MIRED NOMOGRAPH FOR LIGHT SOURCE CONVERSION

ORIGINAL SOURCE IN KTI

10000 MICROSCOPE LAMPS~

9000

8000

7000

6000

SOOO

4000

2S00

2000

MIRED SHIFTVALUE (I'rd)

400

Xenon ArcDaylight Quality

••~Go.:;;:c 860.;~Go>C0V.s:.••'i 8SB2 8S"i)0-

8SC

81A

82A

80D

FILMS

CONVERTED <:IV'-"""-.c:._~,:,.

Daylight Films,KODAKPhotomicrography

Color Film 2483, --=r--~KODAKEKTACHROME-X

Film, etc.

3S0

300

2S0

200

ISO

lOO

SO Type B Films, KODAKHigh Speed EKTACHROMEFilm

(Tungsten), etc.

Metal-Halide Arc

Carbon Arc

Zirconium Arc6-Volt, 5-Amp Coil Filament12-Volt Tungsten-Halogen

6-Volt Coil Filament100-Watt Coil Filament

80C6-Volt 18-Amp Ribbon ••Filament ~ 80B

6-Volt2-~ ~ 80A-Low-Voltage Tungsten .2

••~Go>CoV.r.-; 78-iD

o Type A Film, KODACHROME11Professional Film (Type A)

-SO

-lOO

-I SO

-200

-2S0

-300

-3S0

-400

The Mired Nomograph can be used to find the filter required for a particular conversion by placinga straightedge from an original source (T,) to a second source (T2) as illustrated above by thediagonal line. In the illustration, tungsten illumination at 2900 K requires an approximate - 163rnired shift to convert to daylight illumination at 5500 K. KODAK WRATTEN Filter No. 80A +82B with a mired shift value of - 163 meets this requirement.

"Many microscope illuminators are rated at 2700 to 3200 K depending upon the voltage being used. However,these values are approximate and should be used only as a guide. For critical work a practical test shouldbe run. Consult your microscope dealer for lamp information.

COVER PHOTOGRAPH:

LEFT: Hippuric crystals with polarized lighting- x 150.

Center: Ovary of amphiuma- x 100. Hematoxylin and eosinstains. KODAK EKTACHROME Film 6116, Type B (Process E-3). Adidymium filter enhanced the eosin stain color.

Right: Cross section of lily bud- x 200. KODAK EKTACOLOR Profes-sional Film, Type S.

S

6000

7000

8000

900010000

PHOTOGRAPHY THROUGHTHE MICROSCOPE

PageCONTENTS . .. 1

INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . 4

UNDERSTANDING THE COMPOUND MICROSCOPE 4GENERAL PRINCIPLES . . . . . . . 4OBJECTIVE. . . . . . . . . . . . . . . . . . . 5

Types of Objectives 5Tube Length . . . . . . . . . . . . . . . . . . . . . . . . . .. 6Optical Aberrations ' 7Numerical Aperture 7Working Distance of Objectives . . . . . . .. 8Resolving Power ..... . . . . . . . . . . . . . . . . . . . . . . . . .. 8

EYEPIECES . . . . . . . 9CONDENSER . . . . . . . .10ACCESSORIES. . . . . . .. . . . . . .10

Mechanical Stage . .. 10Field Finder . . . . . . . . . . . . . . . . 10

MAGNIFICATION . . . . . . . . . . . .11DEPTH OF FIELD 12SPECIAL TECHNIQUES 13

Microtomy . . . . . . . . . . .13Selection of Cover Glass 13Use of Oil-Immersion Objectives . 14

CAMERAS IN PHOTOMICROGRAPHY 14GENERAL CONSIDERATIONS 14CAMERAS WITH INTEGRAL LENSES 15

Magnification with a Simple Camera 16CAMERAS WITHOUT INTEGRAL LENSES. . . . . . . .. . .16

Reflex Cameras .. . 16Bellows Extension Camera.. . . .17

PHOTOMICROGRAPHIC CAMERAS .1735 mm Eyepiece Cameras 17Sheet-Film Eyepiece Cameras 18Trinocular Eyepiece Cameras . . . . . . . .. . .. 18Cameras with Adjustable Bellows 19Light Lock 20

THE GROUND GLASS 20CAMERA VIBRATION 20

MICROSLlDES . . . . . . . . . 21HOW MICROSLlDES ARE PREPARED 21

Slides and Cover Glasses . . . . . . . . . .21Tissue Sections 21Smears 23Whole Mounts 23The Mounting Media 23Chemical Crystals .24

HOW MICROSLlDES ARE ILLUMINATED 25

ISBN: 0-87985·019-1©. Eastman Kodak Company, 1974SIXTH EDITION, 1974-First Printing

Light Sources 25Illuminators . 26Methods of Illumination 27Adjusting the Aperture Diaphragm 28Adjusting the Field Diaphragm 28Kohler Illumination Diagram 29Condenser, . . . . . . . . . . 30Built-In Illumination 30Image Brightness and Neutral Filters 30

COLOR PHOTOMICROGRAPHY . .31FILMS. . . . . . . . . . . . . . . . . . . . . . . .. . 31

Kodak Color Films for Use In Photomicrography 31Selection of Color Films. . . . . . . . . . . . . 31Rendition of Stain Color 33Processing Color Films ' 34Increasing Color-Film Speed 34Kodak Processing Service for Increased Speed 34

FILTERS 35KODAK Light Balancing Filters , .. 35KODAK Color Compensating Filters 36Neutral Density Filters 36Color Plates. . . . . . . . . . . . . . . .. . 37-40

FACTORS AFFECTING COLOR BALANCE. . 41Color-Ternperature Variance .. . . . . . . . . . . . . . . .. 42Heat Absorbing Filters 42Ultraviolet Radiation . 42Neutral Density Filters . . . 42Biological Stains .. . 42Mounting Media 43Chromatic Aberration . . . . . . . . . . . . .43Film-Emulsion Variance ... 43Reciprocity Effect .44Miscellaneous Factors. . 44Color Balance Correction Chart. . . . . . . . . . . . . . .46

BLACK-AND-WHITE PHOTOMICROGRAPHY 47PROPERTIES OF PHOTOGRAPHIC MATERIALS 47

Resolving Power and Graininess . . . . . . . . . 48Exposure Latitude . . . 49Development Latitude . . . . . . . . . . . .. . 49Film Speed. . 50

