CANCER RESEARCH

VOLUME 13 FEBRUARY 1953 NUMBER 2

Microscopy. I: A Review

ROBERT C. MELLORS*

(Division of Experimental Pathology, Sloan-Kettering Institute for Cancer Research,and Pathology Laboratories, Memorial Center, New York, N.Y.)

INTRODUCTION Finally, Parts I and II will attempt: Since the origination of the cell theory, its estab d) To indicate the application of these technics lishment by Schleiden and Schwann, and its appli in the study of the size, shape, mass, elementary cation to pathology by Virchow, the microscope and organic composition and ultrastructure of the has played an important and historic role in nucleus, nucleolus, chromosomes, mitotic appara biology and medicine. The microscope has now tus, and cytoplasmic particles. been developed to utilize the several properties of In general, the analytical methods reveal local the light wave—the amplitude, the frequency, the structural and optical inhomogeneities which are phase and the plane of vibration—and to employ, accompanied by alterations in the optical path in addition to electromagnetic radiations, other length (product of thickness and refractive index) forms, or carriers, of radiant energy such as an or by differences in the absorption and the scatter electron beam. It is the purpose of this review: (a) of photons, electrons, and x-rays. The require to describe some of the recent advances in methods ments for good imagery include the presence of of microscopy of the cell and (b) to indicate the contrast and the resolution of fine detail in cellular results obtained and their manifest or potential formed elements which have several orders of significance in cancer research. dimensional magnitude that are very approxi The plan of the presentation is: mately given by: [email protected] nuclei; 1@sfor nucleoli, Part I: chromosomes, and mitochondria; and 0.1k for mi a) To present the microscopic methods of analy crosomes. It will be shown subsequently that for sis that utilize the phase (phase, interference, and certain types of quantitative work a departure is polarizing microscopy), the plane (polarizing mi made from conventional methodology by the croscopy), the dispersion (dispersion microscopy), preparation and the analysis of optically homo and the amplitude and the frequency (microspec geneous cellular material. An excellent general re troscopy) of light waves in the optical spectrum. view on recent progress in practical microscopy is Part II (to be published): that of Barer (8). b) To describe the use of other carriers of radi PHASE MICROSCOPY ant energy in microscopy (electron microscopy and historadiography). Principles.—Upon passing through a transpar c) To discuss briefly the automatic counting and ent object in an ideal microscope, a light wave, which may be treated as a sinusoidal wave motion, measurement of microscopic particles (microscan ning). does not undergo a change in amplitude as com pared to that of a wave passing through the adja

* This work was done under a Damon Runyon Senior cent medium or surroundings. Consequently, pho Clinical Research Fellowship of the American Cancer Society, tosensitive surfaces, such as the retina, photograph Inc., and was supported in part by a research grant from the ic emulsion, and cathode of a photo-cell which are National Cancer lnstitute of the National Institutes of Health, Public Health Service. amplitude- or intensity-detectors, record no differ ence between the object and its surroundings. Received for publication September 6, 195g. However, if there is a difference in the re 101 Thie One IIIIIIII@IIIlIHhII@I@III@IIHIIII@ 5OHC-NGZ-WOQH

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fractive index' of the object and the medium, tide 15dark for n0 > @mandlight for n0 < nm; and then the optical path (linear path multiplied in a bright-contrast microscope the particle is light by refractive index) will be dissimilar for light for n,., > ne,. and dark for n0 < nm. waves that pass through the object (de@viated The literature on the theory and the more prac wave) and the medium (undeviated wave), and tical aspects of phase microscopy includes, aside there will be a displacement in the phase of one from the work of Zernike that of Kohler ant Loos wave in relation to the other. With annular illumi (1@O), Burch and Stock (39), Bennett et at. (16, nation, the deviated and the undeviated waves 17), Payne (179, 180), Osterberg (174, 175), Barer pass through different areas of the back aperture (,5, 6, 9, 10), Oettlé (169), Richards (193) and of the microscope objective, and there, upon trans Francon (8@). mission through the appropriate phase-altering Appli.ation@.—The necessity for brevity pre and amplitude-decreasing plates, the two waves cludes a listing of publications relating to the which initially differed in phase but not in ampli phase microscopy of viral inclusions, rickettsia, tude are brought into phase with differing ampli bacteria, fungi, algae, higher plants, protozoa, and tude. When focused at the image plane of the ob higher invertebrate animals and requires a selec jective, the waves may then interfere construc tion of work pertaining, for the most part, to the tively (addition of amplitudes) or destructively living cells of higher vertebrate animals (Figs. I, (subtraction of amplitudes) to produce either 3). bright or dark contrast in the object in relation to The unique value of the phase microscope is the surrounding medium. A conversion from that it, as no other method, provides a means for phase- to amplitude-differencesunderliesthe the structural analysis of the nucleus and the phase contrast principle which was originally for cytoplasm of living cells without the hazard of in mulated by Zernike and applied by him to phase jury or the uncertainty of artifact. A typical, living microscopy (@5@—254). normal interphase cell in its medium of growth, The most important attribute of the phase mi such as a mouse fibroblast in tissue culture, is ob croscope is to enhance the visibility of particles served in dark contrast to have : a delicate, or which, relative to their surrounding medium, have nearly invisible cell membrane ; formed elements a small difference in optical path. The difference, in the cytoplasm which are of two structural cate @, in optical path is a linear distance given by the gories—smaller, pale gray, rod-shaped and fila expression mentous structures (the mitochondria) and larger,

@= (n@—n,,,)t, ( 1) dark gray to black, spherical elements (“fat―drop lets, etc) ; occasionally, a juxtanuclear, spherical where n0 and n,,, are, respectively, the refractive or crescentic, granular mass designated as the cen indices of the object and the medium and t is the tral body (131, 139) and thought by some (139) to thickness of the object. It is customary to express form the Golgi apparatus after fixation ; a delicate @ as a number or a fraction (CF)of wavelengths (X) nuclear membrane ; spherical nucleoli ; irregular so that masses of chromatin not always distinguishable (2) from nucleoli (see 257, however) ; and a ground and substance in the nucleus and the cytoplasm, the (n@,—n,,,)1 details of which are at, or below, the limit of opti x (3) cal resolution. (See section “ElectronMicroscopy― —to be published in Part II.) where 4 is called the phase difference or the phase The mitotic division of living embryonic cells of the chick in change. tissue culture is recorded on movie film with time-lapse phase For a given thickness, the value of @,which is photomicrography by Hughes and Swann (11@). The sequence of the order of X/@Oor less (17) for many cellular of events in mitosis includes: the dissolution of the nucleoli and particles, depends upon the difference between n0 the nuclear membrane within a period of one minute; appear ance of the chromosomes as radially disposed threads during and nm; and the contrast of a transparent particle the next 5 minutes; irregular motion of the chromosomes and relative to the medium is determined by this dif formation of the metaphase plate; partial rounding of the cell ference, together with the nature of the phase body and withdrawal of its processes; movement of the chromo altering and amplitude-reducing plates of the mi somesin anaphase, after one or more “falsestarts,―producing a @ croscope. In a dark-contrast microscope, the par separation between the sets of chromosomes of the order of 10 or more within S minutes; and telophase, with flattening and ‘Therefractive index of a medium is equal to the ratio of the migration of the daughter cells and the appearance of nucleoli velocity of light in air to that in the medium, a,,,. = and nuclear membranes within 10 minutes after cytoplasmic at a particular wave length and temperature and is a property cleavage. During mitosis the filamentous mitochondria shorten dependent upon and predicted from elementary composition. to become thick, sometimes branched, rods which, together

