, 408 714–717 222–226, 227, 231 Non-confocal microscopy vs. confocal, 746. man-made collagen matrix, 717 for tandem-scanning, 141, 215 high content screening, table, 811 signal-producing structures, table, 715 visualization, of cells, 458, 667, 754, Non-critical phase matching (NCPM), spindle apparatus, 717–718 784 114–115. zebrafish embryo, 716, 718 Nipkow, Paul, 5–6, 109 Non-descanned detection, for MPM, 185, optically active plant structures, 710–714 NIR. See Near infrared. 201, 218, 381, 447, 456, 507, 542, Canna, 710 Nitrogen lasers, 112. 552, 559, 643, 646, 727, 750, 779, Commelina communis, 712 nanosurgery using, 219 904, 909, 910. emission spectrum of maize, 710, 711 NLO. See Non-linear optical effects. for CARS, 559 maize stem, 711, 714 NMR. See Nuclear magnetic resonance. No-neighbor algorithm, 476–477, 496. potato, 712 Noise, 21, 28, 74–77, 83, 87, 114, 190, 232, Non-laser light sources, chapter, 126–144. rice leaf, 712, 715 256–259, 442–444, 495. See also, arc sources, 130, 132, 140 polarization dependence of SHG, 717, Signal-to-noise ratio; Poisson noise; commercial systems, table, 143 719 Quantum noise. comparative performance, table, 140 setup for, 708–710 background, 443–444 control, 138 spectra, 415, 417, 435 of CCD detectors, 30–31, 77–78, for disk-scanning confocal, 141 Euphorbia pulcherrima, 710 232–233, 256, 262 filament sources, 135–136 maize leaf, 710 equations, 256 LEDs, 132–133, 135, 138–139, 143 Pyrus serotina, 711 table, 256 light scramblers, 131–132 STED microscopy, 571–579. See also, vs. photomultiplier tube detectors, 74, measured performance, 139–141 STED microscopy. 77 results, 142 structured illumination, 270, 276 CIC, clock-induced charge, EM-CCDs, solar, 126–127, 131, 135 Non-radiative dipole-dipole interactions, 234, 926 stability, 136–137 790. in counting quantum-mechanical events, Nonlinear constrained iterative Non-specific staining, 27, 44, 74, 303, 345, 21 deconvolution, 68, 458, 475–476, 357–358, 442, 467, 472, 617, 660, deconvolution reduced noise, 39–40, 114, 496–497, 499, 520, 568. 667–668, 760, 820, 878, 882. See 392, 495, 498, 667, 783, 835–836 Nonlinear conversion, tunable laser, 114. also, Background. detector, 28 Nonlinear crystals, frequency multiplying, Non-tunable solid-state laser, 103. fixed-pattern, 74, 76, 278, 924, 927, 931 109. Normal, free-running, pulsed laser, 111. in fluorescence microscopy, defining, Nonlinear optical (NLO) effects, in Northern Eclipse, software, 282. 74–75 microscopy, 90, 114, 163, 165, 177, Notch filter, to transmit laser line, 49. in lasers, sources, 85–86 179, 188, 190, 195, 416–417, Novalux Inc., Protera 488 laser system, 107. reducing, 87 426–427, 430, 442, 504, 535, 507, NSDC. See Nipkow spinning-disk confocal. limits grey levels, 443 703–720, 728, 741, 751. See also, Nuclear import analysis, 802. measurement, 74–75 Multiphoton/microscopy; Harmonic Nuclear magnetic resonance (NMR), 618. multiplicative, 28–33, 51, 77–78, 224, signals; SHG, THG. Numerical aperture (NA), 1, 4, 24, 28, 61, 234, 256–258, 262, 275, 443, 460, absorption, 188, 415–418, 426–427, 430, 126, 141, 145, 148, 168, 180, 195, 633, 661, 667 705 198, 239–250. in photon detectors, 256–259 bleaching, 536, 550, 558, 645, 680–685, affects surface reflection contrast, 180 noise currents table, 256 693, 697, 707, 729. See also, defined, 1 photo flux, 257–258 Bleaching; Photodamage determining axial resolution, 4, 241–242, photoemissive devices, 256–257 CARS, 595–598, 600 657 pixel value represented, 258–259 DIC, 473–474. See also, Differential determining lateral resolution, 1, Poisson. See Poisson noise interference contrast 241–242, 656 polarization, in laser systems, 83 fluorescence, 172, 179 diffraction orders accepted by, 61 read, and readout speed, 77 focus shift with spherical aberration, 409 effect on self-shadowing, 168, 198 Index 967

in fiber-based mini-confocal endoscopes, axial shift, 243–245, 657–658 Olympus, confocal manufacturer, 52–53, 54, 509 chromatic registration, 657–658 119–120, 161, 184, 187, 204, 229, image brightness, 126 cleaning, 642 230, 234–236, 419, 421, 427, 557, matching to CCD pixel size, 62, 928 confocal performance, 145–161, 652–660 708–709, 727–730, 797, 908, objective lenses with high, 145, 239–250 contrast transfer function (CTF), 16, 35, 912. empty aperture, 248 37–39, 59–62, 656, 747 Fluoview-1000, 119–120, 184, 187, 204, with oil-immersion vs. water objective, coverslip thickness, table, 654 908, 912 148 dipping lenses, 161, 209, 411, 429, 568, DSU disk-scanning confocal microscope, pinhole size as function, 28 613, 727, 737, 870, 872 229–230, 234–235, 908, 913 and refractive index mismatch, 147–148. dry, high-NA, aberrations, 15 FRAP system, 210 See also, Spherical aberration in field illumination, 34–35, 127–128, 139, FRET, 797 tandem scanning confocal 461, 627, 658 high content screening, 811 microscopy, 141 flatness of field, 145, 151–152, 154, 418, objectives, 557, 727–730 vertical shadowing, 195 457, 639, 659 stick, in vivo objectives, 806 and zoom setting optimal, 24 Focal CheckTM beads, 657 TIRF objectives, 183 Nyquist criterion, and digitization, 38–39, high-NA planapochromat, 13, 145, transmission, table, 159, 161 64–68. 239–250 SIM scanner, 52–54 Nyquist digitizing, 65, 67. infinity correction, 155–157, 166, 239, tube-length conventions, 157, 239 Nyquist filtering, 70–79, 281. 405 On-axis reflections, artifact, 171. Nyquist frequency, 64, 301. See also, advantages, 49 Onion epithelium (Allium cepa), 390. Shannon sampling frequency. lateral chromatic registration, 657–658 Online confocal community, Listserv, Nyquist, Harry, 64. lateral resolution. See CTF 390. Nyquist noise, 256. light, vector nature, 267 OPA. See Optical parametric amplifiers. Nyquist reconstruction, limit output mounting media. See Mounting media OpenLab, 282. bandwidth, 59, 66–67, 69, 70, 173, photon efficiency losses, 25–26 Operational lifetime, of laser tubes, 102. 235–236, 280–315, 458, 468–469, plan objectives, table, 152 OPFOS, Orthogonal-plane fluorescence 474–475, 496–497, 563, 585, 603, point spread function of high NA, sectioning, 672–673. 607, 610, 615, 635, 672, 675, 239–250 OPO. See Optical parametric oscillators. 677–678, 690, 772, 730–731, 762, measuring, 240–242, 455, 462, 471, OPT. See Optical projection tomography. 77, 774–776, 778, 784, 883. 656 Optical aberrations, 109, 542. See also, Nyquist sampling, 24, 37, 39, 40, 53, 60, polarization effects, 249–250 Aberrations. 64–70, 73, 75–76, 78–79, 142, 146, pupil function, measured, 245–248 thin-disk laser optics, 109 152, 205, 222, 258, 271, 273, 289, 3D point spread function restored, Optical layout of confocal microscopes, 386, 391, 448, 635–636. 247–248 212–213. See also, Optical paths blind spots, 38 empty aperture, 248 by class, 213 for CCD camera, 70, 233, 273, 928 Mach-Zehnder interferometry, 245 evaluation, 212–213 and deconvolution, 59, 65, 67–68, phase-shifting interferometry, 245 class 1 systems, 212 222–223, 635 Zernike polynomial fit, 245–247 class 2 systems, 212–213 diagram, 60 table, 247 class 3 systems, 213 optimal, results of deviating from, 24 resolution test slide, 169, 656 Optical bandwidth/electronic bandwidth, 32. practical confocal microscopy, 448, spherical aberration. See Spherical See also, Bandwidth. 635–636 aberration Optical breakdown, 198, 680, 682, 685, 687, reconstruction, see Nyquist correction, 654–655 703, 705. reconstruction. sub-resolution beads, 181–182, 196, 454, Optical coatings, maintenance, 116. relationship with Rayleigh-criterion and 477, 493, 499, 527, 652–656, 784, Optical coherence tomography (OCT), PSF, 39, 60, 64, 66 900, 904, 930 609–610. signal-to-noise ratio, 67, 448 images, 656 of human retina, 609 subpixel, resampling, 478–479 table of suppliers, 653 schematic, 610 temperature variations, 248–249 Xenopus laevis embryo, 610 O transmission, optical, 154, 158, 159–161. Optical components, chapter, 43–59. Object scanners, image quality, 216. See Transmission, objective Optical density (OD), 71, 81, 416. Objective lenses, 13, 15, 25–26, 34, 49, 145, table of objective lenses, 159–161 filters, 43, 49–50 152, 156, 239–250, 652–660. See water-immersion, 145, 149–150 Optical disks, 586. also, Aberrations. dipping objectives, 161, 209, 411, 429, Optical efficiency, improvements, 143–144, apodization, 250 568, 613, 727, 737, 870, 872 216. See also, Photon efficiency. axial chromatic registration, 287, 658 use and limitations, 15 of disk scanners, 216 axial resolution measurement, 656–657 working distance, 5, 9, 129, 145, 154, of light-emitting diodes, 143–144 vs. pinhole size, 656 157, 198, 249, 511, 568, 598, 643, Optical elements, 43–58, 128, 211. chromatic aberrations 14, 145, 152–156, 673, 678, 727–728, 747, 774, 779, confocal light beam affected by, 211 160, 177–178, 209, 242–243, 641, 781, 872 of Köhler illumination components, 128 659 x-y and z resolution using beads, 656 light beam characteristics affected by, apparatus in measuring, 243–244, 654, OCT. See Optical coherence tomography. 211 659 OLED. See Organic light-emitting diodes. chapter, 43–58 968 Index

Optical excitation, diagram, 82. Optical performance, practical tests, point spread function, 490–491. See also, Optical fiber. See Fiber optics. 652–660. Point-spread function Optical fiber, for scanning by moving fiber axial chromatic registration, 658 STED comparison, 578 tip, 213–214. axial resolution using mirror, 656–657 Optical tweezers, 89–90, 110, 218, 383, 385. Optical heterogeneity, specimen, 22–23. chromatic aberration, 659 setups for integrating, 218 reflection, refraction, scattering, 192–197 chromatic registration, 657–658 table, 385 Optical images, electronic transmission, 5–6. contrast transfer function (CTF), 656 trapping wavelength, 89–90 Optical materials, 158, 501. coverslip thickness vs. RI, table, 654 Optics, general, 12, 125, 156–157. thermal properties, 158, 248–249 field illumination, 658 finite vs. infinity, 156–157 Optical parametric amplifiers (OPA), flatness of field, 659 Optiscan confocal endoscope, 213–214. 100–101, 112, 114–115, 118, 124. Focal CheckTM beads, 657, 658 Organic dyes, 109, 203, 342–343, 353–356. components, 115 lateral resolution, 655 See also, Dyes; Fluorophores; table, 101 resolution test slides, 655–656 Fluorescent labels; Fluorescent Optical parametric oscillators (OPO), specimen self-lensing artifacts, 659 probes. 100–102, 111–112, 114–115, 118, spherical aberration correction, 654–655 AlexaFluor, 353–355 541, 600. Optical power, specimen plane, table, 140, BOPIDY, 353–355 for CARS microscopy, 600 644. classes, table, 355 cavity dumped, to increase white light, Optical probes, 737. See also, Dyes; coumarin, 353, 355 113 Fluorescent indicators; Fluorophors; cyanine, 353–355 tunable, 114–115 Fluorescent labels. fluorescein, 353–355 table, 101 Optical projection tomography (OPT), rhodamine, 109, 203, 353, 355 Optical path. of. 610–613. Organic light-emitting diodes (OLED), 143. 4Pi, confocal, 563 lamprey larva, 612 Orthogonal-plane fluorescence sectioning commercial, 566 mouse embryo, 612 (OPFOS), 672–673. acousto-optical device, 55 refractive index, 613 Oryza sativa. See Rice. compound light microscope, 156–157 setup, 611 Oscillating-fiber scrambler, 8. CARS, 599, 601, 907 Optical pulse length, 537–538. See also, Osmotic permeabilization, 359. CARV-2 disk scanner, 230 Pulse broadening. OTF. See Optical transfer function. confocal, 10, 208–209, 212, 632, 681 group delay dispersion, 537–538 Out-of-focus light. beam-splitter, 213 group velocity dispersion, 537 deconvolution vs. confocal microscopy, disk-scanner, 12, 216 measurement, 115, 901–903 461. folded, 166 Optical resonator in laser, 81–82, 116. information, 26, 32, 487, 644–646. scanning systems, 214 laser, 81–82 Output amplifier, reconstructing analog fiber-optic confocal, 508 maintenance, 116 signal, 64. interferometers, 243, 245 Optical sectioning, 9–10, 13, 180, 182, 222, Output modulation, of semiconductor lasers, Kino single-sided disk scanner, 229 223, 236, 268–270, 469, 748, 108. LaVision-Biotec, Trimscope, 907 763–764, 772, 774, 775, 784. See Overheating, of filters, 43. See also, Leica, TCS AOBS, 910 also, Deconvolution, Confocal, Thermal variables. magnetic resonance imaging, 621 etc. Overlap alignment protocol, montaging, Minsky confocal, 5, 25 algorithms for widefield, 763–764 732. for measuring photon efficiency, 34 of A. Thaliana root, 772, 775 Over-sampling, 60, 70, 728. multi-photon, 540, 681, 708–709 with confocal laser-scanning microscope, vs. duplicate-and-smooth process, 70 multi-focal, 552, 555 9–10 reasons for, 68 spectrometer, 424 example, 182, 463, 471, 492, 656 subpixel, resampling, 478–479 Nikon C1si, 911 dynamic imaging, 784 Oxygen sensor, 45, 347. Olympus DSU disk-scanner, 230 improvement, with deconvolution, 752 Olympus Fluoview-1000, 912 latex bead, 3D image, 196 P optical coherence tomography, 610 limiting excitation, 223 Pack-and-go mode, Power Point, 842, 844. optical projection tomography (OPT), near surface of living embryo, 748 Paeonia suffruticosa, 421. 611 near to refractive index interface, 180 Panda pattern, polarization-preserving fiber, Petran tandem scanner, 228 selective plane illumination, 748 88. selective plane illumination (SPIM), 613, structured illumination, 268–270 PAS. See Periodic-acid Schiff. 673 with widefield phase-dependent imaging, Passively mode-locked lasers, 111. or simultaneous BSL and fluorescence, 13 Patch clamp, for loading dye, 360, 726–727, 128 Optical system, losses, 25–32, 217. 734, 738–740. surface 3D imaging, SIM/EFIC, 608 Optical transfer function (OTF), 164–165, Patch pipette, 738. surface spherical aberration, 405–406 490–491, 562, 563, 567, 569–570, Pattern analysis. See Automated STED, 573 578. See also, Point-spread function; interpretation of subcellular patterns. structured-illumination, 266 Contrast transfer function. Patterned-illumination microscopy, see Visitech VT-Infinity and VT-eye, 914 4Pi microscopy, 562, 563, 567 Structured illumination microscopy Yokogwawa dual-disk-scanner, 231, contrast, 164–165 PC. See Personal computer. 915 deconvolution for image enhancement, PCA. See Principal component analysis. Zeiss LSM-510, META, 916–917 490–491 P-CARS, Polarization-sensitive detection Zeiss LSM-5-Live, 50, 232, 916 I5M, 569–570 CARS. Index 969