SHEET FILM AND PLATES 50ROLL FILMS. . . . . . . . . . . . . . 50FILTERS IN BLACK-AND-WHITE PHOTOMICROGRAPHY 51

Increasing Contrast . . . . . . . . . . . . . . . . 51KODAKWRATTENFilters 52

PROCESSING THE NEGATIVE 52PRINTING . . . . . . . . . . . 53

EXPOSURE METHODS 54EXPOSURE TESTS 54

Roll Film . 54Sheet Film . . . . , ,55

EXPOSURE CALCULATION . 55Judgment of Exposure 55

2

Filter Factors 56Exposure Meters 56Making Exposure Readings 57

EXPOSURE RECORD 58COMMON FAULTS IN PHOTOMICROGRAPHY 58

MORE-COMMON FAULTS 58Unsharp Image 58Hazy Image .. . . . . . . . . . . . . . . . 59Uneven Illumination 59Low Contrast. . . 60

LESS-COMMON FAULTS 60Too Much Contrast 60Poor Resolution 60Bright Spot in Field. . 60Shutter-Blade Image 60Out-of-Focus Spots 60

SPECIALIZED TECHNIQUES AND APPLICATIONS 61SPECIALIZED TECHNIQUES 61

Contrast Systems. . 61Darkfield Method 61Stop-Contrast Method 62Phase-Contrast Method 63Interference-Contrast Methods 66Francon System 66Polarizing Method . .. 67Stereo Method . . . . . . . . . . . . 68

SPECIALIZED APPLICATIONS 69Fluorescence Photomicrography 69Ultraviolet Photomicrography .70Infrared Photomicrography 71Photomicrography of Chromosomes 72Photomicrography of Autoradiographs 72Metallographic Photomicrography 73Photomicrography of Microelectronic Circuits 74Recording Replicas . . . . . . . . . . . . . . . . . 74

GLOSSARY OF TERMS USED IN PHOTOMICROGRAPHY .. 75

REFERENCES 76

Please Note: Since the last edition of Photography Through theMicroscope was published, several significant developments inKodak films for photomicrography have occurred. In this editionyou will find applicable technical information on KODAKPhotomicrography Color Film 2483 and KODAKPhoto-micrography Monochrome Film (ESTAR-AHBase) SO-41 O.Complete technical information regarding processing, physicalproperties, and sensitometric data is available on requestfrom Department 412-L, Eastman Kodak Company,Rochester, New York 14650. Ask for Publications No. P-302 (for2483 Film) and No. P-304 (for SO-410 Film).

THUMB INDEX.

Punched to fit the Binder for Kodak Technical Information (W-4),sold by photo dealers.

"'!"

UNDERSTANDINGCOMPOUNDMICROSCOPE

CAMERAS

MICROSLlDES

COLOR PHOTO-MICROGRAPHY

BLACK-AND-WHITEPHOTO-MICROGRAPHY

EXPOSUREMETHODS

COMMONFAULTS

SPECIALIZEDTECHNIQUES

GLOSSARY

REFERENCES

INTRODUCTIONThe technique of making photographs by means ofa compound microscope is called photomicrography.It involves the coupling of a camera to a microscopeand the efficient use of illumination. The quality ofa "photomicrograph" depends almost entirely on thequality of the image produced in the microscope. Acomplete understanding of the principles and opera-tion of the microscope is therefore the first prerequi-site to producing good photomicrographs.

Photomicrography involves the production of largerecorded images of very small, microscopic objects.Photomicrography should not be confused with mi-crophotography, which involves making extremelysmall photographic images of large objects.

Although any type of microscope can be used tomake photomicrographs, the most common is the"brightfield" illuminated microscope. The specimen,or subject, is either dark or colored and appearsagainst a bright, almost white, background. Hencethe term "brightfield." This microscope is found inall biological laboratories and in all schools wherebiology is taught.

In order to use a microscope efficiently, you musthave a good working knowledge of the instrument.This knowledge includes the capabilities and limita-tions of the optical components, how the microscopeis adjusted, how the illumination of the specimen isapplied, and even how specimens are prepared forexamination. If you intend to make photomicro-

graphs, you should have some knowledge of pho-tography, including an understanding of sensitizedmaterials and how a camera can be suitably attachedto a microscope.

Photomicrographs are made for a variety ofreasons,but the principal one is for teaching. It is much easierto project a color slide for class viewing than to haveeach member peer into the microscope. A photomi-crograph is often used as a research record, or toillustrate a particular phenomenon or condition ina published article. Photomicrographs of metals arevery often made to supplement engineering or metal-lurgical reports.

Photomicrography is used in practically every fieldin which a microscope is the fundamental tool-when-ever an enlarged, recorded image would prove useful.Photomicrography is important to teachers and stu-dents in high schools and colleges for laboratorystudies in biology, botany, zoology, anatomy, etc. Itis probably of even greater importance in industry,medical research, pathology, criminal investigation,agriculture, and forestry.

Whatever the purpose of a photomicrograph, youshould remember that the recorded image is no betterthan the image produced in the microscope, exceptthat contrast can be enhanced photographically.Thus, the first step in photomicrography is tounderstand the compound microscope and how touse it to best advantage in visual applications.

UNDERSTANDINGTHE COMPOUND MICROSCOPE

GENERAL PRINCIPLESA "simple" magnifying system uses a single lens unitto form an enlarged image of an object for viewingor for projecting. An example of the latter is slideprojection where a transilluminated transparency isenlarged to form a real image on the screen. If thescreen were removed and a suitable lens locatedbehind it and focused in the plane of the screen, thelens would form another image of the slide. This isthe basic principle of a "compound" magnifyingsystem-the one found in a compound microscope.The observer looks at the first, or primary, image witha lens that produces an enlarged secondary image,called a virtual image. That is the image the eye

4

perceives. (See Figure 1.) Another feature of micro-scopes is that the lenses are of relatively short focallengths. The shorter the focal length, the greater themagnification at a given image distance. In a micro-scope a high, two-stage enlargement is attained overa relatively short optical path with such short focuslenses.

The first lens in a microscope is called the objective,since it is near the object. This lens projects amagnified image to a fixed position. The amount ofmagnification that the objective produces at this fixeddistance is called its "magnifying power." The magni-fying power of an objective is classified as 5x, 10x,20x -up to 100x. The projection of the primary

EYE - ~ - ~ 'OBJECTIVE 0-ALA ~- - r ><---O<, :::;l.-::::-r:, - LIGHT~-- ~- -~/ .-~EYEPIECE - --::. SUBJECT----

- - _ CONDENSERIMAGE FORMED - - _BY OBJECTIVE

VIRTUAL IMAGE

FIGURE 1-Simplified Diagram-Principle of compound micro-scope. The intermediate image formed by the objective is enlargedby the eyepiece. The virtual image is seen by the eye. Alternatively,the eyepiece can project a real image into a camera.

image takes place within the body tube of the micro-scope; the distance from the back focal plane of theobjective to the primary image is called the "opticaltube length."