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with other cytoplasmic elements, bound the nuclear region. The cytological fixative of choice in terms of producing the These elements encroach upon the spindle area and constrict least phase-contrast structural alteration from the natural the intersonal region prior to the formation of the cleavage state is osmium tetroxide. The double-fixation procedure of furrow. The spindle itself is not seen. (See section on “Polaris Ornstein and Pollster (178), utilizing this chemical component ing Microscopy.―) There is a characteristic formation of cyto together with formaldehyde, and the preparation of very thin plasmic bubbles, often prominent in late anaphase and telo tissue sections (about 1 p thick) provide material with maxi phase, which has been observed by earlier workers. mum resolution and optimum contrast in phase microscopy. In a study of mitosis in living embryonic cells of the mouse Phase microscopy may be performed with ultraviolet (8, 18, in tissue culture Fell and Hughes (79) observed the forma 288) and infrared light (8) and with plane polarized light (8) tion ofchromosomes,tracedthe courseofanaphasemovement, and may be combined with interference microscopy (10). For described the swelling and unraveling of the chromosomes in other applicationsconsultthe excellentmonographby Bennett the reconstruction of the daughter nuclei, and demonstrated et at. (17). Discombe (66) describes the use of the phase micro the origin of polyploid nuclei by the mitosis of binucleate cells. scope in clinical pathology. Other notable studies of mitosis in the living cell include those of Michel (157),Hughes (109, 110), and Firor and Gey (80), POLARIZING MICROSCOPY utilizing the phase microscope and the classical observations of Principles.—The refractive index of a transpar Lewis (138) with the ordinary light microscope. ent object may be identical in all directions, as in Viable cells in spreads of mouse tissues mounted in physio logical solutions were studied in the phase microscope by Lud an isotropic object, or the refractive index may ford and Smiles (159, 14@).The fibroblasts of subcutaneous vary with the direction of propagation and the connective tissue and tumor stroma are compared to sarcoma plane of vibration of light, as in a birefringent or cells of the rapidly growing Rb tumor of the Rifi strain of anisotropic object. There are two or more values, mice. There is an increased content of cytoplasmic mitochon dna, a greater amount of nuclear chromatin, typically larger n4, ne,.,,etc., for the refractive indices of a bire nucleoli, and commonly an enlargement of the central body in fringent object, and the term, n. —n@,which is the sarcoma cells. The mitochondria are more numerous in the called the birefringence is an optical constant that cytoplasm of fibroblasts derived from tumor stroma than from depends upon the space orientation and other subcutaneous connective tissue. Mitosis is studied particularly in the sarcoma cells, in which it appears that both the nucleolar characteristics of the molecular structure of the and the chromatin material enter into the formation of the object. It is a characteristic property of a bire chromosomes at the prophase. The mitochondria of the ma fringent object that when it is rotated between a jority of dividing cells are spherical or rod-shaped, and they crossedpolarizerandanalyzer,theobjectappears may form clusters as though originatingby the segmentation of alternatelylightanddark (positionofextinction) filaments into strings of granules. In the later stages of cell cleavage, the mitochondria return to a more filamentous form, at 90°angularintervals.Itcan be shown that and, while the chromosomes are still visible in the daughter plane-polarized light, when incident upon a bire nuclei and before the appearance of the nucleoli, there is a con fringent object, is divided into two rays, desig current increase in the number of the mitochondria and in nated as the ordinary and the extraordinary ray, the volume of the cytoplasm. which vibratein mutually perpendicularplanes Observations such as the foregoing are of considerable in terest in view of the fact that the mitochondrion, a structural with differing refractive indices, n@ and n, re unit whose isolation and early analysis is due to the work of spectively. After passing through a birefringent Claude(45,47)andofBensleyandHoerr(@0),isacomplex object of thickness, t, and emerging, the two rays (94) of probably several hundred enzymes which catalyzes, recombine to form an elliptically polarized ray among other reactions: the oxidation of metabolites in the citric acid cycle (95, 104, 105, 118, 201); fatty acids (92) and certain with two components which have a difference, @, amino acids (219,220,280); phosphorylation (55); and various in optical path called the retardation and given by synthetic processes (49, 50). the expression Since phase microscopy provides a means for the structural analysis of living cells without the risk of injury or artifact, it A= (n0—n@)l. (4) affords a method for the study of the structural and optical (path length) alterations produced by chemical and physical Utilizing equations (4) and (s), the difference in agents or conditions, among which are anisotonicity, hydrogen optical path may be expressed as the phase differ ion concentration, fixative solutions,cytotoxins, temperature, ence, cf, by the relation pressure, electromagnetic and other radiations. In general, nox ious agents produce an increase in the visual phase-contrast of (n.—n@)l the cell membrane,nuclear membrane,and nuclearchromatin, (5) andafinegranularityoftheprotoplasmicgroundsubstance.A x peculiar type of cytoplasmic budding or blister formation, Polarizing microscopy permits the visualization termed pinocytosis (182) or potocytosis (255), occurs under certain conditions, among the least rigorous of which is the sus of birefringent objects, the establishment of the pension of the cell in isotonic saline solution for several mm opticalaxes,thecharacterizationofthe birefrin utes. The work of Zollinger (255—258), Buchsbaum (87), gence,andthemeasurementoftheretardationand Hughes and Fell (111), and Crawford and Barer (54) are to be therefractiveindices.Theopticalaxisservesasan consulted for the phase-contrast, morphological changes pro axisofreferenceandisthedirectioninwhich light duced by chemical agents and the studies of Kimball and Gaither (119) and of Tahmisian and Adamson (281, 282) for passesthrougha birefringentobjectwithoutalter the structural alterations invoked by physical agents. ationand,inan orientedbiologicalobject,usually

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coincides with a distinctive direction or axis of chromosomes but not the spindle are visible in the phase mi symmetry, such as an axis parallel to or perpen croscope, and the birefringent spindle and, less readily, the chromosomes are discerned in the polarizing microscope. The dicular to the length. spindle is first observed in early metaphase as a small circular Birefringence is designated as being either posi or ovalregionofpositivebirefringence(withrespectto the long tive or negative with respect to the optical axis, axis of the cell) which enlarges rapidly and soon assumes the depending upon whether the refractive index par typical spindle configuration. A small birefringent structure, the aster, radiates out from the centrosome at either pole. The allel to that axis is the greater or the lesser one. In retardation of the fully developed metaphase spindle has an the case of biological objects of interest, the sign average value of about X/500 and a maximum value at the of birefringence is conventionally referred to the poles. Soon after anaphase begins, the spindle starts to don long axis, even though this may not be the optical gate, and the retardation in the equatorial region decreases. During cytoplasmic cleavage as the equatorial region of the axis. Birefringence is further characterized as in spindle is constricted, positive birefringence develops in the trinsic birefringence in which the anisotropy is due cytoplasmic bridge and attains a retardation of X/500. Ulti to a preferential asymmetrical alignment of the mately the cytoplasmic strand appears to snap, and the bire oscillating atoms and molecules within the object fringence fades away. The actualconstricting collar is also bire and is totally independent of the refractive index fringent with a positive sign. Quite often there occurs near one end ofthe main body ofthe cella roughlycirculararea ofnega of the surrounding medium ; form birefringence in tive birefringencewith respect to the long axis of the cell. which the anisotropy occurs when asymmetrical Among the possible interpretations of the foregoing ob particles, small with respect to the wavelength of servations, the most logical (112) is that the spindle is fairly light and not in themselves necessarily anisotropic, completely oriented near the poles and less well oriented at the are preferentially oriented in a medium of different equator; and that the centrosomes at the poles exert an orient ing force through which a co-ordinated contractile mechanism refractive index; and atrain birefringence in which for chromosomemovement is built up from isotropic proto a normally isotropic object is subjected to distort plasm. Schmidt (200), who originally observed the decrease in ing forces that produce preferential orientation of the retardation of the spindle of the sea urchin egg as the chro the oscillating elements. Protein fibers, with one mosomes moved toward the poles, emphasized the analogy to a feature of a muscular contractile mechanism in which the re important exception, have both an intrinsic and a tardation decreases during isotonic contraction. The modern form birefringence which are positive with respect interpretation of the spindle fibers is that of an oriented mass to the long axis and attributed to the longitudinal of elongated molecules formed by molecular aggregates, or orientation of the protein chains (198—@00).An micelles, and having the property of contractility which is re exceptional property is shown by nucleo-protein lated to the folding of molecular (polypeptide) chains (108, 184) during anaphase. fibers: while the form birefringence is positive as in Swami (225, 226) observed a close correlation between the other protein fibers, the intrinsic birefringence is position of the chromosomes and the regions of decreasing bi negative due to the nucleic acid moieties which are refringence in the sea urchin egg and advanced the hypothesis presumably oriented perpendicular to the protein that the change in birefringencewas brought about indirectly chains. by the chromosomes themselves. Accordingly, the chromo somes would be responsible for their own movement by con The conventional polarizing microscope de trolling the structural changes in the spindle. The mediation of signed for petrography is, in general, not suitable this action might be due to the release of an “activesubstance― for the detection and the measurement of the lesser which diffuses away from the chromosomes and produces its magnitudes of retardation, the range of which is of effect as it goes. Inoué(118,114), with an improved polarization microscope the order of X/5,000 to X/500 (0.1—5m@i)for the and an ingenious method (114) for the measurement of small constituents of many living cells (114). Recent im retardations, studied the structure of the mitotic spindle in the provements in instrumentation, notably those by living cells of the animal (Chaetopterus egg) (Fig. 2) and of the Swann (@5, @6),Swann and Mitchison (@7), plant (Easter lily pollen mother cell). The spindle was ob and Inoué(113, 114) make the detection and the served to be composed of several fibrous components, some of which correspond to categories proposed by Schrader (202): measurement of such magnitudes of retardation “continuousfibers―connecting the two poles; “chromosomal possible and provide new potentialities for the fibers―connected with the kinetochores of the chromosomes; biological application of the polarizing micro and “interzonalconnections― between the separating chromo scope. The literature on the theory and the appli somes in anaphase and telophase. The effect of colchicine on the spindle was investigated by cations of polarizing microscopy includes the Inoué(118).When the Chaetopterus egg is immersed in col publications by Schmidt (@00), Schmitt (198, 199), chicine sea water, the spindle length begins to shorten; the bire Bear (15), Chamot and Mason (44), Frey-Wyss fringence of the continuous fibers and the astral rays decreases ling (88) and the excellent review by Bennett (19). and then completely disappears, and finally the thick chromo somal fibers lose their birefringence. The chromosomes remain Applwationa.—Hughes and Swann (11%), by means of “bi on the equatorial plate until the birefringence of the spindle frame recording,―which is a term applied to alternate serial completely disappears, and then the chromosomes begin to photography by phase and polarizing microscopy, studied the scatter. This indicates that there is a close relation between the relations between chromosomal movement and the changes mechanical integrity of the spindle and the maintenance of the within the spindle in the living, dividing chick embryo cell. The metaphase plate of chromosomes.