PCF. See Photonic crystal fiber. Phase and intensity determination from multi-photon microscopy (MPM), 545 PE. See Photoelectrons. correlation and spectrum only Perrin-Jablonski diagram of bleaching, Pear (Pyrus serotina), spectrum, image, (PICASO), 115. 697 711. Phase contrast, 9, 171, 368, 372, 453, 506, photocycling, fluorescent proteins, 698 Pearson’s correlation coefficient, 668. 643, 649, 731, 851, 854, 890, 892. propidium bound to DNA, plot, 695 Pellicle beam-splitter, 216, 228–229, 231, coherent light for, 130 reactive oxygen species, 341–342, 346. depth of field, 13 362–363, 390, 544, 682–684, 691, Peltier cooling. and holography, 7 693–694, 852–853 CCDs, 234, 447 scanning, 9, 13, 386 reduction in, 693–696 cell chamber, 387–389 Phase fluorometry, 518–519, 526. antifade agents, 36, 341, 368, 375, 499, lasers, 85, 106–108, 111, 117 comparison of FLIM methods, table, 526 694 Penetration depth, 177, 343, 643, 672, 731, excitation/emission signals, 519 disk-scanning microscopy, 224 765. fluorescence lifetime imaging, 518–519 quantum dots, 694 of dyes, 360, 387, 731, 739, 882, 874 Phase randomization, to scramble light, 8, results, in living embryos, 759 of fixative, 369–370, 376, 857 13, 84, 131–132, 143, 507. of single molecules, 696–698 FRET sensors, 798–799 Phase-dependent imaging, depth of field, 13. structured-illumination methods, 275 long laser wavelengths, 109, 416, 418, Phase-shifting interferometry, 245. two-photon excitation microscopy 427–428 Phenotyping, 623–624. (TPEM), 690, 697 multi-photon, 381, 418, 433, 435, 439, Phong shading, 308–309. Photocathode, PMT, 28–29, 232–233. 543, 545, 558, 646, 684, 708, 714, Phong/Blinn models, 308. quantum efficiency, 232–233 728, 749, 904 Phosphoinositide signaling, 799. to reduce transmission losses, 28–29 in plant imaging, 779 Photo efficiency. See Photon efficiency. Photoconductivity, in photodetectors, 252, in scanning electron microscopy, 847 Photoactivatable dyes. See Photoactivation. 253. in SPIM, 613, 675–678 Photoactivation, 187, 224, 383, 385, 541, Photocycling, fluorescent protein molecules, TIRF, 177–178 543–545, 693, 759. 698. Peony flower, autofluorescent petals, example, 759 Photodamage. See Phototoxicity. 173–174, 176, 421, 423. genetically encoded Photodetector. See Detectors; Light Peptide-mediated uptake, 359. Kaede, 187, 383, 385 detectors; Perfusion. Kindling, 574, 760 CCD; EM-CCD; PMT etc. chambers, 381, 386–389, 394, 726, 729, PA-GFP, 187, 383, 385, 752, 759–760 Photodiode, 134–135, 253–255, 610, 769, 870–873 table, 385 707–708. fixation, 376 Photobleaching, 174, 218, 224, 275, feedback, to stabilize laser, 87, 682 Periodic grating. See Grating. 341–342, 362–363, 545, 690–700, feedback, to stabilize arc/filament, Periodic-acid Schiff (PAS) reagent, 262, 729, 747–748, 759. See also, 134–135, 137 369, 770, 774–775, 778. Bleaching, and Chapter 39. in hybrid PMT, 29, 30 maize pollen grain, 202 autofluorescence, 698 infrared sensitive for IR lasers, 707 Periodically poled (PP) waveguides, defined, 218, 691 photometer sensor, air space, 26 114–115. dynamics, as a source of contrast, quadrant, for alignment, 87, 134 Perrin-Jablonski diagram, 516, 517, 697, 202–203 of self-aligning source, 134–135 792. effect on contrast, 174 for testing display software response, 830 photobleaching, 697 fluorescence intensity loss, 691, 694, 696, vacuum avalanche, 254, 255 Personal computer (PC), performance 698+ Photoelectric effect, and LED operation, needed for image processing, fluorescent image of single protein, 699 137. 289–290. fluorescent probes, 362–363 Photoelectrons (PE), 29, 30, 62–63, 77, Perspectograph, early studies, 789. fluorescent recovery vs. irradiation time, 232–234, 254–255, 257, 259–264, Petrán disk, 2, 6, 11, 135, 141, 215, 699 339, 633, 863. 223–224, 228, 251, 265, 381, 387, fluorophores signal optimization, 341–342 amplification of, 62–63 447, 458, 554. choice of fluorophore, 342 in the CCD, 232–234, 495, 918, 931 Petrán, Mojmir, 2, 6, 11, 215, 223, 228. fluorophore concentration, 342 production in PMT, 30 pH imaging, 188–189, 221, 346, 348, 359, light collection efficiency, 217, 341 single-PE pulse-height spectrum, 29, 77 386, 421, 517, 529–530, 664, protective agents, 36, 341–342, 363, secondary electrons, as source of PMT 739–740, 743, 744. 368, 375, 499, 694 multiplicative noise, 77 calibration, 421, 530, 745 spatial resolution, 341 Photoemissive devices, 256–257. display, 287 in four-dimensional imaging, 747–748 Photoemissive effect, 254. intensity image, 529, 530, 739, 740, green fluorescent protein (GFP), 690, 692, Photographic recording systems, 6–7, 744 698 11–12, 20, 22, 30, 71–72, 132, 139, lifetime image, 530 intentional See Fluorescence recovery 141, 162, 207, 217, 263, 280, 488, pH indicators, 346, 739–742. after 581+, 588, 590–591, 593–594, 607, pH shift/formaldehyde fixation, 370–371, photobleaching (FRAP) 613, 628–629, 633, 640, 643, 712, 373. kinetics, 695 829, 862, 865–867. Phalloidin, as correlative marker, 235–236, mechanisms, 340, 691–693 “toe” response, quadratic, 71 344, 376, 378, 694, 696, 756, 804, FRET, 691 Photometer paddle, to measure light beam, 854–856. multi-exponential fluorescent bleaching, 26, 35, 139–140, 159, 391, 650–651, Pharmacological screening, 813–814. 697 665. 970 Index

Photometric response, and HD curves, 71. thermal effects, 252 to move optical fiber, 84 Photomicrography (Loveland), 139. vacuum avalanche photodiode, 254, 255 to move scanning mirror, 57, 215, 238, Photomultiplier tube (PMT), 9, 28–31, work functions, table, 252–253 510, 555, 610 35–36, 51, 62–63, 74–75, 222, 232, Photon efficiency, 24–36, 215, 217, 341, to move stage, 215, 567 251, 254, 255, 258–261, 443, 527, 631. phase-shifter 661–662. defined, 24 in 4Pi confocal, 609 after pulsing, 257 as a limitation of confocal systems, 24, in structured illumination, 268 Bio-Rad, 260–261 223 optical coherence tomography, as confocal detectors, pros/cons, 222 measuring, 26, 33–36, 217 609–610 for epi-fluorescence confocal microscope, practical confocal microscopy, 631 stretching optical fiber, 609 9 of scanners, 215 Pile-up, of pulses. functioning, 62–63 table listing photon losses, 217 in avalanche photodiode, 253 GaAs photocathode, 28–29, 232, 252, Photon flux, statistics, 256–258. in photomultiplier tube output, 32–35 255, 263, 527, 931 Photon interactions, 252–256. measuring risk of, 34–35 gain from collisions at first dynode, 31 Photon (shot) noise, 660–661. See also, p-i-n diode, 253. grey levels, 443 Poisson noise. Pinhole, 26–28, 33–35, 149, 150, 154, 201, hybrid, single-pixel signal levels, 31, Photonic crystal fiber (PCF), laser delivery, 210, 213, 215, 224–228, 395, 254–255 1, 88, 109–110, 113, 504, 541. 631–632. linearity, 661–662 for white light source, 113 advantages and disadvantages, 26–28 microchannel plate, 232–233, 255, 262 Phototoxicity, 112, 363–364, 390–391, 651, calibrating diameter, 33–34 mini-PMT arrays, 51, 667 729, 746, 770. confocal, proper use, 28 multiplicative noise, 28–30, 77, 633, chapter, 680–689 disk-scanning, 224–228 677, 926. See also, Multiplicative in brain slices, 729 mini-image detection, 32 noise damage is higher to either side of raster, optical fiber as, 506–507 in multi-channel detection systems, 51 54 optimal size, 226–227, Chapter 22 noise and gain, 74–75 factors influencing, table, 363 Fraunhofer formula, 225 number of photons striking per unit time, fluorescent probes, 363–364 position in confocal microscope, 210 35–36 live cells, 390–391 practical, in confocal, 631–632 optical enhancer to increase QE, 28–30 reduction, 391 radius, effective, 35 photon counting, 21, 29–30, 32–35, from uneven scan speed, 651 ray paths, different sizes, 226–227 258–259, 260–263, 542 Photo-uncaging, 187, 210, 383, 385, 541, single-mode polarization preserving fiber, quantum efficiency, 527 544–545, 692, 729, 760, 912. See 213 vs. cooled CCD, 26–28 also, Photoactivation. small pinholes, effect, 225 signal variation with time, 232 Photovoltaic effect, 252–253. of tandem scanners, 215 transit time spreads, 527 Phycobiliproteins, 338, 341, 343, 355–357, vibration shifts relative positions, 201 Photon(s), 20–21, 30, 33–36, 63–64, 132. 693. Pinhole disks, critical parameters, 224–228. counting precision, 20–21 Physical limitations, 20, 24, 63–64. Pinhole energy, with spherical aberration, uncertainty, 63–64 on accuracy and completeness of data, 149, 150, 154, 631–632. interactions with photomultiplier tube, 30 20 penetration into water, 149, 150 lost, 33–36 Poisson noise, 63–64. See also, Poisson defocus and NA, 150 Photon counting, 21, 29–30, 32–35, noise defocus and wavelength planapochromat, 258–259, 260–263, 542. Physiological fluorescence imaging, 383, 154 circuits, 33–34, 258, 521 385. Pixel clock, digitization, 62, 64–65, 201, digital representation of optical data, PICASO. See Phase and intensity 234–235, 258, 903, 923, 929. 32–33 determination from correlation and CCD, table, 929 effects, 34–35 spectrum only. Pixels, 38–39, 60, 62–63, 65, 258–259. examples, 35, 263 Piezoelectric effect, defined, 57. defining, 60 hybrid PMT, 29–30 Piezoelectric focus controls, 166, 215, 219, digitization, 62–63 pile-up losses, 32–33, 35, 78, 521, 523, 222, 231, 241, 245, 268, 468, 754, optimal, 63–64, 66 526–527 909. representing intensity, 258–259 with PMT, 29–30, 32–35, 258–259, 260, Piezoelectric scanning systems, 57, 215, and resolution, 38–39 263 238, 510, 555, 610. and Abbe criterion resolution, 38–39, 65 Photon detector types. See Detectors and Piezoelectric devices. PKzip, 580. entries by each detector type. AOD driver, 54–55, 57 Plan objectives, Zeiss, field diameter, table, CCD, 254 acousto-optical components, 54–55, 57 152. direct effects, 252 to align objective, 166 Planapochromat, 152, 155. See also, image dissector, 254–255 dithering to increase CCD resolution, Objectives. microchannel plate, 232–233, 255, 262 70 flatness of field and astigmatism, 152 MCP-CCD, 262 effect described, 57 lateral chromatic aberration, 155 gated, 519, 523–524, 527, 532 to focus objective, 166, 215, 219, 222, Plancks law, energy of photon, 35, 137, 252, photoconductivity effects, 252, 253 231, 241, 245, 268, 468, 754, 909 424. photoemissive, 254 laser alignment, 87 Planar illumination, SPIM, optical photovoltaic, 252–253 light scrambler, 84 sectioning, 751. Index 971