The second lens is placed in the body tube abovethe primary image. This lens is called the "eyepiece,"and forms a secondary, further enlarged image withinthe microscope. The eyepiece (often called the "ocu-lar"), like the objective, is also classified in terms ofmagnifying power, and could be 5X, 10X, or higher-upt025X.

The total amount of image enlargement, or"magnification," produced within the microscope isfound by multiplying the magnifying power of theobjective by that of the eyepiece. A 10x objectiveand a 10x eyepiece then produce a "visual"magnification of 100x, or x 100 as it is commonlywritten.

As light rays emerge from the eyepiece they con-verge to a point called the "eyepoint." This is theposition which the eye normally seeks when you lookinto a microscope to see the whole image field in themicroscope. The eyepoint is also often referred to asthe "Ramsden disk" or "Ramsden circle." The dis-tance from the eyepoint to the virtual image, or finalimage, within the microscope system is 250 mm (10inches).

Instead of looking into the microscope, you canallow the image to be projected from the eyepieceto the film plane. If the film plane, or camera, is 250mm from the eyepoint, the magnification at the filmplane will be the product of the eyepiece and objectivemagnifications.

The very fine details in a micro-specimen must bedistinguished by the objective lens. This lens mustbe of high enough quality to "resolve" these details

and produce an efficient primary image. The mainpurpose of the eyepieceis to further enlarge the imageformed by the objective. This image can be degradedby an eyepiece of low quality, but it cannot beimproved (as far as image resolution is concerned),regardless of eyepiece quality.

OBJECTIVETypes of ObjectivesMicroscope objectives are usually classified in termsof magnifying power, mechanical tube length, nu-merical aperture, and their degree of optical correc-tion, that is, whether they are achromats, apochro-mats, or fluorites. Some of this information and thefocal length is usually imprinted on the objectivemount.

The achromat is the most common type ofobjectiveused on any microscope. It is also the least expensive.An achromat is corrected for spherical aberration forone color only, usually yellow-green. IDis correctedfor chromatic aberration for two colors. If an achro-mat is used with white light, color fringes may appearin the outer margins of the image. When black-and-white film is used, these fringes may contributetoward a fuzzy image. If light of one general color(such as green) is used, the image will be muchsharper. When monochromatic green light is used-that is, light of a single wavelength or a narrow bandof wavelengths-the image will be much better.Therefore, the best image will be obtained in photo-micrography when a green filter is placed in the lightbeam. Images ofinferior quality may result when lightof longer or shorter wavelength is used.

An apochromat represents the finest microscopeobjective available. It is corrected for spherical aber-ration for two colors (blue and green) and for chro-matic aberration for the primary spectral colors ofred, green, and blue. Because of this high degree ofcorrection for aberrations, the apochromat is particu-lady suitable for color photomicrography and for thebest resolution of fine details and the finest imagequality. Apochromats, however, achieve their finestcorrection when used with matched compensatingeyepieces.

The third type of objective is known as a fluorite,or "semiapochromat." Objectives of this type arebetter than achromats, but not quite as good asapochromats. They represent a compromise, both inquality and in cost. Like the apochromats, theyshould be used with compensating eyepieces for bestperformance.

All types of objectives mentioned above exhibit acertain amount of "curvature of field." The imagemay be sharp in the center of the field, but sharpness

5

FIGURE 2-Elastic fibers in human artery, x 40, (a) Curvature of field; (b)Corrected curvatu re of field.

falls off toward the periphery. This effect can beovercome to some extent in photomicrography bylimiting the recorded area to the center of the field,when possible. It can also be reduced appreciably byusing a special eyepiece, described further on.

However, it is better to use "flat-field" objectives,made in the above types. Their names are precededby a prefix, "plano" or "plan" -such as plano-achromat, plan-achromat, plano-apochromat, orplan-apochromat. Curvature of field has been cor-rected in the optical design of these objectives, andthey are particularly suitable for examination orphotomicrography of large fields.

"Flat-field" objectives are used in conjunction withcompensating eyepieces or eyepieces designatedspecifically for them. Focus is then relatively uniformfrom the center to the periphery of the field. (SeeFigure 2.)

Most objectives intended for use with transmittedlight must be used with "covered" objects. That is,a "cover glass" must be mounted over the specimen.The thickness of the cover glass is specified as either0.17mm or 0.18mm, depending on the objective. Thespecification for cover-glass thickness results from thefact that objectives are corrected for spherical aberra-tion only when used with a cover of the rightthickness. Deviation from this thickness can result

6

in appreciable overcorrection or under correction forspherical aberration, particularly with high-aperture,dry objectives.

Objectives intended for use with reflected lightrequire "uncovered" objects. No cover glass is usedover the specimen. This principle applies mainly tometallurgical microscopes and metal specimens.

Most of the objectives used in a brightfield micro-scope are considered "dry." This means that only airexists between the front of the objective and thespecimen slide. Some objectives are of the "immer-sion" type; a liquid medium, usually a special oil,is placed between the objective and the specimen slide.

Tube LengthThe objective is screwed into the bottom part of themicroscope body-tube, while the eyepiece is insertedinto the top "draw-tube" of the microscope. Thedistance between the insertion position for the objec-tive and the top of the draw-tube is called the"mechanical tube length."

Some manufacturers specify a mechanical tubelength of 160 mm, whereas others specify 170 mm.Objectives are usually designed optically for a specifictube length. If they are used at a different tube length, ~they will not retain their intended optical efficiency.They should not, therefore, be interchanged on

different microscopes if the specifications are notknown. Image quality may suffer if this practice isfollowed. Some microscopes are equipped with ad-justable draw-tubes, so that mechanical tube lengthcan be altered to comply with the specification foran objective. These microscopes are rare, however.Most modern microscopes have permanent tubelengths designedby the manufacturers of their optics.The best practice for a novice is to use objectivesof one make only, on a microscope of the samemanufacture.

Some microscopesemploy objectiveswith "infinity"correction. Mechanical tube length is not fixed. Suchobjectives should never be used on a microscope ofother manufacture, nor should other objectives beused on a microscope designed for infinity-correctedoptics. Image quality will be very poor in either case.

When an objective is used at other than its specifiedtube length, its magnifying power will be differentthan indicated. For example, a 10X objective designedfor 160mm tube length will magnify more than 10X

if used at a 170mm or longer tube length; conversely,magnifying power will be reduced at lengths shorterthan the intended tube length.

In addition to erroneous magnifying power, poorimage quality will also result if correct tube lengthis not used. Too long a tube length will result inovercorrection and too short a tube length will resultin undercorrection; both will result in poor definition(imagesharpness).