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@ InouéandDan (115), in a study of the birefringence of the ones; and a value, = X/4, occurs between an astral rays in dividing eggsof the medusa and the sea urchin, adjacent bright and dark fringe. emphasized that the birefringence of cellular structures is not a static characteristic but a dynamic, changeable one. The sign Applicaiions.—As shown above, the difference, @,inoptical of the birefringenceof these structures is dependent on the di path is related to the refractive indices, n0 and n,,,, of the object rection from which forces act upon them. and the medium, respectively, and the thickness, t, of the ob ject by the expression

INTERFERENCE MICROSCOPY i@= (floflm)t (6)

Principles.—If a beam of monochromatic light The observation of fringe patterns by multiple-beam interfer at nearly perpendicular incidence to a microscope ometry makes possible an estimation of the optical path differ field undergoes multiple reflections between two ence. In the case of an optically homogeneous (isotropic) oh plane, parallel semi-reflecting slides enclosing a ject, the term (n0 —ii,,,) is a constant for a given condition, and the optical path difference is related directly to variations in medium of refractive index nm, the intensity of the the thickness of the object. The resolution of the interfer light which is transmitted will go through maxima ometric method is extremely high (237). For example, if the and minima depending upon the separation of the separation of two bright fringes is 5mm. in a cell-image,' then a reflecting surfaces. When the field is bright, the tenth of the distance between fringes is readily estimated (0.5 optical path difference between an incident ray mm.) and is equivalent to a path difference of X/20 which for green light is 0.027 p. Now, for the interferometry in air (ii,,, = and a reflected ray is such that constructive inter 1) of an essentially aqueous (n0 = 1.33) cell-object, the expres ference (addition of amplitudes) occurs ; and when sion (n0 —n,,,) in equation (6) becomes 0.83, and the thickness, the field is dark destructive interference (subtrac t, which corresponds to an optical path difference of 0.027 p, is tion of amplitudes) is present. If a small object of 0.081 p. A resolution of 0.081 p and a magnification of 6,200 X (500/0.081) in the third, or the vertical, dimension ofa homo different refractive index, n0, is placed in the me geneous cell object, therefore, falls in the range of that achieved dium between the slides, the optical paths for the by the electron microscope for the lateral dimension (see sec rays which pass through the object and through tion on “Electron Microscopy,― to be published). the medium will differ; and, if the spacing of the An important use of interference microscopy in biology is described by Barer (11) and Davies and Wilkins (63). Since the slides provides a bright background, the object organic mass of a cell is principally protein, then the term will appear in dark contrast, or, if the background (n.—a,,,)inequation(6)maybeconsideredtobetherefrac is dark, the object will be bright as in phase con tive index increment between a protein solution with index a0, trast. Furthermore, if there are differences in and its solvent with index n,,. For proteins such as serum al optical paths through the object, then loci of iden bumin and globulin it has been shown by Adair and Robinson (1) that the relation between the refractive index increment tical paths will be shown by alternate bright and and the concentration is given by dark lines, called interference fringes, which form a no nyu contour-like map of the object. This is a method of a (7) multiple-beam interference microscopy as origi nated by Frederikse (87), developed by Merton where a is a constant called the specific refractive increment (154, 155) for biological application, and used by and C is the number of gm dry protein/i® ml of solution. Richards (195) and Barer (10). Combining equations (6) and (7), the optical path difference The work of Tolansky (Q87, @38)defines the is mathematical conditions which are to be fuffilled L@ a'C't, (8) in order to permit the interpretation of the images where t is the thickness of the cell in cm. in multiple-beam interference microscopy. The The equivalent mass, M, of dry protein@i4gm/cell is given physical requirements include the use of: an axial by bundle of monochromatic illumination with a very M= V'1@j, (9) small angular aperture; slides of high reflectivity; very close spacing and small angle of inclination and an objective of sufficient angular aperture. where V is the volume of the cell in ml. and C is expressed in gm/i® ml. Under these circumstances a necessary mathe As an approximation, matical condition is satisfied, and the following V = A ‘1, (10) interpretation of the interference fringes is per mitted. The difference in total optical path repre where t is the thickness in cm. and A is the area of the cell in sented by two bright fringes, or two dark ones, is square centimeters. X; but in each interreflection there is a double Combining equations (9) and (10), we@have passage through the object or the medium so that the optical path difference for one passage is X/@. M= A @ Therefore, an optical path difference, = X/@, is represented by two bright fringes, or by two dark ‘R.C. Mellors, unpublished observations.