Plane of focus, distortion, 16, 23. pollen grains, 202, 305, 313, 420, Plumbago auriculata, fluorescence spectra, by beam deviations, 16 431–433, 553, 558, 781, 783 422. by refractile cellular structures, 23 protoplasts, 195–196, 203, 416, 421, PMT. See Photomultiplier tube. Plant cell imaging, 769–785. 423–427, 429–431, 438–439, 693 p-n diode, 253. See also, Photodiode. autofluorescence, 770–772 root, 172, 174, 303, 307, 421, 429, PNG (Portable network graphic), 581, 584. birefringent structures, 162–164. 430+, 438, 464–465, 556, 772–773, Pockels cell, variable beam attenuator, 25, 420–421. See also, Birefringence 775, 777, 779–783 54, 57, 87, 116, 543, 701, 903–904. chamber slides for plants, 429 culture chamber, 429 Pockels effect, in crystals, 57. clearing intact plant material, 166, starch granules, 202, 420–421, 428, Point-spread function (PSF), 4, 10, 23, 27, 417–418, 420, 439, 610, 624, 432–433, 435, 703, 710–712, 715, 39, 68–70, 145–146, 189–190, 208, 774–775 719 223, 239–250, 271, 275, 330, 378, computer visualization methods, 778 stem, 168, 172, 180, 417–419, 421, 405, 407, 409, 446, 448, 453–457, deconvolution, 784–785 424, 429, 556, 707, 710–711, 485–486, 489–494, 536, 562–564, direct imaging, 772–773 713–714 570, 574, 578, 635, 656, 674, 750, dynamic imaging, 783–784 storage structures, 435–436 784, 830, 895. effect of fixation, 195, 428 suspension-cultured cells, 189, 3D, 68–70, 247–248 Equisetum, 774 429–430 4Pi microscopy, 562–563 fluorescence properties, 421–428 tapetum, 433–434, 779 additional information from, 570 emission spectra, 421–423 waxes, 420, 428, 434–435, 714–715 space invariance of PSF for, 564 microspectroscopy, 421–426 new spectral tools, 770 apodization, 240, 243, 249, 250, 272, 567, fluorescence resonance energy transfer, obtaining spectral data, methods, 772 889 425. See also, FRET penetration values, 779 blind deconvolution, 468, 485 harmonic generation See Harmonic photodamage, 770 in botanical specimens, 772, 784 signals fungi, 438–439 point spread function, 722, 784 in brain slices, 729 genetically encoded probes, 769, 773, refractive index heterogeneity, 192, calculations, RI-mismatch, 407 783 418–420 for glycerol, table, 409 green fluorescent protein fusions, 773, single-photon confocal excitation, for water, table, 409 783 772–778 CARS, 596 of green tissues, 770 specific methods, 769 comparing widefield with confocal, 27, hairs, 434–435 spectral unmixing, 770 453–457, 493, 644–647 history, 769 examples, 665–666 confocal, 10, 12, 208+, 212, 216, 405, light attenuation in plant tissue, 414–418 staining, 774 632, 681 A. thaliana, example, 416 technological developments, 769 vs. deconvolution, 27, 453–457, 493, absorption spectrum, 415 textbooks, 769 644–647 effect of fixation, 428 three dimensional, 771 deconvolution, 189–190, 223, 489, maize stem spectra, 417, 418 clearing agents, 166, 417–418, 420, 490–494, 784. See also, M. quadrifolia spectra, 416 439, 610, 624, 774–775 Deconvolution M. quadrifolia optical sections, 419 deconvolution protocols, 784 quantifying PSF, 492–494 Mie scattering, 162–163, 167, 417–418 reconstruction, 775–776 deformation caused by RI anomalies, nonlinear absorption, 416–417 segmentation, 776–778 22–23 Rayleigh scattering, 162–163, 167, 417, two-photon excitation, 415–419, 421, Fourier transform, 489, 490 703 423 lateral resolution. See Lateral resolution light effects on, 770 advantages, 778–779 measuring, 240–242, 455, 462, 471, light-specimen interaction, 425–428 best conditions, 781 656 living plant cell specimens, 429–439 compared with one photon, 421 amplitude/phase, 242 calcofluor staining procedure, 424, cell viability, 779–782 fiber-optic interferometer, 240–241 438 deconvolution protocols, 784 images, 246–248 callus, 429 dyes, 782 high-NA objectives, 239–250, 492, 656 cell walls, 168–169, 188–189, 303, green fluorescent protein, 782–783 pupil function, 240 306, 416–417, 420–421, 428–431, light-specimen interaction, 425–427 for 3D deconvolution, 145–146 435–136, 438, 439, 710–711, microspectrometer, 424 non-linear, 552, 750 713–715, 769–776, 779–781 pitfalls, 782 and Nyquist, 635, 636, 751, 752 chamber slides, use, 429 thick specimens, 779 optical transfer function, related to, cuticle, 434–437, 715, 717, 779 in vivo, 781 490–491 fungi, 438–439, 624, 782, 870 Plasma membrane, microscopy. See Total polarization effects, 249–250 hairs, 431, 434–436, 772 internal reflection microscopy pupil function, 245–248. See also, Pupil meristem, 168, 420, 430, 770, 776–778, (TIRF). function 783 Plasma light sources, spectra, 44. Rayleigh-criterion and Nyquist sampling, microsporogenesis, 431–432 Plasmid DNA, nick-damage, 684, 724, 39 mineral deposits, 163, 420, 436–438, 802–804. See also, Microinjection; refractive index mismatch, 405, 407 703 Electroporation; Biolistic spherically aberrated, 148–150, 407, 492 pollen germination, 420, 433–434, 781, transfection. shape in telecentric systems, 208 783 Plasmodesmata, 777. SPIM, 674 972 Index

Point-spread function (PSF) (cont.) to reduce reflections, 6, 25, 141, 158, 171, Pol-scope, 13, 188, 432, 468, 479–480. STED, diagram, 574, 578 516, 229. See also, Antiflex system deconvolved, 479 structured illumination see Structured scramblers, 8, 84, 132, 143 images, 15, 188, 479, 717 illumination microscopy Polarization effects, 211, 249–250, 503. Portable network graphic. See PNG. temperature effects, 25, 85, 248–249, 630 birefringence, 188, 420–421, 431, 434, Position, accuracy in CLSM, 40. terminology, 405 436, 438, 480, 503. See also, Position sensors, galvanometer, 53–54. Wiener filtering, 494 Birefringence Posterizing, 591. Points, defined, 59. blind deconvolution, 479 Potassium titanium oxide phosphate (KTP) Point-source, for measuring photon and CARS microscopy, 595, 600–604 crystal efficiency, 33. high-NA objective lenses, 249–250, for non-linear optical frequency conversion, Poisson noise, 20–21, 29, 37, 63–64, 67, 69, 267 107. 74–75, 81, 164–165, 211, 232, 234, interaction with nucleus, 23 Potato (Solanum tuberosum) SHG signal, 442, 456, 460–463, 468, 487, 495, optical fibers, 503, 507 712. 497, 633–636, 647, 651, 655, 660, stereo image displays, 299, 589 Power requirements, for lasers, 65, 80–81. 693, 784, 835, 923–924, 926. See Polarization microscopy, 43, 50–51, 154, Power spectrum. See Contrast transfer also, Quantum noise, Shot noise. 156, 162, 188, 288, 348, 438, function. bleaching, 693 479–480, 513, 555, 711, 714–715, Power supply, laser as noise source, 86. of CCD 717, 719, 891, 894. PP, Periodically poled waveguide, charge transfer, 920 centrifuge microscope, 8 114–115. dark charges, 921–922 of collagen fibers, 164, 188, 717 Practical confocal, 2-photon microscopy, CT imaging, 615 DIC, 10, 14, 127, 146, 468, 473 tutorial. See also, each topic as a and display linearity, 72–73, 588 and FRET, 793 major entry. digitization, as part of signal, 65, 69, and harmonic generation, 179, 428, 2-photon 633–636 704–706, 717, 719 excitation duty cycle, 644 of EM-CCD, 233–235, 262, 927–928 MFMP, 555 peak power level, 644 and FLIM, 524–525 mitotic apparatus, 15, 717 photodamage vs. penetration, 645 and gray levels, 74 p- and s-, and incidence angle, 50–51 power vs. penetration, 646 importance of deconvolution, 38–41, 60, Pol-scope, 13, 188, 432, 468, 480 3D microscopy methods compared, table, 69, 189–190, 222–223, 320, 399, PSF, 406–407 647+ 471–472, 481, 495, 751–753, 835 to regulate light intensity, 43 best 3D method for, 644–647 intensity spread function, 75–78 STED, 578 biological reliability, 631 photomultiplier tube, 74–75 Polarization noise, in lasers, 83. bleaching pattern, 627–628 affects effective QE, 31 Polarization-preserving fiber, 49, 87, 503, quantum efficiency, 628 multiplicative noise, 29, 647, 660 505, 507. chapter, 627–649 in photon detection, 63–64 as a pinhole, 213 confocal images with few photons, 634 and pixel size, 64, 68, 633–636, 928 Polarization-sensitive detection CARS (P- deconvolution, factors, 646 practical effects, 67 CARS), 600, 601, 604. filling back-focal plane, 210, 509, 629, single-pixel noise, 65, 67, 190, 635, 832, adipocyte cells, 604 633 835–836 Polarized light, 7, 14, 83–85, 146, 158, 162, focus, compensating drift, 395, 732 spectral unmixing, 667, 770 171, 229, 406–407, 420, 479, 894. getting a good confocal image, 629–631 examples, 665–666 deconvolution, 479 alignment of optics, 629–630 structured illumination, 278 image formation, 406–407 back-focal plane (BFP), 210, 509, 629, uncertainty in contrast, 74, 164–165 PSF, 479 633 and visibility, 37, 667 Polarizer, 83, 128, 188, 249, 268, 275, 420, focus, 629 Polarization, 13, 49, 57, 83, 88, 89, 479, 711, 903–904. low signal, 631 211–212. for antiflex, 6, 84, 141, 158, 229 mirror test specimen, 630 attenuator, 43, 543, 907 for attenuation, 43, 85, 87–88, 543, no signal, 631, 660 beam-splitter, 13, 50–51, 57, 85, 87, 100, 903–904 simultaneous BSL/fluorescence, 171, 217, 513, 631, 904 for CARS, 601 631 to avoid spectral distortion, 49 Glan-Taylor, 85, 87, 100, 171 getting started, 627 circular or phase randomized, 211–212, Glan-Thompson, 85, 904 Köhler illumination for transmission, 34, 229 LCD, 589, 715 127–128, 131, 229, 627, 648–649 effect on AODs, 55 micro-wire, 85 multi-photon vs. single-photon, 646 effect, of dichroic beam-splitters, 34, structured illumination, 264 new controls, 631–636 49–50 tunable, 715 biological reliability, 631 Kerr cell, 111, 113, 516 Pollen germination, 433–434. pinhole size, 631–632 of laser light, 8, 83, 88–89, 113, 478, Pollen grains, 202, 305, 431–433, 438, 553, pixel size, 62, 634–635, 784, 928 558 556, 558, 678, 781, 783. Nyquist reconstruction/deconvolution, optical components, 57, 155, 211 germination, 433–435, 783–784 635–636 optical fibers, 213 multi-focal multi-photon imaging, 556 over-sampling, 635 Pockels cell, 25, 54, 57, 87, 116, 543, Pol-scope, 13, 188, 432, 468, 479–480 photon efficiency, 24–26, 215, 217, 341, 701, 903+ test specimen, 195, 269, 313, 553, 556, 631 rectified DIC optics, 846 678 pinhole summary for, 26–28, 633 Index 973

pixel size, 62, 634–635, 784, 928 measuring display speed/sensitivity, Pulsed lasers, 81, 96–100, 110–114, 120, measuring, 635 830 137. See also, Lasers; Ultrafast summary for, 636 random color dot image, 836 lasers. poor performance, reasons, 640–643 reference images, 830–831 broadband tunable, table, 120 air bubbles, 643 removing distortion, 835 diode, table, 96–97 curvature of field, 641 resolution, 832–835 DPSS, table, 98 dirty objective, 642–643 rotating, 835 dye, table, 96 imaging depth, 643 scaling, 835 excimer, table, 96 under filling objective pupil, 642 screen capture, 830 for FLIM, 537 optical problems, 640–641 static image performance, 831 kits, table, 98, 100 sampling problems, 640 step image, 833 nitrogen, table, 96 singlet-state saturation, 643 under-sampled image, 835 scanning only region of interest, 237 under-sampling, 635 up-sampling, example, 834 for 2-photon excitation, 81 schematic diagram, 632 viewer, 830 ultrafast, table, 99–100 statistical considerations, 633–634 Preventive maintenance, lasers, 115–116. vapor, table, 97 stray light, 201, 632, 904 Principal component analysis (PCA), Pulse-counting mode, 21, 29–30, 32–35, test specimen, 636–640 731–732. 258–259, 260, 263. description, 636–637 Printers, 591–593. Pump sources, for dye lasers, 103. diatom, 638–640 aliasing, 592 Pumping media, maintenance, 116. figures, 637–640 color images, 592 Pumping power vs. frequency cubed, 65, 82. reasons for, 636 grey levels, 592 Pupil function, 211, 245–248. widefield vs. beam scanning, 647 ink jet, 593 3D point spread function restored, Prairie Technologies, LiveScan Swept Field laser, 593 247–248 design, 237. posterizing, 591 4Pi, 566–567 Pre-amplifier, in digitizing analog signal, scaling techniques for, 592 AOD, 56 64. Prionium, MMM image, 556. empty aperture, 248 Precompensation, in fiber optic cables, 88. Probe, mismatch with pixel shape, 39. of human eye, 72, 128 Presentation software, 829–845. Processor performance, 3D-image display, intermediate optics, 211, 222, 225, 250 helpful URLS, 844–845 289+. Köhler illumination, 34, 127–128, 131, movies, 837–844 Projection/compositing rules, 3D-image 229, 251, 627, 648–649 artifacts, 839–840 display, 302–304, 763–764. Mach-Zehnder interferometry, 245 coding limitations, 838 alpha blend, 302, 304 measurement, 246–248 compression of large movies, graph, average intensity, 302 images, 246–248 843 first or front intensity, 302 objective, 24, 155, 158–159, 211, compression of PAL TV movies, table, Kalman average, 304 239–240, 242, 492, 551–552, 554, 842 maximum intensity, 302 566–567, 650 digital rights management, 844 Propidium iodide, 344, 355, 360, 426, orthonormal Zernike polynomial for, entropy, 841 651, 693–695, 773, 778–779, 782, table, 247 frame count matching display cycle, 812. phase-shifting interferometry measuring, 838–839 dead cell indicator, 426, 651, 875, 877 245 MPEG display formats, 840–841 Proteins, 195, 756, 760, 794–795, 804. See polarizing effects, 249 overlaying, 844 also, Green fluorescent protein, etc. pupil plane, 50 See also, Back-focal plane Pack-and-go mode, 842, 844 chimeric fusion, 794 transfer lens, 728 performance benchmarks, 841–842 fluorescent, FRET, 794–795 view of pupil image, 629 region code, 844 Kaede, 187, 383, 385 Zernike polynomial fit, 245–247 remote use, 842–844 Kindling, 760 Purkinje cells, Golgi-stained, 167–168. rules 837–838, 844 microinjection, 804 Pyrus serotina. See Pear. up-sampling, 838–839 PA-GFP, 187, 383, 385, 752, 759–760 very high resolutions, 841 tagged, 756, 758 Q precautions, 829–830 translational fusions, 756 QE. See Quantum efficiency. testing, 830–836 UV absorption, 195 Q-switched pulsed laser systems, 111, aliasing gallery, 834 Proteomics, 237, 790, 804, 809, 818, 867. 114–115. aligning images, 835 location, 825 Quantitative analysis, flying-spot brightness, 832 Protoplasts, 195, 416, 429, 430, 431. microscope, 6–7. changing display size, 832–835 A. thaliana, 195–196, 203, 416, 421, Quantization, limitations imposed by, 37–39. codecs, 831 423–427, 429–431, 438–439, 693 See also, Chapter 4. compression, 835–836 Proximal tubule, labeled, 744. Quantum dots, 221, 343, 357–358, 360–361, compression artifacts, 837 Pseudo color display, 173–175, 190, 291. 656, 694, 696, 757, 801, 814, 846, cropping, 835 PSF. See Point spread function. 853. See also, Semiconductor digital rights management (DRM), 830 Pulse broadening, 88, 111, 210, 537–538, nanocrystals. down-sampling in PowerPoint, 834 543, 606, 609, 728, 903. assays for, 814 fast graphics cards, 831, 832 Pulse length measurement, 115, 901–903. in electron microscope, 852–854 gamma, 832–833 Pulse spreading. See Pulse broadening. FRET, 801 974 Index