Optical AberrationsIf a simple, positive lens were used to project theprimary enlarged image, the quality of the imagewould be very poor due to inherent optical aberra-tions. Aberration is a failure of a lens to produce exactpoint-to-point correspondence between an object andits image. In order to improve image quality it isnecessary to design lenses so that this aberration iscorrected as much as possible. Microscope objectivesare corrected for spherical and chromatic aberrations.

Spherical aberration occurs when light rays pass-ing through the central and outer portions of a lensare not brought to focus at the same distance fromthe lens. This condition arises because light is refract-ed more at the edge of the lens with gradual reductionto zero at the optical center. The image of a pointis not reproduced as a point but as a larger, circulararea. The image of an object that is com-posed of an infinite number of points cannot possiblybe sharp as long as spherical aberration is present.Also, white light is composed of all colors-which arerefracted and focused differently, depending onspecificwavelength. It is possible to correct spherical

FIGURE 3-Left, a simple, uncorrected lens focuses three primarycolors at different points on the optical axis. Right, an achromaticlens will focus two primary colors, usually green and red, at thesame point on the optical axis.

aberration for one color, but it might not be correctedfor another color. This aberration can be partiallycorrected, however, by formation of an image at afixed distance; thus the specification of a mechanicaltube length. See Figure 3 for a diagram.

Chromatic aberration occurs because the focallength of a simple lens varies noticeably with wave-length. Blue rays are shorter in wavelength and focuscloser to the lens than green or red rays. The lensis unable to bring light of all colors to a commonfocus.This effect can be corrected to a practical degreein the design of a microscope objective. The amountof correction is specifiedfor each type of objective.

The sharpness of the image produced in a micro-scope is limited by the degree of correction for spheri-cal aberration. If chromatic aberration is present inthe microscope objective, color fringes will be evidentin the image.

Numerical ApertureIn conventional photography, photographic lenses arealways classified in terms of {-value, which is anindication of light-gathering power. A "fast" lensmight be fll.4 or f/2.0. Slower lenses might havef-values such as fI4.5, fI5.6, or fiB. The (-value ofa photographic lens is determined by dividing thefocal length by the apparent diameter of the dia-phragm opening.

Microscope objectives, however, are not classifiedin terms of f-value. We are not concerned in micros-copy with light-gathering power, but with the abil-ity of the lens to distinguish fine structural detailsin a specimen. This is the principal reason for obtain-ing a magnified image-to see the details within thespecimen. This ability is expressed in terms of nu-merical aperture, or "N.A." as it is usually called.

Mathematically, numerical aperture is expressed asthe product of the refractive index (n) for the mediumin which the lens operates and the sine of one-halfthe angular aperture of the lens (u). The formulathen reads: N.A. = n sine u. The sine relationship.is a simple means for expressing the size of an angleas a number, the ratio of two sides of a right triangle.

7

The numerical aperture for any objective is alwaysimprinted on its mount. A 10 X achromatic objectiveusually has an N.A. of 0.25, and a 20 X achromatwill usually have an N.A. of 0.50. Apochromaticobjectives have higher numerical apertures thanachromats of similar magnification.

Some objectives are to be used "dry"; air is themedium between the front of the lens and the speci-men slide. Air has a refractive index of 1.00, so thatthe formula for numerical aperture for a dry lens willread: "N.A. = 1.0 times sine u" or just "sine u." Thelargest numerical aperture for a dry lens is about 0.95;it never reaches 1.00.

In order to have an objective of numerical apertureequal to or greater than 1.00, a liquid medium mustbe placed between the lens and the specimen slide.An "immersion oil" is often used for this purpose;its refractive index is usually about 1.51. Objectivesintended for immersion always have an indication onthe lens mount. There are also special immersionlenses, which require distilled water (n = 1.30) orglycerin (n = 1.48). These are rare, however, and arenot generally used with brightfield microscopes. Thenumerical aperture for oil-immersion objectivesranges from 1.00 to 1.40.

Working Distance of ObjectivesThe distance between the front lens of an objectiveand the top surface of the cover glass on a specimenslide is called the "working distance" for the objective.Objectives are designed optically for use with a coverglass of specific thickness, usually either 0.17 mm or0.18 mm, depending on manufacture. Any deviationfrom the specified thickness when a dry objective isused will introduce spherical aberration, which mayaffect image sharpness. A cover glass that is too thickwill cause overcorrection for spherical aberration.Conversely, too thin a cover glass introduces under-correction. The effect is most noticeable with high-aperture, dry objectives.

Working distance is directly related to the allow-able movement of the objective in obtaining criticalfocus of the specimen image. It decreases rapidly asthe focal length of the objective decreases; for oil-im-mersion objectives, the working distance is measuredin fractional parts of a millimeter. Cover-glassthickness is of lesser importance as far as introducingspherical aberration is concerned, since the oil hasabout the same refractive index as the cover glass.The allowable movement of an immersion objective,however, is affected by cover-glass thickness. Moreworking distance is obtained when the cover glassis thinner than specified or when no cover glass isused, as is often the case with blood smears. A thicker

8

cover glass will decrease the working distance; thethickness can then approach the point where focusof the specimen may become impossible. Other minorfactors which can affect the allowable working dis-tance for an immersion objective are the viscosity ofthe oil and the amount of mounting medium betweenthe cover glass and the specimen. The effects of thesefactors are small, but when they are added to thecover-glass thickness, they contribute toward a re-duction in working distance.

The following table shows the average workingdistances for different objectives of various focallengths. The figures can only be considered approxi-mate, since objectives of different manufacture willdiffer in characteristics. They are, however, fairlytypical.

TABLE INumerical Aperture (N.A.) vs Working Distance

Focal WorkingObjective Type N.A. Length Distance

(mm) (mm)

Achroma! 10 x 0.25 16 7.70Apochromat 10 x 0.30 16 4.85Achromat 20 x 0.50 8 1.60Apochroma! 20 x 0.65 8.3 0.50Achromat 45 x 0.85 4 0.30Apochromat 47.5 x 0.95 4 0.18Achromat 97 x oil 1.25 1.8 0.13Apochromat 90 x oil 1.30 2.0 0.12

Resolving PowerThe ability of the optical system in a compoundmicroscope to distinguish and separate fine structur-al details in a specimen is known as "resolvingpower.':" It is limited by the N.A. of the objective,but it also depends upon the working N.A. of thesubstage condenser. The higher the N.A. of the sys-tem, the greater will be the resolving power. Resolvingpower is also dependent on the wavelength of light.The shorter the wavelength, the better the resolvingpower. The process of making fine details visible iscalled "resolution," and is usually expressed in termsof micrometers, formerly called microns. (One mi-crometer, !lm, equals 0.001 millimeter.)