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and, substituting the value of C from Equation (8), this be hydrating it and mounting it in various organic comes and inorganic liquids (immersion media) of known A ‘i@ (11) refractive index (44). The immersion medium M lOOa' which renders the object completely invisible in a where M is the equivalent mass of dry protein per cell in gm., bright-field microscope has a refractive index iden A is the area in square centimeters, i@is the opticalpath differ tical with that of the object at the temperature ence in cm., and a is the specific refractive increment. and the wave length of observation. The mean value of a is about 0.0019 for a variety of proteins Crossmon (56, 57) describes an immersion meth and for desoxyribonucleic acid and does not vary a great deal for many organic constituents of the cell. Thus, the equivalent od for the determination of the refractive index in dry mass computed by equation (ii) is not markedly influ which the object when viewed with dark-field il enced by reasonable, qualitative variations in organic composi lumination appears in color—predominately blue tion. By the foregoing method of analysis, Barer (11) obtains a with a small amount of red—when its index value of Si X i0― gm of hemoglobin/red blood celland i20 X iO—'2gm of protein/nucleus in the epithelial cell of the oral matches that of the mounting medium. This color mucosa. ation, called “dispersion staining,― is due to the It can be shown that a more general expression for the fact that an organic immersion liquid, in general, equivalent dry mass of protein in a cell is given by has a dispersion curve (rate of change of index M_@1A1@2A2 . .+A,,A,, with wave length, dn/dX), which rises more steep

— lOOa ( 12) ly at shorter wave lengths than does the curve for the solid object examined ; and, thus, spectral which, for & = @,,= X/Q, becomes colors are produced from white light by dispersion @ M ___ ( 13' at the interfaces of solid and liquid. When the im _i lOOa' ‘ I mersion medium and the object have the same re fractive index in yellow light (X 589.3 mz being a wherein L@i= I@*= X/2, is the path difference between sue standard wave length for refractometry), the yel cessive bright fringes, A,, is the area enclosed by each bright fringe, and ZA, is the summation of the areas enclosed by the low light in the obliquely incident illuminating bright fringes.If the optical path differencebetweenneighbor beam passes straight through the object without ing structures is but X/4, as is frequently observed between the bending and does not enter the objective. How aqueous medium and the cytoplasm or between the cytoplasm ever, the refractive index of the object and the and the nucleus, then equation (13) takes the form, M = X/4 X A,,/iOOa, where A,, refers to the area of the structure whose medium are not now matched at other wave anhydrous mass is computed. lengths, and blue light (being of shorter wave Of considerable interest is the fact' that the hydrous length and undergoing greater dispersion) and a protoplasmicmassofthe nucleusand the cytoplasmofaceilcan small amount of red light are bent at such an angle be measured by multiple-beam interferometry in air, a method that they enter the objective. The object, there readily applicable to living cells in tissue culture (FIgs. 4—9).If the assumption is made that the density (gm/mi) and the re fore, appears predominantly blue with a small fractive index of the cell-object are comparable to that of water, amount of red when its refractive index matches then the hydrous mass, M1@,ofthe cell is given by the expres that of the medium in yellow light. sion The proximity to the matching index is also in 1 @A,,. (13 a) dicated by color. If the refractive index of the ob 2 n0—n,,1@ ject is slightly above the matching index, the ob Thus, by this method it is found that the average hydrous pro ject appears predominantly red; for successively toplasmic mass of the nucleus of living sarcoma cells of the higher increments in index, the object is orange, mouse is 240 X 10― gm., while that for embryonic fibroblasts yellow, and pure white, respectively; if the object isaboutone-halfofthisvalue.itistobeemphasizedthatthe is very slightly below the matching index, it is equations used above, such as equation (13), are rigorously ap plicable only to optically homogeneous objects. (For an addi colored pure blue; and for successively lower in tional discussion of mass determination see the section on dices, the object is light blue and pure white, re “Historadiography―tobe published.) spectively. By the use of polarized light in disper A further presentation of the theory, the methods, and the sion microscopy, birefringent objects, if close in applications of interference microscopy is to be obtained in the comprehensive works of Tolansky (237), Merton (155), Dyson index to that of the mounting medium, exhibit two (70, 71), Faust (77, 78), Francon (83, 84), Philpot (183), and contrastingcolorsthataredependentupon there Ambrose (2). The instrument designed by Dyson is among lation of the two refractive indices to that of the those soon to become commercially available. Barer (10) de medium. Isotropic objects have but one color scribes the use and the advantages of combined phase and in under the same condition of observation. terference microscopy. The method of dispersion microscopy, there DISPERSION MICROSCOPY fore, provides a means for the determination of The refractive index of a transparent micro the refractive index and the birefringence of a scopic object is conventionally determined by de biological object without reference to the optical

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path or to the object thickness. The difference in enced by the concentration, a condition which is index between certain structures such as collagen by no means universally applicable. and elastin fibers, or cell nuclei and other tissue An instrument for the analysis of spectra is components, is such that these structures appear composed of a dispersive element (prism or grat in contrasting color, depending upon whether they ing) for the separation of the various frequencies are equal to, above or below, the refractive index or wavelengths of light and a photosensitive dc of the medium. It may be possible' to identify the ment—retina (as in a spectroscope), photographic toxic dusts such as free silica, asbestos, and beryl plate (spectrograph), and photoelectric or radio hum oxide by dispersion staining. The further bio metric device (spectrometer)—for the measure logical application of this method is awaited with ment (spectrophotometer) of spectral intensities. considerable interest. The use of a microscope in spectral analysis con stitutes microspectroscopy. The pioneer develop MICROSPECTROSCOPY ment and biological application of ultraviolet ab Principles.—The extensive treatment elsewhere sorption microspectroscopy was accomplished by (88) of various aspects of the subject precludes Caspersson (40—4@).In recent years, there has more than a brief presentation of the fundamental been an extension of spectral microanalytical basis of spectroscopy. Molecular spectra arise methods to include absorption microspectroscopy when the motions of the constituent electrons and in the ultraviolet (Figs. 10—13),visible, and infra atoms of a molecule are energized by incident red regions (the entire optical spectrum), together radiant energy : the electrons move about the posi with emission and fluorescence microspectroseopy. tively charged atomic nuclei, and the atomic nuclei This is due in part to the development of a reflect themselves move together in a linear translation ing microscope by Burch (38), Brumberg (34), and rotate and vibrate periodically about their Grey (96, 97, 98, 100), Kavanagh,4 and others center of gravity. The frequency of the absorbed (69, 116, 134, 167), (Chart 1) which utilizes pairs radiant energy must be equal, or very nearly of reflecting mirrors rather than refracting ele equal, to the frequency of the molecular vibrations ments and, therefore, remains in focus at the same that it excites: ultraviolet and visible light ener mechanical setting throughout the entire optical gize the motions of electrons and infrared light spectrum; and to activities in research at Oxford excites the vibrational-rotational motions of atom @ (7, 1@,13), King's College (86, 167, 168, 19@, ic nuclei. It is found empirically and substantiated Massachusetts Institute of Technology (136, @07, theoretically that certain groups of atoms and @48),Columbia (185—190), and Memorial Center chemical bonds in a molecule absorb particular (145—148,15@)and in commercial development by frequencies in the infrared, visible, and ultra Willcocks, Ltd., Bausch and Lomb, Polaroid (@4, violet regions, and it is on this basis that the iden @5,1@5),and the American Optical Companies. ti.fication of a molecule can be made from its char The usefulness of the microscope in systems for acteristic absorption spectrum. By the measurement of the attenuation, or the absorption microspectroscopy lies in the small order of magnitude of the analytical sample. In absorption, of a parallel beam of monochromatic accordance with diffraction theory, the resolving radiation as it rectilinearly traverses a solution of power, d, of a microscope is given by an isotropic absorbing material, or solute, which is molecularly dispersed in a nonabsorbing medium, d 1.2X 0.6X the concentration, C, of the solute is given by the (1@) — NAO+ NA@ — NA0' Lambert-Beer equation, where NA0, the numerical aperture of the objec tive, is the same as that of the condenser, NAG, ki' (14) and X is the wavelength. The wavelength in where C is in gm/liter; E@ is the extinction or microns is 0.@—0.4pin the ultraviolet, 0.4—.75p in the optical density given by E,@= logio 1o/I@with the visible, 0.75—@.5pin the very near infrared, L the intensity of the incident light and I that of and @.5—15pin the near infrared regions; and NA0 the transmitted light; k is the specific extinction is of the order of unity. While the theoretical mini coefficient which is characteristic of the absorbing mum transverse diameter of the analytical sample material and is a function of the wave length, X; as set by the resolving power of the objective is of and I is the path length in cm. This equation is de the order of 0.15 p in the ultraviolet at X 0.@5p, rived under the assumption that the absorption and 6p in the infrared region at X lOp, other limit per molecule of the absorbing material is uninflu ing factors (@4, @5)such as the spectral radiance of ‘G.C. Crossmon, personal communication. 4A. J. Kavanagh, personal communication.