Quantum dots (cont.) Raman spectroscopy, 48–49, 90, 167, 254, interossi muscles, SNARF-1, pH image, labeling, 853 339–340, 507, 545, 697. See also, 739 toxicity, 357, 694 CARS. intervertebral disk, 310–311 Quantum efficiency (QE), 25–30, 74–78, CARS, 204, 550, 577, 595–605 kidney, 511, 803 222, 232–234, 238, 251, 254–255, chemical imaging, 90 leukemia cells, 347, 520–521 349, 355, 375, 383, 390, 442–443, hard-coating on interference filters used, FLIM image, 521 459, 516, 527, 575, 628, 646, 556, 48–49 neuron, membrane potential, 205 703, 751, 793, 920–922. image contrast, 167 tooth, 667 of back-illuminated CCD, 77–78 Ramp-up, for light sources, 136, 137. Rate, imaging, limited by signal level, 73. charge-coupled device (CCD), 26–28, and long-term stability, 137 Ratiometric imaging, 189, 346–347. See 74–76, 142, 215, 232, 234, 257–258, and short-time stability, 136 also, Calcium imaging, pH, etc. 261, 644, 707, 751, 754, 810, Rare earths, for doping fiber lasers, 110. bleach ratio, 697–698 920–921 Raster, 62–64. calcium, 736–737, 850. See also, Calcium comparative among CCD cameras, 76 convolution, 485–486 imaging effect on Poisson noise, 74–75 dimensions, in specimen, 63 CARS, 600, 602, 604. See also, CARS effective QE, of photon detectors, 28, 29 retrace, 25, 33, 53–54, 219, 338, 389, concentration calibration, 742–745 of electron-multiplier CCD, 4, 30, 59, 543, 628, 651, 908. See also, to detect colloidal gold labels, 167 234, 920 Retrace, raster scanning shape, to determine ionic concentration, 36 FLIM, 516–517, 520, 523, 526–527, 529, 63 FLIM, 516–532. See also, FLIM 530 size, vs. pixel size and light dose, 64 FRET, 174, 184, 790, 794–795, 797–798. FRET, 792 temporal limitations, 141 See also, FRET of human eye, 251 Raster scanning, 5–6, 25, 141–142, 223, glutaraldehyde autofluorescence assay, and intensity spread function, 74–75 540, 596. 369 and multiplicative noise, 77 alignment, 629–630, 651 HCS, high-content screening, 813, optical enhancer, to increase QE, 28–30 assymmetrical sampling, 38–40 823–824. optimal 3D microscopy, 644 bleach pattern, 3D, 538, 628, 693 indicator choice, 738 photomultiplier tube (PMT), 26–28, 51, chromatic aberration limitations, 156, interpretation, 740–741 77, 222, 257, 262, 527, 707 640–641 live/dead assay, 875 graph, 29 damage is higher to either side of raster, pH, 739–744. See also, pH imaging table, 707 54 structured illumination. See Structured signal-to-noise ratio, 263, 442–443 display, 830–831, 835 illumination microscopy variation with wavelength, 29 distortion, 40 water-immersion objectives, 737 vs. wavelength, 922 and electronic bandwidth, 70, 238 Rayleigh criterion (Abbe criterion), 1–3, 9, Quantum noise, 21 63–64, 69–70, 468, 472. for fast confocal imaging, 223 37–39, 60–61, 66, 129, 146, 486, See also, Poisson noise. fiber-scanning, 214, 508 703, 822, 928. and approximation, for reconstruction, galvanometer limits, 52–54, 223, 651 breaking the Abbe/Rayleigh barrier, 69–70 limitations imposed by AODs, 56 571–573 Quantum wells, as absorbers, 111. MPEG formats, 840 Nyquist sampling, 39, 60, 66 Quantum yield, of fluorescent dyes, 172, Nyquist sampling, 38, 41, 59–60, 62, of two point images, 1–3, 146 180, 338–345, 353, 360, 363, 383, 634–635 Rayleigh scattering, 162–163, 166, 167, 421, 543–544, 574, 661, 683, 690, off-axis aberrations, 151, 640–641, 339, 342, 417, 703, 747. 710, 737, 792, 794–795. 659–662 compared to Mie scattering, 163 Quartz-halogen lamp, control, 138–139. See pattern on Nipkow disk, 5–6, 223–225. in embryos, 747 also, Halogen lamps. See also, Nipkow disk scanning by colloidal gold labels, 167 retrace gating, 25, 54, 56, 219, 389, 543, light attenuation in plant tissue, 417 R 628, 651, 908 wavelength dependency, 162–163 Rabbit, 237, 744. scan angles, 209, 214 Rayleigh unit, 147. antibodies, 855, 877–878 stability, 708 Reactive oxygen species (ROS), 341–343, kidney proximal tubule, pH, 744 sampling in time and space, 141–142 362–363, 390, 544, 682–684, 691, Radiance, of non-laser light sources, 126, timing, 33, 53, 753 693–694, 852–853. See also, 132, 137–139, 141. zoom, raster size and magnification, 11, Bleaching; Phototoxicity. measuring with radiospectrometer, 24, 37, 63–64, 66, 70, 79, 317, 389, as basis of correlative TEM staining, 139 493, 627, 634–636, 655–658, 683, 852–853 Table, 1140 731 Readout noise, 74–75, 77, 232. See also, Radiospectrometer, radiance vs. wavelength, Rat, cells and tissues, 205, 320, 323, 330, Noise. 139. 398, 739, 813. and readout speed, 77 Raman background, in glass fibers, 88, 90, brain slices, 393, 398, 686 Real image, disk-scanners, 224. 162, 506–507. CA1 region, 323 Real-time 2D imaging, 12–13, 167–168, lower in large-mode-area, fiber, 110 cardiac muscle, 498, 529, 556 215, 222–224, 232, 235, 307, 496, Raman scattering, 162–163, 167, 339–340, cerebellar granule neurons, 813 542. 348, 506–507, 545, 697. EDL muscle, calcium, 740 Real-time 3D imaging, 154. and bleaching, 697 fixation, 370, 372, 393 Receptors. defined, 162 hippocampus, 268, 317, 341 cholera toxin, 790–791, 796–797, 802 Index 975

deconvolution, 495 of optical glass vs. wavelength, 152 mirror position, 40 EGF, 533 optical projection tomography (OPT), 613 photometric, 312 ERD2, 791, 796 self-shadowing, 198 spectral detectors, 662 fibrinogen, 846–847, 850 temperature, 148, 248–249, 411 Removable storage media, 585–588. high-content screening, 809, 812–814 of tissue/organs, table, 377 random-access devices, 586–588 KDEL, 790, 797 Refractive index mismatch, effects, compact disks (CD), 586–587 ligands, 354 404–413. See also, Spherical digital video disks (DVD), 587–588 lipid, 790, 791 aberration. floppy disks, 586 proteins, 357 table for glycerol, 409 magnetic disks, 586 Streptococcus, 879 table for water, 409 MO (magneto-optical) disks, 586 transferin, 819 calculation, 404–407 optical disks, 586 uncaging, 545 dependence of focal shift, 410 WORM (write once, read many) disks, Reconstruction, 3D. diagram, 404 586 definition, 280. dry objectives, 410–411 Rendering, of 3D views, 280, 285, 290, 301, Nyquist and filtering/deconvolution, 59, experiments, 409–410 307, 309, 311, 377, 749, 762, 764. 66–67, 69, 70, 173, 235–236, water/glycerol results, table, 410 definition, 280 280–315, 458, 468–469, 474–475, field strength calculation, 405 voxel speed, 290 496–497, 563, 585, 603, 607, 610, other considerations, 410–413 RESOLFT microscopy, 571–574, 577. See 615, 635, 672, 675, 677–678, 690, spherical aberration correctors, 15, 151, also, STED. 772, 730, 731, 762, 77, 774–776, 147, 192, 411–412 breaking diffraction barrier, 571–573 778, 784, 883 terminology, 405 concept, 571–573 Recording times, 141–142. actual focal position (AFP), 405 different approaches, 573–574 in widefield microscopy, 141–142 focal shift, 405 ground state depletion (GSD), 573 using LED source systems, 141–142 nominal focal position (NFP), 405 STED, 573–574 Recovery curve, after bleaching, 187. theory, 404–407 outlook for, 577 Red fluorescent protein (RFP), 221–222. Region code, for MPEG-encoded movies, resolution, new limiting equation, 571 Reference list. 844. triplet-state saturation, 573 historic, 889–899 Region-of-interest (ROI), 835. Resolution, 1, 4, 13, 16, 24, 36–41, 59, 61, lasers, 123–125 brain slice, 726, 733 65–67, 210. See also, PSF; FWHM. Reflected-light images, 180, 181. See also, diagonal, 658 adequate levels, 36–41 Backscattered light. display presentation, 835 axial, 13 confocal, of integrated circuit, 180 embryos, 747, 759 axial-to-lateral ratio vs. NA, 4 of glass bead, in water, 181 FRAP, 51, 187 back-focal plane diameter, table, 210 Reflecting objectives, constraints, 156. FRET, 797, 801 confocal vs. non-confocal, 16 Reflection contrast technique, Antiflex, 159. in image processing, 289, 300, 323, 330, and contrast transfer function, 37, 59, 61 Reflection mode, low coherence light, 130. 676 estimating, 65–67 Reflectivity, optical surfaces, 159, 163, labeling, 353 measured, widefield, 16 167–171. must be smaller at high resolution, 577 minimum resolvable lateral spacing, 1, 16 anti-reflection coatings, 158 nanosurgery, 219, 686 spatial and temporal, 24 on-axis, artifact, 168–171 photobleaching, 690 sufficient, 36–37 refractive index, 159, 163, 167 preprocessing, 676 Resolution, structured illumination. Refracting regions affect imaging beam, rapid acquisition, 236–237 Fourier-space, 270–271 15–16. structured illumination, example, 272 linear image reconstruction, 271 Refractive index, (RI), 14–15, 23, 45, 148, viability studies, 683 Lucosz’s formulation, 273 152, 163, 198, 377, 404–413, Registration synthesis method, 328–331. methods, 270–276 418–420, 613, 654. See also, defined, 328 Moiré effects, 270–271 Spherical aberration; Dispersion. landmark-based, 328–329 photobleaching, 275 anomalies in, effect on PSF, 23, 418–420 multi-view deconvolution, 330 reconstruction results, 272 of biological structures, 163, 377 Relationships, in fluorescence microscopy, standing-wavefield microscope, 275 table, 277 80. thick samples, 274, 275, 278–279 of botanical specimens, 418–420 energy per photon, 80 Resolution scaling, STED comparison, 578. coverslip thickness, importance, table, flux per pixel, 80 Resolution test slides, 16, 656. 654 photons/s vs. wavelength, 80 Resonant cavity, laser, 81–82, 111, 115. of immersion medium, 277, 411 Relative motion, objective vs. specimen, Resonant scanners, 52–54, 56–57, 223, 447, effect on PDF, 23, 418–420 39–40. 543. effect on sharpness, 14–15 Relaxation, in laser energetics, 82. acceleration distorts mirror shape, 53 effect of wavelength and temperature Relay optics (telan lenses), 145, 157, 214, blanking, 25, 218, 338, 389, 543, 628, on, 148, 248–249, 411 455. 651, 908 and intensity, and spectral broadening, Reliability. compared to acousto-optical deflector, 56 111 of 3D image, 461, 517 duty cycle, 52 of layers in interference filters, 45 biological, vs. damage, 24, 68, 631, 633 galvanometer, 52 of mounting media, table, 198, 342, lasers, 80, 102, 115 multi-photon excitation, 543 370–371, 373–377 living cell work, 387 raster-scanning, 33, 53–54, 56 976 Index