The formula for computing resolving power is

R = 2 ~.A. where R is resolving power, in microm-

eters; A is wavelength, also in micrometers; and N.A.is, of course, numerical aperture. When the opticalsystem is correctly aligned and adjusted, the N .A.of the objective can be used in the formula.

"However, tills value is subjective. An image may be unsharp butmay still be considered resolved.

With regard to wavelength, the finest resolvingpower is obtained with ultraviolet radiation, whichrepresents the shortest usable wavelengths. In thevisible region of the spectrum, blue light has the nextshortest wavelength, then green, and then red. Ifwhite light is used, the applicable wavelength is thatfor green-the middle of the visible spectrum andregion of highest visual acuity.

If the dominant wavelength for green light (.550micrometer) and an achromat of high N.A. (1.25)are applied in the formula, the resultant resolvingpower is 0.22 micrometer. With green light, then,it is possible to resolve particles as small as 0.22micrometer (0.00022 mm) using an achromat. Thefollowing table shows the effective resolving powerwith several achromatic objectives of different nu-merical apertures when green light is used.

Resolving Power for Achromats

Magnifying Power 4x 10x 20x 45x 100xoil

Numerical Aperture 0.10 0.25 0.50 0.85 1.25':'

Resolution (micrometers) 2.75 1.10 0.55 0.32 0.22

"Hiqhest N.A. for an achromat.

The resolving powers for apochromatic objectivesare better than those for comparable achromats, sincethey are more highly corrected optically and havehigher numerical apertures. The following tableshows pertinent, comparable data for apochromats,again with green light. (Apochromats lower than 10X

usually are not manufactured.)

Resolving Power for Apochromats

Magnifying Power 10x 20x 47.5x 90x oil

Numerical Aperture 0.30 0.60 0.95 1.40':'

Resolution (micrometers) 0.92 0.46 0.29 0.19

*Highest N.A. for an apochromat.

Resolving power for an apochromat of the highestN.A. (1.40) .with light of different wavelengths isshown in the following table. This indicates theimprovement of resolution with shorter wavelengthsof light.

Change in Resolution with Wavelength

Green Blue Ultraviolet

Wavelength (micrometers) .546 .365.436

Resolution (micrometers) 0.130.19 0.16

Therefore, it is possible to resolve particles as smallas 0.13micrometer (0.00013mm) by using ultravioletradiation.

Although it is not expected that everyone will needto determine the exact resolving power for any objec-tive, it is important to understand the capabilitiesand limitations of a lens in order to use it to bestadvantage. This knowledge should help in the selec-tion of a lens or of the type of light needed tophotograph a specimen most efficiently.

EYEPIECESThe purpose of the eyepiece in a compound micro-scope is to enlarge the primary image formed by theobjective, and to either render it visible as a "virtual"image in the microscope or project it as a "real" imagethat can be recorded in a camera.

For visual examination of the image in the micro-scope, any type of positive eyepiece can be usedsatisfactorily. The most common eyepi ces are called"Huygenian" and "wide field." Although Huygenianeyepieces can be used with low- and medium-powerachromatic objectives in photomicrography, colorfringing and poor image quality may result if theyare employed to make high-power photomicrographs.The principal advantage of "wide field" is in visualwork, to scan a slide in order to find a suitable fieldfor photography.

Whenever an apochromatic objective is to be uti-lized for photomicrography, the eyepiece should beof the "compensating" type in order to provide thehigh image quality and flat field ofwhich the objectiveis capable. This is especially true of plan- or plane-ob-jectives. Apochromats inherently produce what iscalled "chromatic difference in magnification." Com-pensating eyepieces are overcorrected for this effectand will produce images free from color defects. Thistype of eyepiece can also be used effectively withsemi-apochromats and the high-power achromats.

Eyepieces designated as "Hyperplane" and "Peri-plan" are excellent for black-and-white photomicrog-raphy, particularly with filtered light. They will pro-vide reasonably flat fields, thus overcoming the effectof "curvature of field," which is inherent in image-for-mation by most objectives.

Some eyepieces are intended specifically for photo-micrography; they are, appropriately, called "photoeyepieces." Besides producing fair flat-field results,they are also color-corrected and, therefore, of advan-tage in color photomicrography.

Eyepieces, generally, are produced with differentmagnifying powers, ranging from about 4 X to 25x ..The most common are those with a magnifying powerof 10X or 15x.

9

CONDENSERThe third optical component of a compound micro-scope is called the "substage condenser." The speci-men slide is placed on a platform beneath the objec-tive. This platform is called the "stage"; the substagearea is beneath the stage. In some microscopes, lightis reflected from a mirror, and through the substagecondenser to illuminate the specimen. Many micro-scopes today have built-in substage illumination, soa separate mirror is not used. In either case, however,the light is directed through the substage condenserand converges to a very small area at the positionof the specimen. The light rays diverge as they passthrough the specimen and form an inverted cone,whose base is just large enough to fill the apertureof the objective. The size of the light beam enteringthe condenser is controlled by the opening of avariable diaphragm beneath the condenser. This iscalled the "aperture diaphragm." Both the focus ofthe condenser and the opening of the cliaphragm areadjusted for Kohler illumination. (See page 29.)

Three types of substage condensers are available.The Abbe condenser is the simplest and least expen-sive type. It is provided on most microscopes unlessanother type is specified.This condenser is uncorrect-ed for optical aberrations, and may have either twoor three lens elements. The top element can some-times be removed, either by unscrewing it or byswingingit away on a pivot, for low-powerwork (10X

objective or lower). For medium- and high-powerwork, it remains in place. If the top element is notremoved, it may be impossible to illuminate the entirefield seen by the low-power objective. Consult thesection, "Chromatic Aberration," on page 43 for acliscussionof condenser adjustments to obtain goodcolor balance.

The second type of condenser is called "aplanatic,"and is optically corrected for spherical aberration.This type is not available from all manufacturers,but is ofbetter quality than the Abbe condenser.

The "achromatic" condenser is the best availableand is corrected optically for both spherical andchromatic aberrations. It is highly recommended forphotomicrography, particularly with color film. Boththe aplanatic and the achromatic condensers aredivisible, like the Abbe condenser, for use in low-,meclium-,and high-power work.

Any substage condenser should have centeringscrews so that it can be correctly aligned with respectto the objective. This is an important feature inphotomicrography and should definitely be consid-ered when a microscope is purchased.

The numerical aperture of a complete substagecondenser should always be, and usually is, equal to

10

or greater than the largest numerical aperture of theavailable objectives. The opening of the substagecliaphragmcan then be adjusted to match the workingN.A.to that of the objective in use.