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the source, the detection power of the microspec phosphoric acid groups for the estimation of trometer, and the magnitude of the extinction co nucleic acids. Kurnick (124) comprehensively efficients determine the minimum size of the sam evaluated the selective staining of nucleic acids by ple. At present, the spectral analysis of various methyl green and pyronin. Dempsey and collabo compounds of biochemical interest requires a rators (64) have investigated the affinity of a va weight of sample of the order of 1O_8_1O_12gm. in riety of tissue components for basic and acidic the ultraviolet and JO@—10@gm. in the near infra dyestuffs by the method of micro-absorption color red region (1@, @4,147). The theoretical detectable imetry. concentration of biochemical compounds which Recent attempts are made to circumvent the will give an optical density of 0.1 in a path length unfavorable factors (91, 172) inherent in the cell of 1 p can be computed from equation (14) using object as well as in the optical system of micro their specific extinction coefficients for absorption spectroscopy. Methods designed to avoid the er maxima in the ultraviolet and the visible regions. rors in the analysis of inhomogeneously dispersed The values when computed as the concentration systems (117) and objects of indeterminate thick in gm/mI, or the quantity in 1012 gm/p', are of ness for ultraviolet absorption microspectroscopy the following order: for purines and pyrimidines, include the preparation of isolated, unfixed cells 0.01; nucleic acids, 0.05; cyclic amino acids, 0.03— or nuclei (129), suitably mounted (3) and proved 0.09; porphyrins, 0.002; steroids, 0.02—0.10, and (151)' by phase, interference, and absorption mi

MAKCUTOV ++BRLJM8ERG BURCN GRaY (AVANAGH Ca&wr 1.—Variousdesigns of reflecting microscope objectives

conjugated proteins, 0. 14—0.53.These quantities croscopy to be optically homogeneous ; and the and concentrations of compounds are of the order fluorescence microphotometry (149, 172) of mdi of magnitude of those which may occur in individ vidual, not necessarily homogeneous, cells that are ual cells or in parts of cells. stained with a basic fluorochrome. The use of The theory, practice, and limitations of ‘ultra coated (166) and reflecting optics (147) and the violet microspectroscopy in cytology are dis limitation of fields of illumination in micro-absorp cussed in the comprehensive works of Caspersson tion colorimetry (166) and microspectroscopy (41) and Thorell (234, 235) in symposia (7, 51, 62, (147)todiametersofa fewmicronsaremeasures 76, 99, 145, 192, 206, 208, 241, 242) and in other taken to minimize the defects that lead to an publications. Land and co-workers (125, 211) at anomalous distribution of energy by light scatter the Polaroid Corporation utilize the color-trans at the interfaces of optical elements and by diffrac lating concept of Brumberg (34, 35) and have de tion effects in the object. signed an ingenious ultraviolet microscope for Ultraviolet and visible ab8orption.—Asshown by biological applications (144), in which a visible the extensive work of Caspersson and his collabo three-color image is obtained by simultaneously rators (41), as well as by the earlier observations of projecting onto a single screen three black-and Lucas (137, 138) and Wyckoff (249—251), the white photomicrographs taken at any three de nucleus of a typical cell, when photographed in sired wavelengths. Stowell (221, 223) and Pol the ultraviolet region, is transparent at longer lister and co-workers (185—190)utilize the method wavelengths (> 300 mp) and absorbing at shorter of visible micro-absorption colorimetry of cells in (260—280 mp wavelengths). While Soret (216) which measurements are made at or near the ab originally suggested that the ultraviolet absorp sorption maximum for pigments that characterize tion in the region of 280 mp shown by protein solu such tinctorial reactions as that of Feulgen for tions was due to the constituent cyclic amino desoxyribonucleic acid; and the Columbia group acids, and Dhéré(65)attributed the ultraviolet also employs the Millon reaction for tyrosine as a absorption of yeast cells at 260 mp to the purine measure of proteins and the basic dye-affinity of and the pyrimidine moieties of ribonucleic acid,

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1953 American Association for Cancer Research. MEu@oP@s—Mieroscopy. I. A Review 109 it remained for Caspersson to show that a greater 260 mis. Larionow and Brumberg (36, 126) maintained that absorption maximum in cells at 260 mp and a nuclear structural elements (exceptnucleoli)do not absorb at 254 m@i,unless the cell is damaged or killed. Ris and Mirsky lesser maximum at 280 mp are due, respectively, (196) stated that the living interphase nucleus of uninjured to the presence of nucleic acids and proteins. By cells absorbs diffusely at 254 m@and that chromosomal (chro such means as (a) the ultraviolet absorption meth matin) structures are not visible in the ultraviolet unless they od, (b) the use of the visible Feulgen reaction for are also distinct in the visible region. The qualitative and the desoxyribose, which is a constituent of desoxyribo quantitative observations of others (62, 75, 148, 241), however, show beyond a doubt that there is localized absorption at 260 nucleic acid (DNA) but not of ribonucleic acid mM in the nuclear chromatin, nucleolus, and cytoplasm of liv (RNA), (c) the employment of specific nucleases lag, uninjured interphase cells. The maximum tolerated dose of (123, 143) for hydrolysis, and (d) the use of isola ultraviolet radiation imposes a considerable restriction on the tion procedures, the work of Caspersson (40-42), type and number of observations permissible in this wave length region (62, 135, 148). Injury by radiation or by other Claude (45—48),Brachet (29—Si),Mirsky and co means modifies the light-absorbing properties of both nucleus workers (158—160, 196, 197), and others (59—61, and cytoplasm, and, as shown by Bradfield (32), the response 90, 204) demonstrate that in the interphase cell varies with the type of cell. there are appreciable quantities of DNA in the Ludford, Smiles, and Welch (140-142) have some re nuclear chromatin and of RNA in the nucleolus markable ultraviolet photographs at 257 and 275 m@ of fresh tissue spreads prepared from a variety of sarcomas and carcino and in the cytoplasm and that in the dividing cell, mas of the mouse. These show that the sarcoma cells, in com particularly at metaphase, there is a great concen parison with stromal fibroblasts, are larger; their nuclei are tration of DNA in the chromosomes. larger, have more chromatin, and bigger nucleoli; and their cytoplasm has a greater mitochondrial content which absorbs The observations that nucleic acids are prominent constitu ents of biological elements such as chromosomes, nucleoli, intensely. The cells from the most rapidly growing tumors cx mitochondria (45-47) and, presumably, genes, and of viruses hibit the most intense ultraviolet absorption. (217), growth organizers (29) and mutation or transforming Recently, the results obtained by the microspectroscopic factors (4) focus great interest upon the role of these com analysis of individual nuclei for nucleic acids are being com pounds and their chemical moieties in the mechanisms of pared to the data obtained by the biochemical analysis of growth, reproduction, heredity, and the genesis of cancer. The known numbers of isolated nuclei. The one method gives the majority of the applications of ultraviolet and visible micro frequency-distribution of values for individualnuclei, while the spectroscopy in biology and cytology are in theseSfields of in other yields the average for many nuclei, which, depending terest. Since the subject of ultraviolet microscopy is thoroughly upon their tissue of origin, may be cytologically very diverse. covered in the comprehensive article by Fitzgerald and Eng The concept of Boivin, Vendrely, and Vendrely (28, 239, 240) strom (81), this review will present only those observations and the confirmatory observation of Mirsky and Ris (160) that which appear to be of immediate pertinence to cancer research. the quantity of DNA in each set or fundamental number of Caspersson and co-workers (41, 42), as a result of microspectro chromosomes is constant in accordance with the genetic re scopic studies of nucleic acids, have presented a theory of cel quirements of relative constancy in the composition of chromo lular growth. Caspersson and Santesson (42) observed an in somes has been extensively investigated by Pollster and his as creased ultraviolet absorption at 260 mi in the nuclear chroma sociates (189), notably, Swift (228, 229) and Leuchtenberger tin, nucleolus, and cytoplasm ofepithelialcancercells of human (127, 130), and by Pasteels and Lison (178) with the method of tissues in fixed sections and interpret this as a manifestation of Feulgen-staining and micro-absorption colorimetry. The rela an altered and accelerated growth mechanism in which the tive quantity of DNA so determined in the nucleus of an inter DNA of the nucleus has a special and determinative role. phase somatic (diploid) cell is very similar for different species Stowell (221, 223), using the Feulgen reaction and visible mi of mammals and is twice that of a germinal (haploid) cell; and cro-absorption colorimetry of fixed sections, found a greater some tissues, notably the liver and the pancreas, contain nuclei amount of DNA in the cells of human epithelial carcinomas as with 1, 2, and 4 times the diploid amount of DNA, although in compared to adjacent homologous normal tissues, and ob some groups of nuclei the DNA does not follow this geometrical served analogous differences in experimental carcinogenesis. relationship (178). High order polyploidy and correlating DNA Opie and Lavin (171) determined by ultraviolet absorption and values are observed in the tissue cells of lower animals (229); enzymatic digestion that RNA accumulates in the cytoplasm studies on the cells of plants show either inconstancy (203) or of liver cells that are hyperplastic and precursors of tumors constancy (228) of the DNA per nucleus; and under certain produced by feeding butter yellow to the rat; Stowell (222) re conditions the nuclear DNA is observed to be altered spon ported substantially similar observations. Catchpole and Gersh taneously (29—Si),experimentally (59—61,121, 122), and func (43), in an ultraviolet microabsorption analysis of isolated cel tionally (130). Among the most interesting observations is the lular components, found an increased amount of nucleic acid in finding (189) that in rapidly dividing tissue the synthesis of small cytoplasmic particles in cells of hepatoma, as compared DNA for the new set of chromosomes takes place not in pro to normal liver. The ultraviolet method is applied to the study phase, as customarily supposed, but in interphase, when there of: tissue sections of human sarcomas by Moberger (41) and by is no morphological evidence of mitosis. Moses (163) studied Santesson, Moberger, and Caspersson (41); sections of brain the nucleic acids and the proteins of Paramecium by visible tumors by Moberger, Hakanson, and Ringertz (161); marrow ultramicrocolorimetry and has substantiated the genetic evi cells of acute leukemia by Thorell (234); marrow cells of multi dence that the macronucleus contains a multiple of the genetic ple myeloma by Olhagen (170); and exfoliated cancer cells in elements possessed by the micronucleus in the diploid state. the vaginal smear (145, 146, 151). The results of these studies Naora (164, 165), in contrast with the work of many and using indicate an alteration in the quantity and the distribution of an improved optical system, found an arithmetical rather than nucleic acids in the neoplastic cell. a geometrical progression in the quantity of Feulgen-stained There is confusion in the literature concerning the ultra DNA in hepatic nuclei. violet absorption properties of the living cell in the region of The quantitative estimation of DNA in isolated individual