Resonant scanners (cont.) S Scanners, 51–55, 57, 214–216. retrace, 54, 56. See also, Retrace, below. Safety, 83, 85, 90, 115, 117–118, 124, acousto-optical deflectors, 55. See also, scan speed, 54 132–139, 900, 903, 904. AODs Retrace, raster scanning shape, 25, 54, 56, arc sources, 132–139 mirror arrangements, 214 219, 389, 543, 628, 651, 908. beam-stop design and use, 118. 903–904 evaluating, 215–216 acousto-optical deflector, 56 classification of laser systems by hazard, mechanical, 51–54. See also, blanking, 25, 219, 338, 389, 543, 628, 117 Galvanometers 651, 908 cleaning objectives, 642 piezo-electric, 57, 215, 238, 510, 555, raster-scanning, 33, 53–54, 56 display geometry, 297 610 Review articles, listing, 889. equipment needed, 900 single mirror/double tilt, 215 RFP. See Red fluorescent protein. eye protection sinusoidal, “tornado” mode, SIM scanner, Rhodamine, dyes, 81, 109, 116, 136, 140, against Brewster surface reflections, 83 52 203, 264, 292, 339, 342–345, 353, goggles, 118 Scanning electron micrographs, 428, 434, 355, 362–363, 375–378, 409, 538, with external-beam prism method, 90 437, 846–848, 850–852. 553, 592, 693, 697–698, 762, fiber optics for transporting laser light, Scanning laser ophthalmoscope (SLO), 480. 783–784, 794, 851, 854–856. 88 Scanning fiber-optical microscopy. See arsenical derivatives, 348 hazardous materials Fiberoptic confocal microscope. bleaching, 697, 698 fluorescent laser dyes, 85, 103, 116 Scatter labeling for tracing lineage, 461, calibration plot, 661, 851 used beryllium oxide tubes, 115 462. excitation of, 181, 109 high pressure Xe lamps, 136 Scanning systems for confocal light fluorescence correlation spectroscopy, monitor power to avoid explosions, microscopes. See also, 693 138–139 Galvanometers; Disk-scanning FRET, 347 in disk-scanning confocal microscope, confocal microscopy; Acousto- photobleaching quantum yield, 363 231 optical deflectors; Linescanning planar test specimen, 538 laser, 117–118, 839, 900, 903–904 confocal microscopes; Raster. power for 1-, 2-photon excitation, 81, 3 installation requirements, 85 Lissajous pattern, circular scanning. 554 41 monitor power to avoid explosions, “tornado” mode, SIM scanner, 52 Rhodamine-123, 374, 389 138–139 Scattering, 162–163, 167–171, 550. resolution measurement, 409 references, list, 123 coherent anti-Stokes Raman (CARS), 550 stability and cost, 116 safety curtains, 117, 904 elastic, Rayleigh, 162–163, 166–167, 339, Rice (Oryza sativa), 168, 171, 414, 415, training, 118 342, 417, 703–747 712, 715. SAM, saturable absorber mirror, 111. Raman, 162, 167, 339–340, 348, absorption spectrum, 415, 706 Sampling. See Digitization, 20, 63–64. 506–507, 545 and reflection contrast, backscattered light image, 168, 171 non-periodic data, 38 167–171 emissions spectra, autofluorescence, 713 optimal, 63 Scattering object, viewed by TIRM 177. See leaf fluorescence images, 714–715 Saponin, formaldehyde fixation, 359, 375, also, Backscattered light. light attenuation in plant tissue, 414 856. Schiff reagents, 262, 369, 770, 774–775, silica deposits, 714–715, 717 Saturable absorber mirror, pulsed lasers, 778. Richardson-Lucy, deconvolution, 497, 568. 111. Schottky diode, photodetector, 253. Richardson Test Slide Gen III, 652, 656. Saturable Bragg reflector (SBR), 111. Scientific thought, four aspects, 789–790. RLE. See Run-length encoding. Saturable output coupler (SOC), 107, 111. Scion Image, 281–282, 395, 730. RNA, microinjection of, 803, 804. Saturation, singlet-state fluorescence, 21–22, Scramblers, light, 8, 13, 84, 131–132, 143, RNA labels, 344, 369, 465, 531–532, 612, 41, 142, 265, 276, 339, 442, 448, 507. 691, 758, 874–875. 450, 643, 647, 899. Screen capture, 830. ROI. See Region of interest. performance limitations, 81, 450, 928 Screens, to enclose laser beams, 118. Room light, as stray signal, 201, 632, 904. SBR, saturable Bragg reflector, 111. SD. See Static discharges. Roots, plants, 172, 174, 303, 307, 421, SBT. See Spectral bleedthrough. SDA. See Stepwise discriminant analysis. 430–432, 438, 464–465, 556, Scaling techniques, 592, 835. Sea urchin, S. purpuratus, 173, 198, 200. 772–773, 775, 777, 779–783. Scan angle, and position in image plane, Second harmonic generation (SHG), 90, maize, image, 432 209–210. 114–115, 166–167, 179, 188, 550, mounting, 429, 431 Scan instability, detecting, 40–41. 552, 556, 703–719, 729–730. See ROS. See Reactive oxygen species. Scan raster, testing, 651–654. also, Harmonic signals. Rose Criterion, 37–38, 68, 164, 633. malfunctioning system, 653 as autofluorescence, 361 relationship with signal-to-noise ratio, phototoxicity from uneven scan speed, cell chambers, 166, 429, 552 164 651 detectors, 706–708, 728 for visibility, 37 sources of fluorescent beads, table, 653 disk-scanning, 552, 556 Rotating, specimen, 188, 568, 835. well-calibrated system, 652–653 double-pass detection, 166–167 micro-CT, 615 x and y galvanometers, 651–652 table, 706–708 optical projection tomography, 610–611 z-positioning calibration, 652, 654 crystals for SHG, 103, 107, 114–115, 188, SPIM, 672–673, 676, 751 stability, 652 703 Rotor, galvanometer, detecting position, Scanned-slit microscopes, table, 224. energy relations, 705 53–54. Scanner arrangements, evaluation, in lasers, 103, 107, 114–115 Run-length encoding (RLE), 580. 213–215. layout, 166, 191, 552, 708–709, 712 Index 977

light attenuation spectra, 706 for plant cells, 774–777 SFP. See Simulated fluorescence process. light sources, 706–708 balloon model, 776 Shannon, Claude, 64–65. brain slices, 729–730 watershed algorithm, 322–325, 777, Shannon sampling frequency, defined, 64, non-linear optical microscopy, 704–705 822 443. optically active animal structures in, region-based, 321–322 SHG. See Second harmonic generation. 714–717 top-down, 322 Shift invariance, deconvolution, 457, 490, brain slice, 729–730 tube-like object segmentation, 324–328 564. collagen structure, 703, 717 mean/median template response, 328 Short-pass filters, 43–44. sarcomeres, 716 skeletonization methods, 324–325 Shot noise, 232, 256–257, 286, 442, spindle in mouse zygote, 717 vectorization methods, 324–327 460–461, 495, 558, 660–661, 831. spindle in zebrafish embryo, 718 validation/correction, 333–334 See also, Poisson noise, Quantum structures producing SHG, table, 715 manual editing, 333–334 noise. table of structures, 715 Selective plane illumination microscopy Signal, 27, 62. See also Speed relationship zebrafish embryo, 716, 718 (SPIM), 613, 614, 672–679, 751. to magnification, 62 optically active plant structures, 428, 3D scanning light macrography, 672 Signal attenuation-correction, 320–321. 710–714 anisotropic resolution, 678 Signal detection, basics, 660–663, 918–931. Canna, nonlinear absorption, 710 applications, 675 See also, Detectors. cell wall, 428, 711, 714 axial resolution, 674–675 coefficient of variation, 660 Commelina communis, 712 vs. CLSM, 678 instrument dark noise, 660 emission spectrum of maize, 710, 711 Drosophila embryogenesis, 675–676, photon (shot) noise, 660–661 Euphorbia pulcherrima, spectrum, 710 747–748, 751–752, 754, 756, 759, PMT linearity, 661–662 mineral deposits, 436 804, 810 signal-to-noise ratio, 660 Pyrus serotina, spectrum, 711 and FLIM, 527 spectral accuracy, 662 rice leaf, 712, 715 images processing, 675–678 spectral resolution, 662–663 starch granules, 433 image fusion, 676–677 wavelength response, 663 maize, 710–711, 713–714 pre-processing, 676 Signal levels, 16-photon peak signal, 73–74. emission spectrum, 710–711 registration, 676 Signal-to-background ratio, of titanium- leaf spectrum, 710 lateral resolution, 674 sapphire laser, 112. pol-microscopy, 711 light-sheet illumination, 672–674 Signal-to-noise (S/N) ratio, 37, 53–54, 67, stem, optical section, 714 light sheet thickness, 674–675 81, 164, 251, 257, 265, 330, 340, stem, spectrum, 710, 713 Medaka, 614–615 386, 391, 442–451, 470, 481, 495, chloroplasts, tumbling, 713 heart image, 614 498–499, 528, 542, 562, 567, 582, membranes of living cells, 90 embryo image, 675 599, 621–622, 660, 690, 696, 699, mineral, deposits, 436 multi-view reconstruction, 675–678 707, 736–737, 740, 753, 769, 772, photodetector suitability, table, 706–707 point spread functions (PSF), 674 778–780, 810, 813. polarization dependence, 71, 717–720 schematic setup, 613, 673 3D imaging, 448–451 potato, as SHG detector, 712 thin, laser light-sheet microscope,TLLSM, 4Pi microscopy, 562–567 pulsed laser suitablity, table, 706 672 bleaching, 391, 442, 690, 696 signal generation, 179, 552, 597, 704–705 Self-aligning arc source, 135. in calcium imaging, 737 spectra, 706 Self-shadowing, 165, 174, 194, 195. chapter, 442–451 spectral discrimination, 421 in confocal optical sections, 174 comparative performance, 256, 448–451 starch granules, 433 spherical structure, 195 bleaching-limited performance, Segmentation, FLIM image, 527–528. in epi-fluorescent mode, 165, 194 448–450 Segmentation methods, 281, 283–285, 290, SEM. See Scanning electron microscope. configurations of microscope, 448, 449 300–302, 304–306, 309, 311–312, Semi-apochromat, pros and cons, 158. disk-scanning microscope, 449 316–319, 321–330, 333–334, Semiconductor lasers, 86, 105–108. line illumination microscope, 449 527–528, 776–778, 812. noise sources, 86 saturation-limited performance, 450 3D, 776, 822, 828 Semiconductor nanocrystals (quantum dots), scanning speed effects, 53, 450–451 automated, 818, 821–822, 828 221, 343, 357–358, 360–361, 656, structured illumination, 265–266, 270, background, 321 694, 696, 757, 759, 801, 814, 846, 275–276, 279–280 blob segmentation example, 322–324 853. S/N ratios for, table, 450 gradient-weighted distance transform, as probes, 221, 757, 759 widefield (WF) microscope for, 450 323 Semiconductor saturable absorber mirror confocal microscope, 444–447, 660 model-based object merging, 323–325 (SESAM), 107, 111. calculations, 444 watershed algorithm, 323–324 for self-starting intense optical pulse detectability, 446–447 bottom-up, 321 trains, 111 methods compared, 450 combined blob/tube segmentation, Sensitivity, video photodetectors, 6–7. noise model N1, 444–445 328–330 Sensitized emissions, of acceptor, 795–796. noise model N2, 446–447 foreground, 321 See also, FRET. deconvolution, 470, 481, 495, 498–499 hybrid bottom-up/top-down, 322 Sequential devices, 585–586. designs, confocal, 212–216, 447–448, 450 integrated, 322 Serial sampling, single-beam confocal, 20. disk-scanners, 221 intensity threshold-based, 321 SESAM, Semiconductor saturable absorber dynamic range, 2-photon, 644, 778–780 object, 321 mirror, 107, 111. high-content screening, 810 978 Index

Signal-to-noise (S/N), ratio (cont.) Sinusoidal modulation, in FLIM, 524–526. Spatial laser beam, characteristics, 89. improvements, 736 SIT. See Silicon-intensified target camera Spatial light modulator (SLM), 266. micro-CT, 615 imaging. Spatial orientation factor, for FRET, magnetic resonance microscopy, 621–622 SLF. See Subcellular location features. 792–793. multi-photon fluorescence microscope, Slice AM-dye-painting protocol, 726–727. Spatial resolution, in confocal microscopy, 112, 427, 447, 542, 779 Slice chamber protocol, 727. 24. See also, Resolution, PSF, CTF. Nyquist sampling, 67, 448 Slit scanning confocal, 12, 25, 37, 50, 51, Special setups, for CLSM, 218–219. optimal excitation power, 81, 340 56, 221–226, 231+, 235, 238, 519, Specifications, general, for scanner, 54. Rose criterion, visibility, 37–38, 68, 164, 522, 664, 741, 914, 916. Specimen, general considerations, 192–197, 633 Achrogate, 50, 212, 231–232, 916 228, 361–362, 779. See also, Living saturation, 442 with AOD scanning, 56, 914 cells, Living embryo imaging. vs. scan rate, 53 commercial, 913–914, 916 fluorescent probes interactions, 361–362 signal level, 67, 75, 528 critical parameters, 224–228 cytotoxicity, 362 sources of noise optical sectioning, 228, 444–449 localization, 361–362 background noise, 443–444 optimal slit size, 225 metabolism, 361–362 grey levels, 443 point excitation, slit detection, 914 perturbation, 362 quantum efficiency, 442–443 SLM. See Spatial light modulator. optical heterogeneity, 22, 23 shot noise, 442–443 SLT. See Subcellular location tree. plants. See Plant cell imaging; Botanical sources of noise, 442–444 Smart media, digital storage, 588. specimens STED, 574 SMD. See Surface mount device. Specimen chambers. See Living cell and visibility, 37 SNARF-1, 345, 346, 531, 739, 744–745. chambers. Silica glass, transmission losses, 502. ratiometric pH label, 744–745 Specimen heating, in 2-photon, 539. Silicon diodes, near infrared emission, 132. stained rat interossi muscles, 739 Specimen holder, for scanning specimen, 9. Silicon-intensified target (SIT) camera, 730. table of variants, 531 Specimen preparation, for automatic 3D brain slices, 730 Snell’s law of refraction, 167, 654. image analysis, 319–321. SIM. See Surface imaging microscopy. SOC. See Saturable output coupler. image analysis, 319–321 Simplicity, as design goal, 43, 66, 220, 229, Software packages, visualization, table, imaging artifacts, 320 387, 508, 647. 282–283. Specimen preservation, general, 368–378. Simulated fluorescence process (SFP), 310. SoftWorx, 3D display software, 282. antibody screening on glutaraldehyde- Single-cell automatic imaging, 809, 812. Solanum tuberosum, potato, 712. fixed specimens, 377 Single-cell calcium imaging, 812. Solid state memory devices, 588. evaluation, 371–374 Single-longitudinal-mode fiber laser, 110. compact flash cards, 588 cell height to measure shrinkage, Single-mirror/double tilt scanner, 215. memory stick, 588 371–373 Single-molecule, 80. smart media, 588 defined structures, distortion, 373–374 biochemistry, 221–222, 575, 690, 693, Solid-state photodetector, 30–31, 918–931. MDCK cell, stereo image, 373 696 See also, CCD; EM-CCD. MDCK cell, vertical sections, 372 bleaching, 690, 693, 696, 697–698, 699 Solid-state lasers, 86, 103–118, 236–237. fixation/staining, 370–371 Single-photoelectron pulse heights, 30. cooling, 108 fixative characteristics, 368–370 Single-photon, energy, equation, 35. noise sources, 86 chemical fixatives, 369 Single-photon counting avalanche thin-disk lasers, 109 cross-linking fixatives, 369 photodiodes (SPAD), 527. tunability, 109 freeze substitution, 369 Single-photon excitation, plant imaging, use, 236–237 microwave fixation, 369 772–778. Source brightness, measure, radiance units, protein coagulation, 369 Single-photon pulses. See Photon counting. 126. formaldehyde, 369–370, 373 Single-scan images measure scan stability, Source optics, reflecting and collecting light, general notes, 374–378 40–41. 134. glutaraldehyde, 369, 370 Single-sided disk scanning, confocal Space invariance, telecentric systems, immunofluorescence staining, 371 microscopy, 132, 141–142, 168, 171, 207–208. improper mounting, 376 175, 215–216, 229, 231, 907, 913. Space multiplexing, in MMM, 555. labeling thick sections, 376–377 See also, Disk-scanning confocal Spacer, material in interference filters, 46. microwave fixation, 377–378 microscopy. SPAD, single-photon counting APD, 527. mounting methods, 370–374 advantages and disadvantages, 215–216 Spatial coherence, 84. critical evaluation, 371–374 basic description, 141 Spatial filter, 89, 107, 391, 542, 708, 729. mounting media, table, 377 commercial, 907, 913 optical devices for, 89, 222–223, 729 pH shift/formaldehyde fixation, 370–371, light source, 132, 141–142 digital, 391–392. See also, Gaussian 373 Singlet state saturation, 21–22, 41, 81, 142, filtering refractive index mismatch, 377 265, 276, 338–339, 442, 448, 450, Spatial frequency, 37, 60, 65, 66. See also, mounting media, table, 377 643, 647, 899, 928. CTF. refractive index of tissue/organs, table, Sinusoidal bidirectional scanning, 25, and contrast transfer function, 37 377 52–54. See also, Resonant scanners. and geometry, 66 tissue preparation, 376 duty cycle, 53, 260 response of microscope, and pixel size, triple labeling, 375–376 Sinusoidal image, 831, 838. 65 Specimen-scanning confocal microscope, fiber-optic confocal, 510 zero, as measure of brightness, 60 9. Index 979