When an objective lens is intended for oil immersionand its numerical aperture exceeds 1.00, immersionoil of the same type should be placed between themicroscope slide and the top lens of the condenser.If this step is omitted in high-power photomicrog-raphy, the numerical aperture of the condenser willbe limited to 1.00,the refractive index of air. Becausethe effective numerical aperture of the objective isalso limited to 1.00,the resolving power of the systemwill be lower than when oil is placed on the condenser.

ACCESSORIESMechanical StageThe mechanical stage is one of the most usefulaccessories for a compound microscope. The stage isa device both for holding a specimen slide firmly inposition and for moving it smoothly, either back andforth or right and left. This feature is of particularimportance, since it enables the user to scan a slideeasily in order to locate a suitable field for pho-tography. Then, if the mechanical stage is graduated,it becomes possible to note the graduation figures forthe position of a specific field for future reference,in case the same fieldmust be relocated and, possibly,rephotographed.

A mechanical stage is not always included with amicroscope, so it is necessary for the buyer to specifythat one be included. If a microscope in use does notinclude this type of stage, it is often possible topurchase an attachable stage separately.

A rotating, mechanical stage is the most useful ofall stage types. The rotating stage enables the userto adjust image composition to better suit the rectan-gular format of the film frame. However, if a rotatingstage is not available and the camera over the micro-scope can be rotated, the same effect can be achieved.

Field FinderIn adclition to a graduated mechanical stage, anotherdevice for accurately relocating fields on a specimenslide is called a "field finder." This is a microscopeslide containing a grid with rectangular coordinates,which are numerically indentified.

When a particular field has been located on aspecimen slide, the slide is carefully removed fromthe mechanical stage and the field-finder slide is putin its place. The coordinates for the visible field arethen recorded for future reference. Then, wheneverthe original field must be relocated, the field-finder

slide is placed on the stage and the stage is adjustedto the recorded coordinates. The specimen slide isthen substituted for the field finder, and the originalfield is visible.

If a graduated mechanical stage is used to locateand relocate fields, the technique is satisfactory aslong as the same microscope or one with an identicalstage is used. Graduated stages, however, vary fromone manufacturer to another, and often betweenmicroscopes of the same manufacture. The field-finder slide provides the advantage of being inter-changeable between microscopes.

Field-finder slides can be purchased either frombiological supply firms or microscopemanufacturers.

MAGN IFICATIONIn photomicrography, image size (or magnification)is controlled by the magnifying powers of the objec-tive and eyepiece, and by the bellows extension (oreyepiece-to-film distance). When you look into amicroscope, the visual magnification is equal to theproduct of the magnifying powers of the objectiveand eyepiece.When the micro-imageis projected fromthe eyepiece, this magnification is reproduced at adistance of 10 inches (250mm) above the exit pupilof the eyepiece. If the distance is greater than 10inches, magnification will be increased proportion-ately. If the distance is less than 10inches, magnifica-tion will be decreased proportionately. For example,if the objective is designated as 20X and the eyepieceas 10x, visual magnification in the microscope willbe X 200.Then, if the image is projected to a groundglass screen (or film plane) 20 inches above theeyepiece, magnification will be x 400. But, if theprojection distance is only 5inches, magnification willbe only X 100,etc.

When the magnifying power of either the objectiveor the eyepiece is unknown, or when the exacteyepiece-to-filmdistance is unknown, it becomes nec-essary to measure magnification. This can be doneby means of a calibrated scale called a "stage microm-eter." Such a scale consists of finely ruled lines ona microscope slide, with a finite separation in decimalparts of either inches or millimeters. In use, the stagemicrometer is placed on the microscope stage and itsmagnified image is projected to the ground glass orfilm plane ofthe camera. By measuring the separationof lines on the ground glass and comparing it withthe original separation on the slide, image size canbe measured directly. When no ground glass is used,the image of the lines can be recorded on film andmeasured there after the film is processed (Figure 4).

It is sometimes desirable to record a microm-

FIGURE 4-Magnified image of eyepiece micrometer disk.

eter-scale image simultaneously with a specimenimage. This can be done in either of two ways. Thestage micrometer image and the specimen image canbe recorded together by double exposure: first oneimage is exposed, then the other, on the same film.In this case, it is advantageous to have a micrometerscale with white lines on a dark background. Anothertechnique is to use a focusingeyepiecewith a microm-eter scale included. In this case, the micrometer scalein the eyepiece is calibrated with a stage micrometerbeforehand so that the line separations will be known.Then, both eyepiece scale and specimen image arerecorded in one exposure.

The usable limit of magnification that can beachieved with any combination of objective, ocular,and bellows draw is determined by the numericalaperture, or N.A., of the objective. Each objective hasits numerical aperture engraved on the mount. Anachromat of 6 mm focal length might have an N.A.of 0.65; a 4.3 mm fiuorite, an N.A. of 1.00. N.A. isimportant in figuring the capability of objectives.

If magnification in the microscopeis carried beyondthe point of resolution-that is, no more detail isresolved-we have a condition called "emptymagnification." The N.A. of an objective can be usedto compute maximum usable magnification by a "ruleof thumb." If the N.A. figure is multiplied by 1000,we will obtain a figure which, although not exact,is closeenough for practical purposes. An N.A.of 0.65,.then, will allow usable magnification up to X 650. Ifmore magnification and better resolving power are

11

A

FIGURE 5-(a) Empty magnification because of insufficient resolvingpower of objective (16 mm achromat N.A. 0.25), x 500. (b) Significantmagnification (8 mm apochromat N.A. 0.65), x 500.

wanted, a lens with a higher N.A. must be used (seeFigure 5).

TABLE 2Usable Magnification for Some Common Objectives

Common ObjectivesInitial

MagnilicationWorkingDistance

UsableMagnification

NumericalAperture

Achromat (40 mm)Achromat (16 mm)Apochromat (8 mm)Apochromat (4 mm)Annchromat 2 mm)

3x10x20x40X90x

x120x250x650xl000x2000

0.120.250.650.951.30

35mm16mm0.7 mm0.12mm0.11 mm

A condition of empty magnification may also resultafter a photomicrograph has been made. This oftenhappens when an enlargement is made from a nega-tive that is already at its limit of resolution. Anyfurther increase in magnification, such as that pro-duced by an ordinary photographic enlarger, willresult in empty magnification. If carried to the ex-treme, say 5 X to 10X enlargement, the print imagewill exhibit very poor quality.