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nuclei from theliver, the thymus, and the sperm ofthe beef and trum, as compared to spectra obtained in the rat is made by the Leuchtenbergers and the Vendrelys other spectral regions, are shown by the fact that (129) with the ultraviolet absorption method of Caspersson. The DNA per nucleus, expressed in units of iO―gm., is found biochemical compounds, such as the carbohy by this method to be of the order of 6 for diploid and S for drates and the lipids (which are essentially non haploid cells—which is in excellent agreement with the results absorbing in the ultraviolet and the visible regions) obtained by chemical analysis. Leuchtenberger, Klein, and and the proteins (which have one similar broad Klein (128) found by means of ultraviolet microspectroscopic absorption band in the ultraviolet region), are analysis that the DNA content of nuclei of the Ehrlich ascites tumor cell of the mouse is twice that found in normal diploid characterized by distinct, many-banded spectra in nuclei, whereas the quantity of DNA per nucleus in the DBA the infrared. Moreover, a study of the absorption ascites lymphoma is similar to that of diploid nuclei. These re spectra with polarized infrared radiation (12, 74, sults are in accord with the chromosome counts of Hauschka 85, 103, 181, 224) reveals the direction, if such and Levan (107) on similar material. The average RNA per nucleus (128),as estimated by the differencein ultraviolet abs exists, for the preferred arrangement (orientation) sorption before and after ribonuclease digestion, is markedly of certain chemical bonds. By this means, for ex increased, however, in both types of ascites tumor cells, in ample, it is observed that in certain types of comparison with that of normalcells. Petermann and Schneider fibrous proteins the carbonyl bonds (C = 0) are (182) observed by biochemical analyses an increase of RNA oriented perpendicular to the axis of the fiber, and, and DNA in the nuclei of transplanted mouse leukemia over that of normal splenic nuclei; and Cunningham, Griffin, and from this fact, and the accepted nature of the Luck (58) found no difference in the chemically determined peptide bond, it can be deduced that the polypep DNA per nucleus in normal (see 106) and neoplastic tissues of tide chains are predominately extended and on the rat. ented parallel to the fiber-axis. The advantages of using isolated nuclei mounted in glycerin for the microspectroscopic determination of DNA are (129) a In the application of infrared spectroscopy to the study of more favorable distribution of absorbing materials and the the biochemical constituents of tissues and the tissues them elimination of the necessity for the measurement of thickness. selves, the cross-sectionalarea of the sample required in current Recently, by means of simplified procedures for the conversion systems of mierospectroscopy (1%,24, 25, 86, 108, 147, 246) is of tissues into suspensionsof nuclei,and with propermounting of the order of a square 10—SOpon an edge, with the smaller (3), for which glycerin alone is less than optimal, nuclei are sample analyzable only in the very near infrared, while for prepared' that are optically homogeneous, as shown by phase macrospectroscopy the linear dimension is 10 or more times and interference microscopy as well as by ultraviolet absorp this value. Barer, Cole, and Thompson (12) were the first to tion microspectroscopy. A factor of uncertainty in quantitation describea system of infrared microspectroscopyand to record for which no immediate answer is in sight is the magnitude of the spectra of minute quantities of antibiotics, therapeutic fan the specific extinction coefficient for DNA as it exists in the tors, biochemical compounds, fibers, crystals, and tissue cells. nucleus. Blout and his co-workers (27) determined the infrared spectra of dehydrated tissue sections and smears by macrospectroscopy Ultraviolet micro-absorption spectroscopy is and observed thepresence of an intense absorption band, which used to estimate the quantity of total nucleic lies at the same position as a strong band in the nucleic acid acids in interphase nuclei of squamous-cell car spectra (26), in a section of a rapidly proliferating cellular cinomas and of normal epithelial cells that exfoli mammary carcinoma, as contrasted with the lesser absorption of a benign adenoma of this gland; and they (28-25) re ate from the mucosa of the human cervix uteri corded the spectra of nucleohistone fibers, steroid crystals, and (151). There is a trimodal distribution of values in tissue sections with both a macro- and a microspectrometer. a group of abnormal cells and cancer cells (classes Wood (247) observed the infrared microspectrum of living III, IV, and V of Papanicolaou [176, 177] com muscle cells (in the spectral region beyond that in which water bined), with an average and a modal quantity of has intense absorption) and found the spectrum to be closely duplicated by that of the protein, myosin. Morales at at. (16%) total nucleic acid per nucleus which is 2, 4, and 8 studied the infrared absorption of proteins and of other con times, respectively, that for the unimodal distribu stituents derived from the contractile system of skeletal tion given by a group of normal and atypical muscle. Fraser and Fraser (85, 243) presented data on the epithelial cells (classes I and II combined). A molecular structure of DNA obtained by the microspectros geometrical progression in values is consistent copic study of oriented samples with polarized infrared radia tion. Schwarz (205) and co-workers found by macrospectros with the general concept of growth (72) by the re copy that the spectra of anhydrous sections of “visceral―tissues duplication of nuclear formed elements (22, 121, (liver, muscle, heart, kidney, adrenal, thymus, spleen, and 236) containing DNA and RNA, and the data lymph node) and of neural tissue have two different types of have cogent bearing upon the development of absorption bands: namely, bands which are common to all tis sues and similar to those in the spectra of proteins and polypep methods for the cytological diagnosis of cancer tides, and so-called “fingerprintbands―which are characteristic (149). for the individual tissue. Stevenson and Bolduan (218) dis Infrared ab8orption.—The infrared spectrum of cussed the use of infrared spectroscopy in the identification of an organic compound is a physical property that dried films of bacteria. Woernley (245), in an investigation of may be used in the identification of the compound the infrared macrospectra of dehydrated tissues and tissue components, attributed the absorption pattern of DNA and and in the investigation of its molecular structure RNA in the wavelength region, 8—11p, to the pentose and (33, 244). The potentialities of the infrared spec phosphoric acid moieties. He observed a strong resemblance in