Speckle, from high-coherence sources, 8, disk-scanning confocal, 141, 216, 224, Spinning filter disk, digital projector, 590. 84, 90, 130–132, 448. 754 Spirogyra, and depth of optical sections, Speckle microscopy, 13, 383, 385, 889. for display, processing, 803, 839, 841- 195. Spectra, emission. 842, 862 Spot scanning, to avoid coherence effects, arcs, 130 factors affecting, 235–236, 482, 496, 84. black body, 136 753–754 Spot size, full-width at half-maximum. See LEDs, 133 of fixation, 370 Pointspread function, Full-width solar, 127 FRET, 795, 805 half-maximum. tungsten source, 153 galvanometer, 52–54, 211, 214 Square pixels, advantage of using, 62. Spectral accuracy, 662. high-content screening, 809–810, 813 Stability, 86, 102, 103, 136–139, 826. Spectral bleedthrough (SBT), 185, 203–204, MMM, 551–555, 563–564 algorithmic, 473 664. need for, in living cell imaging, 222, arc sources, 136–137, 477 in intensity-based FRET, 185 753–754 argon-ion laser vs. krypton laser, 102 Spectral confocal image A. thaliana rendering, 3D display, 831 disk scanners, 215 seedling, 175. SPIM, 613, 678 of DVDs, 587 Spectral detector, 203–204, 662–663, Spermatocyte, crane fly, 15. dye. See Dyes; Bleaching from fiber-optic 666–667. Spherical aberration, 15, 34, 147–149, 151, coupler, 505–506 testing, 662 160, 192–197, 208, 241, 244, 247, galvanometer, 54 Spectral discrimination, filter bandwidths, 330, 395, 404–413, 454–455, 463, halogen sources, 136–139, 346 44. 466, 471, 480–481, 542, 629, 640, interferometer, 240–241, 267 Spectral imaging, 175, 382, 384. 654–655, 657–658, 728, 772, 774, laser, 81, 85–89, 704 table, 384 893, 903–904. See also, Aberrations, diode, 106, 108–109 Spectral leakage, inter-channel signal spherical. fiber output, 505 imbalance, 185, 203–204. blind deconvolution, 471, 480–481 helium-cadmium, (low), 103 Spectral phase interferometry, for direct chapter, 404–413 intensity, 85–87, 113, 116, 136, 477, electric field reconstruction confocal microscopy performance, 654 903 (SPIDER), 115. correction of RI mismatch, 192, 287, 411, measurement, 650–651 for pulse length measurement, 115, 542 pointing, 87, 903 901–903 correction of, figure, 145, 411–412, results, 86, 103 Spectral properties, of filters vs. angle, 49. 654–655 structure, 82–85, 103 Spectral resolution, of detection system, corrector, 92, 395, 398, 411, 477, 640, thermal, 111 203–204, 662–663, 666–667. 654 wavelength, 106–108, 115, 118 Spectral response. deconvolution, 463, 466, 468–469, 471, mechanical, 39, 82, 85, 201, 267, 512, of CCD chips, 29, 234, 922 480, 498–499, 658, 728, 784 652 of eye, 153 effect of specimen, 192–197, 418, 454, objectives, 146 PMT photocathodes, 29 747 photostability, 363, 369, 690–702, 802. Spectral transmission, objectives, plots, index mismatch. See Index mismatch See also, Dyes; Bleaching 159–161. measurement, 145, 407, 455, 471, scan, 40, 638–639, 651 Spectral unmixing, 190–192, 319, 361, 382, 481, 492, 657 shutter, CCD camera, 929 384, 386, 423–425, 431, 664–667, signal loss, 330, 389, 395, 413, 457, 661 thermal, 111, 219, 387, 389, 394, 539. See 770, 905. SPIDER, Spectral phase interferometry for also, Thermal variables detectors for, 51, 667 direct electric field reconstruction, Stage-scanning confocal microscope, 11. examples, 665–666 115, 901–903. piezoelectric scanners, 57, 708 limitations, 51, 382, 667 Spill-over, between detection channels. See Staining, plants, 438, 774. See also, Dyes; overlapping fluorophore emission, 190, Bleedthrough. Livingcells; Botanical specimens; 319, 423–425, 664–667 SPIM. See Selective plane illumination Plant cell imaging; Fluorophors. removing autofluorescence using, 667 microscopy. calcofluor procedure, 438 Spectrofluorimetry, for FRET, 793, 795. Spinning disk, 3, 5–6, 11, 40, 141, 176, 216, of plant tissues, 774 Spectroscopic ruler, 765. 223–224, 231–232, 235–236, Standards, ISO (DIN) microscope design, Speed, in confocal imaging, 7, 11–12, 36, 260–265, 459–460, 464, 468, 156+. 41, 53, 142, 222–224, 235–236, 434, 481–483, 783–784, 810–811. See Standing-wavefield microscope, 275. 447, 450, 458, 460, 482, 526, 536, also, Diskscanning confocal Starch granules, plant, 202, 420–421, 428, 563–564, 748, 753–755, 784. See microscopy. 432–433, 435, 703, 710–712, 715, also, Temporal resolution. commercial, 907, 913, 915 719. 4Pi-MMM, 563–564 FLIM, 519–520, 522 Static discharges, destroy semiconductors, AOD, 55–56 high-content screening, 810–811, 820 109. calcium imaging, 741 MMM, 554, 558 Statistical noise, in counting quantum- CARS, 599–600, 604 performance, 449–450 mechanical. See Poisson noise. charge-coupled device cameras, 77–78, systems for, cytomic imaging, 810 STED. See Stimulated emission depletion. 142, 229, 231–235, 259, 647, 651, vs. TPE imaging, in plant cells, 783 Stem-cells, 623, 678, 762, 790, 813. 754–755, 885 Yokogawa CSU-10/22, 231. 915 Stem, plant, 168, 172, 180, 417–419, 421, data compression, 581–582, 586–588 Spinning-disk light scrambler, ground glass, 424, 430, 556, 707, 710–711, detector, in FLIM, 523, 558 8. 713–714. 980 Index

Stentor coeruleus, backscattered light image, Stimulated emission of radiation, defined, pattern generation, 266–268 168. 82–83, 124. schematic setup, 266 Step index optical fibers, 501–503. Raman scattering, 167 nonlinear, 276 Stepwise discriminant analysis (SDA), 818, semiconductor, 106 resolution improvement, 270–276 820. and stimulated-emission depletion, 573, Fourier-space, 270–271 Stereo Investigator, software, 282. 577 linear image reconstruction, 271 Stereology, 316, 319. STN, supertwisted nematic, 589. Lucosz’s formulation, 273 Stereoscopic image, about, 6–7, 9, 11, 154, Stochiometry, ion kinetics, 741. Moiré effects, 270–271 224, 298–299, 317, 396. Stokes field intensity, 595, 597. photobleaching, 275 biofilms, 880 Stokes laser, in CARS microscopy, 595, reconstruction steps/results, 272 cheek-cell specimen, 23 597–604. standing-wavefield microscope, 275 diatom, 640 Stokes shift, 44–45, 268, 338, 341, 343, test results, 274 Drysophila, microtubules, 752 443–447, 539, 542, 690, 759, thick samples, 274, 275, 278–279 embryo, 200 792–793. Subcellular location features (SLF) in fat crystal, polarization, 479 anti-Stokes, automatic image analysis, 819–820, neurons, 298, 314 CARS, 550, 595–604 822–824, 828. Alexa stained, 330 defined, 44–45 2D, 819–820 backscattered light images, 167 in fluorescence resonance energy transfer, 2D SLF feature descriptions table, 819 eye, optic nerve, 481 792+ 3D SLF, 822–823 Golghi-stained, 298 large, in 2-photon, 539, 646 test results, table, 824 Lucifer-yellow, 314 of quantum dots, 694, 759 Subcellular location tree (SLT), 825. microglia, 396–398 size of fluorophores, 45 Subpixel deconvolution, 478–479. rat-brain neurons, 398 Storage, digital. See Data storage. Subresolution beads, 655–656. See also, transmitted light, 475 Storage structures, plant, 435–436. Beads. lung, 292 maize, image, 436 Sun, microscope light source, 126–127, 131, MDCK cells, 373–374, 378 Stray light, 58, 632, 904. 135. Milium chromosomes, Fuelgen-stained, laser light, 632 spectrum, 127 298 non-descanned detection, 904 Superficial optical sections, living embryo, Paramecium, chromosomes, 298 practical confocal microscopy, 632 748. pea root, RNA transcript, 465 room light, 201, 632 Supertwisted nematic (STN), 589. platelet, high-voltage, EM, 848–849 Streak camera, FLIM detector, 520. Surface imaging microscopy (SIM), sea urchin, S. Purpuratus, 173, 198, 200 Strehl ratio, measure of image sharpness, 607–608. skin, 298 247. mouse embryo, 608 Spirogyra, 195 S. purpuratus (Sea urchin), 173, 198, 200. setup, 608 tandem-scanning confocal microscope, embryo, 173, 198, 200 Surface mount device (SMD), for LED, 6 first mitotic division, 173 133. Stereoscopic views, image processing and image degradation, from top and Surface orientation, affects reflected light, display, 290, 292, 293, 295–299, bottom, 198 181. 451, 764. stereo-pairs of embryo, 200 Surface structures, distortion, 197. color space partitioning, 297 Structural contrast, 188. See also, Harmonic Surface topography, maximum intensity, display, 293, 299 signals. 180. interlaced fields of frame, 297 Structure, optical, 59, 68, 132–135. Surfaces, of interference filters, 47. movie projection, 838 of light-emitting diodes (LED), 133 Suspension-cultured cells, 189, 429–430. pixel-shift/rotation stereo, 297 of microscope sources, 132–135 bacteria, 876, 878 stereo images example, 298 recognizing features in noisy images, 68 image, 430 synchronizing display, 297 chapter, 265–279 frozen, 854 Stick objective, for in vivo confocal, 806. Structured illumination microscopy, Swept-field confocal microscope, 238. Stimulated emission depletion (STED) 265–279. Synchrotron, wide-spectrum light source, microscopy, 3, 539, 561, 568, advantages/disadvantages, 265 135+. 571–578. computing optical sections, 268–270 Synthetic pinholes, in structured- axial resolution increase, 576 vs. confocal microscopy, 265 illumination breaking the diffraction barrier, 571–573 degree of spatial excitation modulation, microscopy, 268, 269. challenges, 577 268–270 images, 269 compared to confocal, 575–576 absolute magnitude computation, SYTO, 396, 874–876, 879–885. diagram, 573 268–269 different approaches, 573 homodyne detection scheme, 268–269 T dyes used successfully, table, 575 max/min intensity difference, 268 Tagged image file format. See TIFF. OTF compared to confocal, 578 scaled subtraction, 269–270 Tandem-scanning confocal microscope outlook, 577 square-law detection, 268–269 (TSM), 2–6, 11, 13–15, 39–40, 141, PSF compared to confocal, 578 synthetic pinholes, 268, 269 167, 215–216, 223–224, 228–229, RESOLFT, the general case, 572–573 experimental considerations, 265–268 447. results, 576, 578 illumination masks for, 266 comparison with other confocals, triplet-state, 573 light source for, 267 13–15 Index 981

description, 6, 141, 215–216, 228–229 cell chambers, 117, 386–389, 394, 727, CARS, 596–597 development, 5–6 790, 810, 814, 885–886. See also, contrast mechanism, 166–167 evaluation, 215, 216 Cell chambers cooling, 108, 133 deposits no energy, 361 observing ciliate protozoa, 141 cryo preparation for EM, 856–857 detectors for, 421, 706–708 rate of data acquisition, 11 on detectors, 29, 252, 256–257, 495 table, 707 real-time imaging of tooth, 167 drift, 16, 115, 219, 386, 567, 489, 652 double-pass detection method, 166–167 sources of vibration, 39–40 compensating, 396, 732 intracellular inhomogeneities tracked, viewing color/depth-coded, real-time, on dye labeling, 359, 361, 738–739 90 stereo effects of anti-bleaching agents, 694 light attenuation spectra, 706 images, 154, 304 effect on bleach rate, 696–689 light sources, 706–708 Tapetum, plant, 433, 434, 779. effect fiber pinhole size, 506 to make more laser lines, 109, 114 TEC, Thermo-electrically cooled, see Peltier fiber-optic, pol-preserving fiber, 503 mechanism, 705 cooling. filament spectra, 135–136 microspectroscopy, 421 Telan systems, 129, 157. fixation, 369–372, 375, 377 MMM, 551, 559 Telecentric plane, 208–209, 211. incandescent lamp emission, 135–136 non-linear optical microscopy, 705 conjugate, 208–209 laser cavity, 34, 82, 85–88, 107, 109, 111, optical sectioning, 704 effect of angular deflection in, 211 541 optically active animal structures, Telecentricity, 207, 214. of LED, 133, 136–138 714–717 of closely-spaced scan mirrors, 214 brightness, 133 collagen mat, polarization microscopy, defined, 207 lensing, in pulsed lasers, 109, 113, 543 717