When a photomicrograph is to be reproduced asan illustration in a book, magazine, etc, the bestpractice is to utilize a higher-than-necessary, butefficient, magnification in making the photographicnegative or original. From this original an illustrationcan be prepared about 1/5 larger than the finaljournal reproduction size. This practice seems tominimize loss of image quality when the illustrationis reduced to 4/5 its size in the reproduction process.

12

DEPTH OF FIELDIn ordinary photography, depth of field is consideredas the distance from the nearest part to the farthestpart of the subject in acceptable focus. When thesubject is a considerable distance from the lens, depthof field is measured in feet, but as the subject isbrought closer to the lens, the depth of field decreasesrapidly. The image of the subject, however, is alwayssmaller than the subject itself.

In photomicrography, as in microscopy, the imageis considerably greater in size than the specimen.Depth of field is exceedingly short and is expressedin micrometers. The higher the magnification and thehigher the numerical aperture, the shorter the depthof field. The following tabulation shows the approxi-mate depth-of-field figures for dry objectives ofvarious numerical apertures. Depth of field also de-creases as wavelength decreases; so computation foronly one wavelength, green, is shown here.

Variation in Depth of Field with Change in N.A.

NA O~ O.~ O.~ O.~ O.~ 0.%Depth (in fLm) 8.00 5.50 2.00 1.00 0.25 0.10

It is clear from the table that depth of field isextremely small for objectives of high N.A. Focus ofthe image with objectives of high N.A. becomes verycritical, and just a touch of the fine-focus adjustmenton the microscope may cause the image to go outof focus. This veTYlimited field-depth also presents

a problem when the specimen is too thick for theobjective in use. The out-of-focus areas within thespecimen will scatter and diffuse the light passingthrough, affecting the recorded image quality in photo-micrography. Ideally, the specimen should be nothicker than the usable depth of field for a givenobjective.

The problem is not so acute when the specimenis examined visually in the microscope, since the eyecan rapidly accommodate for the actual depth. Then,too, the microscopist constantly changes focus fromtop to bottom of the specimen in order to see theentire structure.

In photomicrography, however, the image is re-corded on the film in one plane. Any pronounceddifference between depth of field and specimenthickness may affect the quality of the recordedimage.

Selection of Cover GlassThe quality and thickness of the cover glass arecontrollable factors of great importance for the bestimage quality. For best results in photomicrography,cover glasses should be scrupulously clean and withina thickness range of0.16mm to 0.19mm.

This thickness-range recommendation is made be-cause an objective lens is corrected optically forspherical aberration, so it will render the best-qualityimage when the proper thickness of cover glass is used.This factor is particularly critical with any high, dryobjective lens that is corrected for a cover glass 0.17mm to 0.18mm thick. An excessivethickness of coverglass produces appreciable spherical overcorrectionand loss in image sharpness. Cover glasses that aretoo thin produce undercorrection for spherical aber-ration, with similar loss in quality (see Figure 6). A"correction collar" is usually incorporated in the

FIGURE 6-(a) Spherical aberration induced by too thin cover glass;(b) Aberration reduced by the use of the proper, specified cover glass.

SPECIAL TECHNIQUESMicrotomyOne important fact that is often overlooked in micros-copy and photomicrography is that the specimen isa part of the optical system. The specimen is usuallya stained tissue section, a smear, or some otherpreparation mounted on a microscope slide beneatha cover glass.

It is important that tissue sections be as thin andas uniform in thickness as possible. Although thesections made routinely in most histology laboratoriesare several ias: thick, they would be better for photo-micrography if they were made with a thickness inthe order of 1 or 2 iui», or less. This, however, is aproblem of microtomy and will not be covered indetail here.

mounting of a high, dry apochromatic objective. Itcan be adjusted to compensate for cover glasses thatrange in thickness from 0.15 mm to 0.25 mm. Thiscollar is not included, however, in achromat, fluorite,and most phase-contrast objectives.

Cover-glass thickness is not critical with oil-immer-sion objectives when the refractive index of the oilis equal to, or very close to, that of the cover glass.This refractive index may vary, however. With im-mersion objectives of highest aperture (N.A. 1.3 and1.4), it is still important to use cover glasses withinthe ideal thickness range. Cover glasses of other-than-specified thickness can be utilized in visual micro-scopic examinations, but since you cannot always besure when a photomicrograph will be required, it is'good practice to use a cover glass of the correctthickness whenever a microslide is prepared.

13

Use of Oil-Immersion ObjectivesIn order to achieve high magnification and highresolving power in a photomicrographic system, it isnecessary to use oil-immersion objectives. When anobjective is intended for oil immersion, the word "oil"will appear imprinted on the mount. The N.A. of theobjective is also shown.

A drop of oil is placed on the cover glass of thespecimen slide. If the N.A. of the objective exceeds1.00, a drop of oil is also used between the bottomofthe slide and the top lens of the condenser.

Immersion oil can be obtained from biologicalsupply firms, from microscope manufacturers, or frommicroscope dealers. Many immersion oils have beenfound to contain toxic polychlorinated biphenyls.Extreme care should be taken to avoid skin contactor ingestion of these oils. Some nontoxic oils areavailable.

A bottle of immersion oil should never be shaken,since air bubbles may be introduced. A single, micro-scopicbubble under the objective lens will cause flare,which will lower contrast and affect image quality.Also, the bottle should never be left open to the air,since dust may settle on the oil. Dust or other debrisin the oil will impair image quality.

The procedure for using an oil immersion objectiveis as follows:1.Search for a suitable field on the slide with a

low-power, dry objective. A 10x or 20x objectivewill suffice.

2.Rack up the coarse adjustment of the microscopeand move the oil-immersion lens into position.

3. Place a small drop ofoil on the front of the objectiveand another small drop on the cover glass over thearea of interest. This technique will prevent forma-tion of air bubbles.

4. Using the coarse adjustment, move the objective

lens down slowly until the oil on the lens contactsthe oil on the cover glass. A flash of light will occurat this moment, visible when the eye is close tothe level of the lens.

5.Now use the fine adjustment on the microscopeto accomplish fine focus of the specimen.

If it is impossible to find the specimen focus, itmay be that the cover glass is too thick or that themounting medium is too thick; in this case, correctfocus can never be achieved. Oil-immersion objectiveshave extremely short working distances.

When the N.A. of the objective exceeds 1.00, oilshould be placed on the condenser, as stated. If thisis necessary, proceed as follows, before the objectiveis focused.1.Remove the specimen slide from the stage and place

a drop of oil on the top lens of the condenser.2. Place another drop of oil on the bottom of the slide,

under the specimen.3. Replace the specimen slide on the stage carefully.4. Rack the condenser up until the oil on the condens-

er contacts the oil on the slide.5.Now find a suitable field on the slide with a

low-power objective; focus the condenser; and pro-ceed as above, using the oil-immersion objective.