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1953 American Association for Cancer Research. MELLORS—MiCrO8COpJJ. I. A Review 111 this region between the spectra of nucleic acids and that of clear and cytoplasmic structures, as do conventional dyestuffs. films of isolated nuclei, and correlated, in a general way, the The subject of fluorescencemicroscopyand the employmentof intensities of the absorption bands with the concentrations of fluorochromes are discussed in the comprehensive monograph nucleic acids in the tissues. Thus, the infrared and the ultra of Haitinger (10%) and in articles by Barnard and Welch (14), violet absorption spectra may provide complementary and Ellinger (73), and Richards (194). substantiating information about cellular nucleoproteins. A universalmicrospectrometer for the infrared, visible, and ultra An ingenious method for the microscopic locali violet regions is described (15%);and a ratio automatic record zation of antigenic material in tissue cells, employ ing microspectrophotometer for the entire optical spectrum, ing fluorescence microscopy and antibody conju which is under design and construction by the Perkin-Elmer Corporation,' willbe a most versatile instrument with manifold gated with the fluorochrome, fluorescein, is de applications in biology and medicine. scnibed by Coons (53) and his collaborators. Friedman (89) described the use of three fluoro Fluore,rcence.—When a molecule is brought to chromes in the study of exfoliated cells in the an excited state by the absorption of electromag vaginal smear: berberine sulfate, a basic dye that netic radiations and by The transfer of energy to a imparts a brilliant yellow-white fluorescence to particular part of the molecule, a transition from nuclei; acid fuchsin, an acidic dye that combines this to a lesser energy level may be accompanied with the cytoplasm to provide a red-purple fluores by an emission of light which, if instantaneous, is cence; and acridine yellow, which acts as a nuclear called a fluorescence spectrum (191) and for corn and cytoplasmic fluorescent stain. Cancer cells are plex molecules consists of a continuous emission observed in general to fluoresce more brightly than band with several peaks of higher intensity. While do normal cells and to have cytoplasmic bright the relation between the chemical structure and orange or red-orange fluorescence which differs in the fluorescence of an organic molecule is but poor color from that of normal cells. ly understood, it is known that a closed ring struc The development of a quantitative scanning ture favors luminescence, as is observed in the ma method for the preliminary screening of smears for jority of aromatic hydrocarbons composed entirely the presence of cancer cells has been undertaken of condensed benzene rings, and in many hetero by workers at the Memorial Center (149, 153). cyclic compounds. The underlying chemical and physical principles The use of fluorescence spectroscopy in the investigation of are as follows : cells are stained with a basic fluoro complex mixtures of hydrocarbons in carcinogenic tars is a chrome, such as berberine, under conditions (101, historical methodological step in the isolation and identifica tion of benspyrene as a cancer-producing agent (5%).Graffi (93) 156) that favor selective combination of the dye was the first to employ the fluorescence microscope in the study with chemical constituents, such as DNA, which of the uptake of chemically pure carcinogens by living mam are located in greater concentration in the nucleus; malian cells in tissue culture. The localization and distribution squamous cancer cells, relative to normal cells of cutaneously applied carcinogenic hydrocarbons is a subject of investigation which is facilitated by the fluorescence of car which exfoliate from the cervix uteri, combine cinogens and their derivatives. Doniach, Mottram, and Wei with more fluorochrome and emit on the average gert (67, 68) observed a brilliant violet fluorescence spectrum per unit nuclear area twice the quantity of light in the skin of mice painted with S,4-benzpyrene and in certain energy of normal cells. The light energy derived organs of animals receiving intravenous injections of suspen from the scan of a cell is converted by a photocell sions of the carcinogens. Simpson and Cramer (%1%,%1S)ex amined frozen sections of the skin of mice with a fluorescence into a voltage pulse which, in turn, is analyzed in microscope and found that fluorescing material appears in the terms of its ability to energize an electronic count epidermis within a day after a single painting with 20-methyl ing circuit that is set to respond to a certain volt cholanthrene and persists for several weeks. Setälä(209, %iO) age imput and to register automatically thereby used polyethylene glycols rather than organic solvents as vehicles for the carcinogens, %0-methylcholanthrene, 1,%,5,6- the presence of certain types of cells—e.g., a can dibenzanthracene, 9,iO-dimethyl-1,%-benzanthracene, and l,% cer cell, as differentiated from a normal cell. If it is benzanthracene; and he studied their cutaneous localization by shown by microspectroscopy that cancer cells with fluorescence microscopy. Berenblum, Holiday, and Jope (flu) a characteristic fluorescence hue, when stained by discussed the possibility of seeking carcinogens in individual cells by fluorescence microspectroscopy, an approach which is the polyiluorochrome method of Friedman (89), now a reality in that this analytical method is used (150) in the have a distinct difference in the fluorescence spec studyoffluorescingmaterialthatlocalizesinepithelialcellsof trum when compared to the normal cell, then this the bladder after the ingestion of the carcinogen, fl-naph additional discriminative property may be con thylamine. Sjostrand (%14, %15) described the use of fluores veniently utilized by the scanning device. cence microspectroscopy in the detection of riboflavin and thiamin in tissue cells. Cells and tissues may be stained with fluorescent dyestuffs SUMMARY (fluorochromes) which, because of their basic or acidic chemical A description has been given of some of the re properties and other factors, localize in and differentiate nu cent advances in methods of microscopy of the ‘V.Z. Williams, personal communication. cell which utilize the phase (phase, interference,