Tellurium oxide (TeO2), for use in AODs, and light-source output, 136, 138, 650 mouse zygote spindle, 717 55 noise signal, 254, 257, 232–234, structures producing THG, table, 715 TEM. See Transmission electron 261–262, 495, 660, 734, 921, 924, zebrafish embryo, 716, 718 microscope. 925 optically active plant structures, 710–714 TEM. See Transverse electromagnetic on objective lenses, 248–249 cell walls, 438 modes. in photography, 71 Commelina communis, 712 Temperature, 29, 56, 133, 135–136, 856, properties of ice, 856 Euphorbia pulcherrima spectrum, 710 885. See also, Thermal variables. properties of optical materials, 158, maize, emission spectrum, 710, 711, Temperature tuning, of diode lasers, 108. 248–249 713 Temperature effects on high NA objectives, and photomultiplier tube, (PMT), 29 maize, polarization microscopy, 711 248+. on refractive index, 15, 56, 145, 411 maize, stem section, 714 Temporal aliasing, 39, 41, 391, 836–837, immersion oil, 148–149, 248–249, phytoliths, polarization microscopy, 839. 411 720 Temporal coding, 299–300. retinal exposure, 117–118 potato, 712 Temporal coherence, 7–8, 82–85, 131. sensors, 255–256, 727 Pyrus serotina spectrum, 711 defined, 84 solid-state laser, 86, 108 rice leaf, image, 712, 715, 719 Temporal dispersion, 502. See also, Pulse specimen damage, 84–85, 139, 685 photon interactions, 179 broadening. specimen heating, 539, 545, 681, 685, pulsed lasers suitable, table, 706 Temporal displays, 292–293, 297, 836. 904 STED, 577 Temporal experiments, biofilms, 885–886. temperature tuning, laser, 108, 115 structural contrast, 188 Temporal pulse behavior, pulsed laser, 111. thermomechanical effects, 685 Three-decibel point (3dB), for bandwidth, See also, Pulse length measurement; time constant, 38 59, 65. Pulse broadening. Thermo-electrically-cooled, see Peltier- Three dimensional cell pellet, 815. Temporal resolution, 12, 24, 36–38, 41, cooled. Three dimensional microscopy, 766, 771, 221–222, 322, 334, 386, 391, 399, diode lasers, 85, 107–108, 111, 117 804+. 458, 558, 577, 618, 620, 622, 651, THG. See Third harmonic generation. future perspectives, 804–805 667, 730, 737, 746, 772, 784, 801, Thick samples, 274, 275, 278–279. See also, living embryos, 766 809. See also, Fluorescence lifetime Living embryo imaging; Brain slices; of plant cells, 771 imaging (FLIM). Biofilms. Three dimensional projections, embryo, 763. of photodetectors, 263 background, 278 Three dimensional segmentation, plant, Temporal signals, 162, 286, 331, 383. structured illumination, 274, 275, 776–778. “Test drives,” for living embryo imaging, 278–279 Three-channel confocal microscopy. 752. close focus region, 279 with 4 recombinant proteins, 190 TFT. See Thin-film transistor. distant focus region, 279 assays for, 814 Tetracysteine, labels, 221, 348, 359, 853. in focus region, 278 Three-dimensional diffraction image, 4, 147, Thalamocortical slice protocol, 724. number of collected photons, 279 407, 455, 463, 471, 491. Thermal lensing, pulsed lasers, 109, 113, Thin disk lasers, 109–110. Three-dimensional micro-array assays, 543. Thin Laser Light Sheet Microscope 815–816. Thermal variables, 219, 856. (TLLSM), 672. See also, SPIM. Three-dimensional reconstruction, 775–776, active medium, lasers, 81 Thin-film transistor (TFT), 589. 778, and Chapters 14 and 15. of AODs, 56–57 Third harmonic generation (THG), 90, plant imaging, 775–776 arcs, peak emission wavelengths, 129 166–167, 179–180, 188, 428, 435, A. thaliana, 778 automated confocal imaging, 810 550, 705–718. Equisetum, 774 982 Index

Three-photon excitation (3PE), 88, 415, 447, 4Pi, 563–564, 567 speed, S/N, sensitivity and damage, 221, 535, 550–552, 555, 558, 647, 680, brain slices, 731 224, 232, 556, 644–648 709, 876. CARS, 599 SPIM, resolution and number of views, absorption cross-section, 680 compare to other fast lasers, 112–113 613 damage, 682, 686 Cr:Fosterite, femtosecond pulsed laser, Transcriptional reporters, embryo analysis fiber-optics, 507 109, 114, 415, 541, 706–709, and, 748, 755–756. resolution, 447 712–714 FluoroNanoGold, 854 setup, 708–709 embryos, 750, 756, 759, 731, 764 mRNA, 316–317, 465 TIFF (Tagged image file format), 580. emission stability, 86 plants, 773, 781 Tiled montage, 851, 858. four-level vibronic model, 82, 109 NF-kB, 814 Tiger, ECDL laser system, 90. maintenance, 116 Transfection buffer, electroporation, table, Time correlated single-photon counting multi-photon excitation, 541 802. (TCSPC), 518, 520–523, 526. and OPOs, 114–115 Transfection, cellular, 756–758, 790, 791. for lifetime imaging, table, 526 plants, 415, 423–424, 706–708, 713–714, brain slices, 722, 724–725, 730–731 FLIM, 520–523 717, 781–783 Transfection reagents, for chromophores, FRET-FLIM, 186 popular models, specs, table, 120 358, 360, 362, 556, 682, 790–791, schematic diagram, 521 STED, 575 795, 803. Time multiplexing, of adjacent excitation ultrafast, 112–113 2-OST-EGFP, 566 spots, to reduce flare in MMM, URLs, 124 COS7, 693 553–554. TLB. See Transmitted light bright-field. EB3-GFP, 183 Time-gated detection, FLIM, 522–524, 526, TLLSM. See Thin Laser Light Sheet for FRET, CFP/YFP, 795–796, 798, 528+. Microscope. 801–802 diagram, 522 Tobacco, 116, 189–190, 430, 693. GaIT-EGFP, 566 FLIM methods compared, table, 526 smoke, not around lasers!, 116 GFP-MusculoTRIM, 184 FLIM, image, 528–529 suspension-cells, ligand binding, 348 Time-lapse imaging, 136, 222, 354, birefringence, 189–190 Transfer function, implications for image 382–384, 392–399, 652, 773, GFP expressing cells, 430 contrast, 164–165. See also, CTF. 885–886. photo-bleaching, 693 Transient permeabilization, 359, 373, 375. Amoeba pseudopod, 191 “Toe” photographic response, defined, 71. Trans-illumination, absorption contrast, 166. confocal of plant cells, 773 Tornado mode, SIM scanner, 54. Transistor-transistor logic (TTL), 259. high-content screening, 812 Total fluorescence signal, 742. Transit time spreads (TTS), 527. illumination stability, 136 Total internal reflection fluorescence Translational fusions, 756, 757. See also, image analysis, 286, 320, 333, 732–733 microscopy (TIRF), 90, 160, Transfection agents. mechanical stability, 219 180–184, 223, 477, 801. subcellular specific protein distribution, microspectrometry, maize damage, blind deconvolution, 477 756 424–426 vs. confocal image, 184 Transmission, 33, 49, 159, 225, 231, 804. rectified-DIC, of platelets, 846 contrast, 180–184 AOBS, 57 SPIM, 613 cytoskeleton, image, 183 contrast, 163–164 table, 384 FRET, 801 disk-scanning micro-lens array, 223–226, three-dimensional plus time, 222 limits excitation to single plane, 223 227–229, 231, 235 two-dimensional plus time, 222 objectives, for epi-TIRF, 161 dispersion, 683 Time-lapse recordings. Total internal reflection microscopy (TIRM), of filters. See Filters Amoeba pseudopod, 191 177–179, 477. linear vs. log plots, 44–49 Ascaris sperm, 846 blind deconvolution, 477 of glass fibers, 501–505 biofilms, 885 evanescent wave generation, 178 illuminator, 201, 127–128 brain slices, 725, 727, 729, 732–733 TPE. See Two-photon excitation. losses due to refractive optics, 33, 217 embryos, 676, 749, 752, 759, 761 TPEM. See Two-photon excitation table, 217 meristem growth, 430 microscopy. of objectives, 154, 158, 159–161, 641 plant roots, 781, 784 Trade-offs, 36, 68, 78–79, 221, 224, relative, measurement, 26, 34, 36 rectified-DIC, of platelets, 846 644–648, 747–748, 825. table, transmission, 158, 159–161 two-photon microscopy, 10 beam power, visibility/damage, 693 of plant tissue, spectra, 416, 422 TIRF. See Total internal reflection blind deconvolution, 483, 488, 499 of Polaroid materials, 85 fluorescence. compression algorithms, 581, 840 SHG signal detection, 707–709, 729–730 TIRM, 177–179, 477. confocal endoscopes, 508 by small pinholes or slits, 225 Tissue specimens, introducing the probe, 360. when digitizing, 68, 78–79 Transmission electron microscope (TEM), Titanium:sapphire laser (Ti:Sa), 82, 84–86, embryo specimens, 747–748 846. 88–91, 94, 100–103, 105, 107, 109, high-content screening, optimal correlated LM-TEM images, 852–855, 111–112, 114, 123–124, 165, 346, clustering, 825 857–859 358, 415, 423–424, 459, 541, 550, living cells, 381, 693 stereo images of platelets, 848–849 551, 645–647, 688, 706–708, 713, micro-CT, dose/resolution, 616 Transmission illuminator, ghost images, 727, 750, 756, 759. See also, Lasers, MRM, time/resolution, 622 201–202. titanium:sapphire and Ultrafast and pinhole size, 265, 267 Transmission intensity, specimen thickness, lasers. processing speed/segmentation, 301 164. Index 983

Transmittance, optical system, measured, of peony petal, cytoplasmic, 175–176 specific specimens, see specimens by 25–26. rat intervertebral disk, 310–311 name imaging multiple labels, table, 217 of zebrafish embryo, 177 904–905 Transmitted light brightfield, 468, 472–473, Two-dimensional imaging, 60, 222, neurolucida protocol, 731 477. 397–398. resolution, 539 blind deconvolution, 472–473, 477 time lapse, 222, 397–398 and speed, 12 Transparency, lighting models, 309–312. Two-photon fluorescence excitation (2PE), vs. spinning disk imaging. in plant cells, Transverse electromagnetic modes (TEM) 156, 160, 218, 535, 536, 750, 783 laser, 83. 778–783. stray light and non-descanned detection, Trends, in laser design, 118. chapter, 535–549 904 Triple-dichroic, 33, 46, 48, 217–218, 678, chromatic correction for, 156 theory, 535, 537 783. for plant cells wavelengths, 538–541, See also, light loss due to, 33 advantages of, 778–779 Botanical specimens performance, 46–48 cell viability, 779–781 Triplet state, 103, 338, 339–342, 348, vs. confocal microscopy, 779 U 362–363, 390, 516–518, 573, 646, dyes, 782 UBC 3D living-cell, microscopy course, 684, 691–693, 697, 698, 704, 852. of green fluorescent protein, 782–783 174, 183, 184, 190, 205, 364, 430, saturation, 339, 573 pitfalls, 782 435, 439, 805–806. as a RESOLFT mechanism, 573 of thick specimens, 779 Ulbricht sphere, for measuring light, 140. Triton X-100, 730, 852. in vivo, 781 Ultrafast imaging, two dimensional, 222. formaldehyde fixation, 370–372, special objectives for, 160 3D, 235 375–377 visible and ultraviolet dyes, 218 Ultrafast lasers, 88, 101, 103, 112–114. True color, 291. Two-photon microscopy, 10–12, 195, 357, Cr:Fosterite. 109, 114, 415, 541, 706, TSM. See Tandem-scanning confocal 535–549, 690, 697, 900–905. See 707–709, 712–713 microscope. also, Multi-photon excitation; Multi- diode-pumped solid-state (DPSS), 112 TTL. See Transistor-transistor logic. photon microscopy distributed feedback (DFB) diode laser, Tube length/chromatic corrections, table, autofluorescence, 545 113 157. basic principles, 535 fiber, 113–114 Tunable lasers, 91, 103, 107, 109, 120. of biofilms, 882–885 table, 101 broadband, table, 120 bleach planes, in fluorescent plastic, 193, fiber-diode, mode-locked, 113 continuous wave dye, table, 91 194 Nd:YAG, 88–89, 91, 95, 97, 103, diode, emerging techniques, 107 caged compounds, 544 107–109, 111, 113–115, 117, 218, solid-state, 106, 109 calcium imaging, 545 245, 514, 680, 798 solid-state ultrafast, 103 chromophores, 543 Nd:YLF, 89, 98, 100, 103, 109, 112–114, Tungsten carbide electrodes, radiance, 2-photon absorption, 543 750, 760–761

