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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 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1953 American Association for Cancer Research. 1o@ Cancer Re8earch 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 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1953 American Association for Cancer Research. MEU40RS—Microscopy. I. A Review 103 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.

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