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and polarizing microscopy), the plane (polarizing discussed. The quantitative estimation of nucleic microscopy), the dispersion (dispersion micros acids in individual nuclei of cancer cells of the copy), and the amplitude and the frequency (ml mouse and the human cervix uteri yields values crospectroscopy) of light waves in the optical that are distinctly greater, by a geometrical pro spectrum. The applications of these methods to gression, than those of the normal cell. the study of the mass, the elementary and organic The use of infrared microspectroscopy in the chemical composition, and the structure of the analysis of the biochemical constituents of tissues nucleus, nucleolus, chromosomes, mitotic appara and the tissues themselves is discussed in terms of tus, and cytoplasmic particles are presented with a the identification of organic compounds and the view toward the manifest or potential significance investigation of molecular structure. of the results in cancer research. Fluorescence microscopy and microspectros The unique value of the phase microscope is copy provide a means for the investigation of the that it, as no other method, provides a means for localization and the distribution of fluorescent the structural analysis of living cells without the carcinogens in tissues and cells. Fluorescent dye hazard of injury or the uncertainty of artifact. stuffs (fluorochromes) may be used to differentiate Outstanding among the applications are the study nuclear and cytoplasmic structures, as do con of the events during mitosis, the formation and the ventional dyestuffs. Exfoliating cancer cells of the movement of chromosomes, and the structural al vaginal smear have distinguishing properties rela terations in the mitochondria. tive to normal cells when stained with fluoro Recent improvements in the polarizing micro chromes. One or more of these discriminative prop scope make it possible to investigate the spindle erties may be utilized by an automatic scanning apparatus in the living, dividing cell and provide device for the detection of cancer cells. information which indicates the spindle to be an orienting force through which a co-ordinated con REFERENCES tractile mechanism for chromosome movement is 1. ADAm, G. S., and RoBINSoN, M. E. The Specific Refrac built up from protoplasm. It has been shown by a tion Increments of Serum-Albumin and Serum-Globulin. study of the effects of colchicine that there is a Biochem. J., 24: 993-1011, 1930. fi. Aaumosn, E. J. An Interference Microscope for the Study close relation between the mechanical integrity of of Inhomogeneous Media. J. Sc. Inst., 25: 134—35,1948. the spindle and the maintenance of the metaphase S. ANDERSON,N. G., and Wnssiu, K. M. Studies on Isolated plate of chromosomes. The contractility of spindle Cell Components. IV. The Effect of Various Solutions on fibers is interpreted in terms of the folding of the Isolated Rat Liver Nucleus. J. Gen. Physiol., 35:781— 96, 195%. molecular (polypeptide) chains during anaphase. 4. Avzai@,0. T.; MACLEOD,C.M.; and McCarn, M. Stud By means of interference microscopy, loci of ies on the Chemical Nature of the Substance Inducing identical optical paths through a cell-object appear Transformation of Pneumococcal Types. J. Exper. Med., as alternate bright and dark lines, called interfer 79:137—57, 1944. ence fringes, which form a contour-like map of the 5. [email protected],R. Some Applications of Phase-Contrast Micros copy. Quart. J. Microscop. Sc., 88:491—600,1947. object. This effect may be interpreted in terms of 6. - Variable Colour-Amplitude Phase-Contrast Mi variations in thickness of the cell-object, with a croscopy. Nature, 164:1087—88,1949. resolution of the order of 0.03 @i,or it may be used 7. - Aspects of Ultra-Violet and Infra-Red Micro to compute the anhydrous organic (protein) mass spectrography with the Burch Reflecting Microscope. Disc. Faraday Soc., 9:369-78, 1950. and the hydrous protoplasmic mass of a living, or 8. - Progress in Practical Microscopy. J. Roy. Mic. unfixed, cell-object. Soc., 71:307—36, 1951. Dispersion microscopy provides a method for 9. - “Phase-Contrast―Methods and Birefringence. the determination of the refractive index of a bio Nature, 167:64%, 1951. 10. - Combined Phase-Contrast and Interference-Con logical object. The difference in index between trast Microscopy. Nature, 169:108, 195%. certain cellular structures is such that, by the use 11. - Interference Microscopy and Mass Determina of this method, structures appear in contrasting tion. Nature, 169:366—67,195%. color due to optical effects. 1%.B@@unn,R.;Coix, A. R. H.; and THOMPSON,H.W. 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244. Wna@uss, V. Z. Infra-Red Instrumentation and Tech 252. ZEmiIKE, F. Beugungstheorie des Schneidenverfahrens niques. Rev. Sc. Inst., 19:185—78,1948. und seiner verbesserten Form, der phasenkontrast Me 245. Wozns@, D. L. Infrared Absorption Curves for Nor thode. Physica, 1:689-704, 1984. mal and NeoplasticTissuesand RelatedBiologicalSub 253. - Das Phasenkontrastverfahren bei der mikro stances. Cancer Research, 12:516-23,195%. skopischen Beobachtung. Physik. Z., 36:848-51, 1985. 246. WooD, D. L. An Infra-Red Microspectrometer for Bio 254. - Phase-Contrast, a New Method for the Micro logicalResearch.Rev.Sc.Inst.,21:764-66,1950. scopic Observation of Transparent Objects. Physica, 9:686-98, 1942. 247. - Infrared Microspectrum of Living Muscle Cells. Science, 114:36—38,1951. 255. ZOLLINGER, H. U. Cytologic Studies with the Phase Mi croscope. I. The Formation of “Blisters―onCells in Sus 248. WYCKOFF, H. The M.I.T. Recording Microspectro pension (Potocytosis) with Observations on the Nature of photometer.Lab.Invest.,1:115—22,1952. theCellularMembrane.Am. J.Path.,24:545-67,1048. 249. WYCKOFF,R. W. G. Ultraviolet Microscopy as a Means 256. . Cytologic Studies with the Phase Microscope. of Studying CellStructure.ColdSpringHarbor Symp. II.TheMitochondriaandOtherCytoplasmicConstitu Quant. Biol., 2:89-44, 1934. ents under Various Experimental Conditions. Ibid., pp. 250. Wycxorr, R. W. G., and EBELING,A. H. Some Ultra 569—89. violet Photomicrographs Made with Different Wave 257. . Cytologic Studies with the Phase Microscope. Lengths. J. Morphol., 55:181—35, 1938. ilL Alterationsinthe Nucleiof“Resting―andDividing 251. WYCKOFF,R. W. G.; EBELING, A. H.; and TEn Louw, A. Cells Induced by Means of Fixatives, Anisotonic Solu L.A ComparisonbetweentheUltravioletMicroscopyand tionsAcids,and Alkali.Ibid.,pp.797—811. Feulgen Staining of Certain Cells. J. Morphol., 53:189- 258. - Morphologic Changes Associated with the Death 99,1932. of Cells in Vitro and in Vivo. Ibid., pp. 1039-53.

FIG. 1.—Living fibroblast cell in tissue culture of embryo mouse skin. Phase photomicrograph, dark contrast, 1,500X, N. A. 1.25. The formed elements in the nucleus are nucleoli; and those in the cytoplasm are dark spherical ‘fat' droplets and elongatedfilamentousmitochondria. FiG. 2.—Livingoocyteof Chaetopieruspergainentacscus. Photomicrograph taken with a special polarization microscope, approximately2,000X,N. A.0.84.Themetaphasespindleis vividlydisplayedbecauseofitsbirefringence.Thepositionof some of the chromosomes can be identifiedinthe equatorial plane.(PhotographobtainedthroughthecourtesyofDr. Shinya Inoué,School of Medicine, University of Washington.) Fm. 3.—Living sarcoma cell in tissue culture of the mouse tumor, Ma 887. Phase photomicrograph with same magnifica tionandopticalconditions asin Figure 1.The nucleusislarge and contains numerous aggregations of chromatin; and spherical and elongated mitochondria are present in abundance. (Photo graphs obtained through the courtesy of Dr. John Biesele, Sloan-Kettering Institute for Cancer Research.)

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1953 American Association for Cancer Research. FIGS. 4—7.—Living sarcoma cells in tissue culture of the mouse tumor, Ma 387. Multiple-beam interference photomicro graphs with monochromatic light, X, 0.546 @,1,000 X, X.A. 0.85,andwiththecellstemporarilysuspendedinair.Theinter ference fringes appear as bright topographical or contour lines, some of which areconcentricwiththe nucleus,andthey may he intei@preted in terms of minute variations in the thickness of the cell or be used to compute the hydrous mass of protoplasm in various parts of the cell. For example, the nuclei of these sar coma cells have an average hydrous protoplasmic mass of @40X 10—12gm., a value substantially greater than that found in embryonic fibroblast nuclei. FIGS. 8, 9.—Squamous epithelial cells of the human buccal mucosa. Multiple-beam interference photomicrographs in monochromatic light, X, 0.546 @,1,000 X,N. A. 0.85, and with the cells suspended in air.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1953 American Association for Cancer Research. Fins. 10—13.—Sarcomacells and nuclei of the mouse tumor, Ma 387. Ultraviolet photomicrographs at X, 0.265 @,1,500 X, Bausch and Lomb (Grey design) reflecting objective, N. A. 0.72. Fio. 10.—Living cell in tissue culture. FIG. 11 —Cell in tissue culture fixed with acetic acid-alcohol 1111(1niounted in glycerin. Fm. 12.—('ells and nuclei isolated in 0.88 M sucrose with 0.0018 M CaCl2 and mounted in glycerin. Fm. 13.—Similar procedure to Figure 12 except cells and nu clei are mounted in 0.88 M sucrose, pH 7.0 buffer. The nucleic aci(l content per nucleus is computed from the product of the ultraviolet absorption (extinction) and the area of nuclei in optically homogeneous preparations such as Figure 13 and is 17.1 X 10-12 gin, for these sarcoma nuclei, a value very much greater than that found in embryonic fibroblast nuclei.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1953 American Association for Cancer Research. Microscopy. I: A Review

Robert C. Mellors

Cancer Res 1953;13:101-118.

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