137–138. diagram, 540 Nd:YVO4, 89, 95, 100, 103, 107–109, Tungsten halogen source, 132, 137, 153. detection, 538, 541 111, 113–114, 541 Turnkey ultrafast laser systems, 118. descanned, 542 solid-state, tunable, 103 Tutorials, lasers by level, 124. non-descanned (whole area) detector, spectrum, 44 Tweezers, optical, 89–90, 110, 218, 383, 541 titanium:sapphire, 112–113. See also, 385. stray light, 904 Laser, titanium:sapphire; Titanium- setups for integration, 218 fluorescence, shadowing, 195 sapphire laser single-longitudinal-mode fiber laser for, group delay dispersion, 5443 Ultrafast pulses, delivery by fiber optics, 88, 110 laser. 540–541 507. trapping wavelength, 89–90 alignment, 900–904 dispersion losses, 502 Two-channel confocal images, 175–177, monitoring, 901–903 Ultraviolet (UV), argon-ion laser lines, 85, 177, 193, 425, 522. mounting, 541 87, 90, 102, 339, 346. A.thaliana, epidermal/mesophyll cells, power level, 903–904 other UV lasers, 111–117 193, 425, 431–432, 434–436 safety, 117–118, 839, 900, use for micro-surgery, 218–219 Amoeba pseudopod, 169 903–904 Ultraviolet (UV) confocal microscopy, 109, colocalization, 667 living cell studies, review, 544–545 174, 195, 571. display, 311, 841 living animal studies, 545 absorption, 707, 713 FLIM, 522 minimize exposure during orientation, autofluorescence, 431–432, 434, 544 harmonic images, 714–716 905 CCD response, 29, 255, 459, 921–922 mouse muscles, 716 mirror scanning, 543 correct imaging with planapochromats, montaging, 331 optical aberrations, 542 14, 154 neurons, 332 photobleaching, 690, 697 damage, 212, 290, 439, 544, 680, 686, microglia, 396–398 practical tips, 900–905 903 eye, optic nerve, 481 beam alignment, 901 disk-scanners, 229 Golghi-stained, 298 bleed-through, 904 DNA-dyes, 782, 874. See also, DAPI; Lucifer-yellow, 314 choice of pulse length, 537, 903 Dyes GFP excitation, 798, 873 rat-brain neurons, 398 pulse length, 109, 112, 115, 507, 537, high-content screening, 811 transmitted light, 475 538, 902–903 ion-imaging, 346, 383, 529, 738, 742 984 Index

Ultraviolet (UV) confocal microscopy Video, 2, 4, 5–7, 11–14, 17, 37, 52–53, dipping objectives, 161, 209, 411, 429, (cont.) 61–62, 88, 219, 237, 261, 263, 346, 568, 613, 727, 737, 870, 872 multi-photon excitation, 535, 538, 544, 372, 430, 451, 505, 539, 554, 556, in fluorescence ion measurement, 737 559, 646, 706, 905 589–590, 593, 604, 860, 885. ion measurement, 737 photoactivation, 759 confocal, 25, 237, 914 living cells, 386–387, 389, 395, 398 safety, 117–118, 839, 900, 903–904 impact on light microscopy, 5–7, 14 performance measured, 47, 655–656 simultaneous with DIC imaging, 846, results, 14 plant cells, 429, 433, 772 850 signal, 258–259 STED, 576 as source of stray signal in PMT Video-enhanced contrast microscopy, transmission curves, 159–161 envelopes, 257 imaging small features, 14, 68. use and limitations, 15 Ultraviolet performance of objective lenses, Vignetting, 210–211, 229, 245–247, 492, Watershed algorithm, 322–325, 777, 822. 154, 159–161, 706. 541. for segmentation, plant cell images, 777 Ultraviolet widefield light sources, 132, 136, objective, off-axis performance, 245–247 Wave optics, 4, 10. 139, 143, 226, 542. Visibility, and signal-to-noise ratio, 37–38, for calculating axial resolution, 4, 146, table, 226 68. See also, Rose Criterion. 154 Ultraviolet transmission of optical fibers, Visilog/Kheops, software, 282, 301–302, Wavefront error, 217. 88. 312. lower, with hard coatings on filters, 45 Ultraviolet (UV) light, effects produced by Visitech, confocal manufacturer, Wavelength, 24, 28, 43–51, 62, 88, 107, multiphoton intrapulse interference, descriptions, 88, 119–120, 226, 237, 114–115, 118, 129–130, 135–139, 88. 908. 165–166. Ultraviolet scanning light microscope, 6–7. VT eye, 119–120, 908, 914 calculation of Forster radius, FRET, 793 Uncaging, multi-photon microscopy, 383, VT Infinity, 119–120, 908, 914 and CCD coupling tube magnification, 62 385, 545, 693, 760–764. See also, Visual cortex, identification of primary, 724. filters for selecting, 43, 44, 88 Photoactivation. Visual observation, magnification for, 146. in multi-photon lasers, 165–166. 415, 750 Unconjugated bodipy/ceramide dyes, 760. non-linearity, 72–73 multiple, dynamic embryo analysis, 756 Under-sampling, 79, 635, 640, 652, 662, Visualization, 280, 282–283. See also, of non-laser light sources, 129–130, 831, 833, 836, 839, 841. Multidimensional microscopy 135–136 example, 640 images; Rendering. and optimal zoom setting, 24 uses, 68 definition, 280, 292 vs. pinhole size, 28 Uniformity, of light source, 127–129. software packages for, table, 282–283 selecting, with interference filter, 88, Unit image body, 3D Airy figure, 147. Vitrea2/Voxel View, software, 282, 335. 165–166 Upright vs. inverted microscope, 140, 157, Volocity (software), 281, 236, 282, 295, stability, in non-laser light sources, 217, 230, 413, 722, 727, 870–872. 299, 312, 757, 762–764. 137–139 Unmixing. See Spectral unmixing; VolumeJ, software, 282, 304, 764. tunability, of lasers, 107, 109. structured illumination. VolVis, 281–282. Wavelength expansion, non-linear, 114–115. Up-conversion, fiber lasers, 110. VoxBlast, 283, 301–302, 309, 312. Wavelength ratioing, 346. See also, FRET; doped ZBLAN, 110 Voxel, defined, 20. FLIM. dual-ion doped, 110 Voxel rendering, speed, 290. Wavelength response, chromatic aberration, UV. See Ultraviolet. Voxx, software, 283, 377, 764. 663. Wavelength-selective filters, 43–51, 88. V W Wavelength-tunable lasers, summary, 107, Vacuum avalanche photodiode (VAPD), 31, WAD. See Whole-area; Non-descanned 113, 116, 118, 550. 254, 255. detection. Wavelet compression, 581–584. definition, 254 Water, as immersion medium, 409, 410. Wavelet de-noising protocol, 733–734, schematic, 31, 255 refractive index mismatch, table, 409, 410 819–820. VAPD. See Vacuum avalanche photodiode. two-edge response curves, 410 Waxes, plant, 420, 428, 434–435, 714–715. Vertical-cavity semiconductor diode laser Water-coverslip interface, spherical Website references, 123. (VCSEL), 108. aberration generated at, 147. 2 photon excitation spectra, 546, 727, Vibration. Water-immersion objectives, 15, 23, 36, 729, 782 compensation, 732 141, 148–149, 154, 190, 235, brain slices, 727 from cooling water, 84, 102, 499 241–242, 247, 261, 377, 386–387, CCDs, 76, 234, 927, 931 of disk scanner, 753 389, 395, 411–412, 513, 542, 552, components, 58 causing distortion, 16, 39–41, 166, 201 556, 562, 567–568, 584, 654–656, confocal Listserve, 390, 901 of galvanometer mirrors, 40, 201 708, 727–728, 737, 747, 772. See deconvolution, 495 high-frequency, of acousto-optic devices, also, Spherical aberration. dyes, 221, 343–344, 782 55, 84 4Pi, 562, 567–568 fluorescent beads, 653 isolation, 85, 201, 219, 541 advantages, 149 FRET technique, 185, 803 measurement, 30–41, 652 biofilms, 870, 872 high-content screening systems, 811 of mechanical shutters, 929 brain slices, 727–728, 730, 737 image management, 865 of objective lens motion, 754 chapter, 404–413 lasers, 104, 115, 120, 123–125 optical fiber isolation, 505, 507 correction-color/flatness/transmission, live-cell chambers, 388–389, 870 of optical fiber scrambler, 8, 84, 131 154 movies related to book, 235, 392 Vibronic laser, Ti:Sa four-level, 109. deep imaging, 395 muscles, 237 Index 985

non-laser light sources, 138, 143 sensitivity, 459–463 Zea mays. See Maize. plants, 769 single point images, 454 Zebrafish, 174, 176, 761. safety, 117–118, 839, 900, 903–904 pros/cons, 644–648 GFP image, 176, 176 software, 282, 376, 594, 734, 762, 764, table, 459 autofluorescence, 174 776, 777, 820, 824, 827, 831–833, temporal resolution, 458 pancreas expressing DsRed, 176 844, 845, 864–862, 865–867, 869 Wiener filtering, 494, 496. See also, scatter labeling/lineage tracers, 761 SPIM, 672 Gaussian filtering. Zeiss, confocal manufacturer, 212, 214, Wedge, compensator, 566–567. image enhancement, 496 217, 226, 231–232, 655, 771, Wedge, rotating, for light scrambling, 84, image restoration by, image, 494 916–917. 131. Windows software, for automated confocal, 510 META confocal microscope, 655, Wedge error, in interference filters, 45–46, 810. 908, 916 151, 211–212, 630. WinZip, 580. Achrogate beam-splitter/LSM 5-Live, 50, in traditional filters, 45 Wollaston prisms, DIC, 156, 468, 473, 475. 119–120, 212, 231–232, 916 Wedged fiber-optics, reduce reflections, 85. See also, Nomarski; DIC contrast. Axioimager system, 217 Well-by-well data, 817. Working distance (WD) of objective lenses, fluorescence correlation spectrometer WF. See Widefield. 5, 9, 129, 145, 154, 157, 198, 249, (FCS), 383, 385, 602, 801, 803, 805, WFF. See Widefield fluorescence 511, 568, 598, 634, 673, 678, 917 microscopy. 727–728, 747, 774, 779, 781, 872. HBO-100 source, self-aligning, 134–135 White light continuum lasers, 88, 109, 113 table, 157–158 high-content screening, 811 continuum, 88, 109 WORM disks (write once, read many), 586. LSM 5-Live line-scanning confocal He:Cd, 113. microscope, 50, 51, 231–232, 237, Whole-area and external detection, 541–542. X 784, 908, 916 See also, Non-descanned detectors. Xenon arc lamps, 44, 132, 137–138, 144. META confocal spectral detector, 51, Whole-cell patch pipet delivery, 360, iso-intensity plot of discharge, 132 119–120, 161, 202, 660, 663, 796, 726–727. pulsed-operation, 137–138 916. Widefield deconvolution, 751–753, 785. See shapes of electrodes, 132 mini-PMT arrays, 51, 667FRET, 706 also, Deconvolution. spectral distribution, 144 tests, 663 botanical specimens, 785 super-pressure, spectrum, 44, 136 objectives, advantages of, 155–156 for living imaging, 751–753 explosion hazard, 136 Infinity Color-corrected System, 155, Widefield (WF) fluorescence microscopy, 3, wavelengths available for detection, 44 217 22–23, 26, 172–173, 219, 453–467, Xenon/iodine fill arc, radiance, 137–138. plan objectives, table, 152 518. See also, Epifluorescence Xenopus laevis, 13, 610, 746, 748–753. transmission specifications, 161 microscopy, Deconvolution. blastomere, 757 tube length conventions, 157, 239 compared to confocal, 453–467, 644–647 confocal/multi-photon comparison, 750 working distance of objectives, table, CCD/confocal comparison, 458–459, embryo 158 465 viewed with confocal, 748–753 Zernike moments, 247–249, 818–820. same specimen, 465, 482 viewed with OCT, 610, 749 Zernike polynomial fit, 245–247. compared to structured illumination, 274 embryo viewed with MRM, 623–264 table, 247 deconvolution, imaging living cells, 23, in situ imaging, 746, 748 wavefront aberration function, 247 392 oocyte wound closure, 749 Zinc selenide (ZnSe) diode lasers, 106. deconvolving confocal data, 461–464, X-Y resolution, confocal/widefield Zirconium arc lamps, 136, 141. 466 compared, 36. spectrum, 136 fluorescence detection, 459–460 Zone System (Ansel Adams), 71–72. fluorescence excitation, 459 Y Zoom magnification, 11, 24, 37, 63–64, 66, fluorescence lifetime imaging, 518 Yellow fluorescent protein (YFP), 221–222, 70. See also Magnification gain-register CCDs, 460–461 429. optimal, 24 images utilizing out-of-focus light, 26 FRET pair with CFP, 791–803 optical vs. electronic bandwidths, 70 imaging as convolution, 453–457 YFP, 221–222, 429 relationship to area scanned, 63 imaging thin specimens, 172–173 Yokogawa disk-scanning confocal system, 6, Z-position and pinhole/slit size, 227. integration of fluorescence intensity, 12–13, 16, 216, 224–226, 231, Z-resolution, 3–4, 22, 36, 149–150, 224, 459 234–237, 458, 754. 225–228, 563, 752. See also, Axial interaction of photons with specimen, CSU-10/22 model, 223, 231, 236, 915 resolution. 22–23 with EM-CCD, 234, 237, 755 4Pi microscopy, 563 light-emitting diode sources, 136 high speed acquisition, 11, 220, 222–226, in confocal fluorescence microscopy, 36 limits, linearity/shift-invariance, 457, 490, 229, 231, 458, 667, 754, 784 effect, of fluorescence saturation, 22 564 results, 236–237, 755, 783 improvement, 752 model specimens, 461 vibration, 16 of pinhole disks, 224 noise, 459–463 Ytterbium tungstate (Yb:KGW) laser, 108. in STED, 576 optical sectioning schematic, 469 Z-scanners, evaluating, 215. optical tweezers/cutters, 219, 89, 383, 385 Z Z-sectioning, imaging brain slices, 729. out-of-focus light, 461 ZBLAN up-conversion glass fiber, 110. Z-stack, 23, 754. point-spread function, 453–457, 459–463 Z-buffering, 304–305. of images of cheek-cell specimen, 23 resolution, 3 Z-contrast, in confocal microscopy, 180. speed acquisition constraint, 754