Correct focus of the condenser is achieved accordingto the Kohler illumination system. (See page 30.)Thethickness of the specimen slide is important in thissystem.

When you are through using the oil-immersion lens,the oil should be removed. Gently wipe the frontsurface with lens tissue, followed by a tissue damp-ened with xylol (Eastman Organic Chemical No.T460). Oil can be removed from the slide and thecondenser, if necessary, in the same manner.

CAMERAS IN PHOTOMICROGRAPHYGENERAL CONSIDERATIONSAn efficient microscope, used to best advantage withproperly controlled illumination, is the image-formingpart of a photomicrographic system. The camera isthe means for recording the image formed by themicroscope. If the quality of the image is the highestattainable, an excellent photomicrograph can bemade. If it is not, then no film, camera, or camerarefinement can improve the image quality in thephotomicrograph. It is important, however, that the

14

image should not be degraded in the camera or byany photographic technique.

Although almost any camera can be used to recorda micro-image, a camera specifically designed forphotomicrography offers more advantages than onewhich is merely adapted for the sake of convenience.A discussion of photomicrographic cameras begins onpage 17. The selection of a camera is most oftengoverned, however, by the amount of photomicrog-raphy to be done and the availability of funds.

CAMERAS WITH INTEGRAL LENSESThe simplest way to make a photomicrograph is touse a conventional, existing camera over the micro-scope. It might be an inexpensive fixed-focus cameraor an expensive 35 mm camera, but it is usually onedesigned for regular photography of people and places.The lens is an integral part of the camera and oftencannot be easily removed. A fixed-focuscamera is thesimplest, having only one shutter speed and usuallyonly one lens aperture. The more expensive type ofcamera offers a range of shutter speeds, various dis-tance settings, and a variety of aperture settings. Thistype, obviously, offers more versatility, particularlyin exposure control. Consider this camera first, sinceit will be more suitable.

When a microscope is focused visually with anormal, relaxed eye, the image is considered to beat infinity. Therefore, the distance setting on the

tFILM PLANE\

\\\\\\\

//

//

//

/I

/

i---- EYEPIECE

FIGURE 7- The proper positioning of a camera (with integral lens)on the microscope. The eyepoint is focused on the front surfaceof the camera lens by moving the camera.

camera should be set at infinity. If the camera isplaced over the microscope in the correct position,the micro-image will be in focus on the film plane.Any other distance setting on the camera may causean unsharp image. Many persons can relax their eyesby staring at a very distant object just prior tofocusing the microscope. Spectacles for aiding distantvision or astigmatism should be left on. Should theinitial results be unsatisfactory in terms of sharpness,you may be focusing your eye at some other virtualdistance such as 15 or 25 feet. When this is the case,a series of photographs should be made which runthe gamut of focus settings on the camera. Thesharpest record will indicate the virtual distance atwhich you are most likely to focus the microscope.Those who cannot focus the microscope consistently

will have to adopt a camera with a ground glass,preferably with a detachable lens.

The lens-aperture settings ({-numbers) on the cam-era do not control exposure in this work as they doin regular photography. They have no effect on imagebrightness. The largest aperture setting should beused-that is, the lens should be "wide open." Theeffect of using smaller apertures will be to "vignette"the image; that is, to reduce the illumination at theedge of the field because of the restrictive action ofthe aperture. Also, a stopped-down diaphragm cutsinto the image field and reduces field size, so onlya small, ci.rcularimage is recorded on the film.

The camera should be positioned over the micro-scope so that the "eyepoint" of the eyepiece is at,or very near, the front surface of the camera lens,as shown in Figure 7. The eyepoint is the positionabove the eyepiece at which light rays converge afterleaving the eyepiece. They then diverge to form thephotographed image. The position of the eyepoint canbe determined by holding a piece of white paper righton top of the eyepiece, then slowly raising it. A brightcircle will appear on the paper. This circle becomessmaller and then larger. The position at which thecircle is the smallest is the eyepoint. The distanceof the eyepoint above the eyepiece will vary withdifferent eyepieces. If the eyepiece is changed, there-fore, the eyepoint may appear in a different position.The distance may only be a few millimeters or it maybe as much as 20millimeters.

The camera can be held in place over the micro-scope by any available means. A vertical stand canbe constructed out of wood or metal; you can usea laboratory ring stand; or you can attach the camerato the upright member of a small enlarger, with theenlarger head removed. In any case, the camerashould be held firmly in the correct position, but youshould still be able to move the camera up out ofthe way or swing it to one side to look into themicroscope and adjust image focus. The rule is: Focusthe microscope and do not change the distance settingon the camera.

When the image appears sharp, the camera can bereplaced in position and the camera shutter actuatedto make an exposure. An arrangement must be madeto bring the camera back to the correct position. Somekind of "stop" on the stand should be devised.

Now to consider the inexpensive fixed-focuscamera.This camera can be placed over the microscope ina similar manner, by using a constructed or impro-vised stand. There is one important exception, howev-er, with regard to the lens and eyepoint positions.A fixed-focuscamera usually has only one, fixed,smallaperture behind the camera lens. If the camera is

15

positioned, so that the eyepoint occurs at the frontsurface of the lens, the small aperture in the camerawill reduce field size; only a very small image willbe recorded in the camera.

In this case, an eyepiece with a "high" eyepointis selected. The camera is positioned so that theeyepoint occurs at the aperture. The correct positionfor a fixed-focus camera is shown in Figure 8. Mostfixed-focus cameras will require an eyepiece with aneyepoint distance of about 15mm.

CAMERA

l

EYEPOINT ATCAMERA APERTURE

+ EYEPIECE

l-------..------

----------+

FIGURE 8-Placing the eyepoint at the camera aperture allowsthe largest field size on film.

Exposure becomes a problem with fixed-focus cam-eras because usually only one shutter speed is avail-able. With some cameras, two shutter speeds can beused. This means that you must have a very brightimage in the microscope and, of course, a very brightlight source. The exposure time, or shutter speed, isusually very short and will be about 1/30 or 1140second.

A simple method of attaching a fixed-focusor othercamera with integral lens is described and illustratedin Kodak Pamphlet No. AN-6, Photomicrographywith Simple Cameras, available on request fromConsumer Markets Division, Eastman Kodak Com-pany' Rochester, New York 14650.

Magnification with a Simple Cameraormally, when an image is formed outside of the

microscope, the magnification, as seen in the micro-scope (objective power times eyepiece power), is re-produced if the image is 10 inches (250 mm) fromthe eyepoint. An integral camera lens always has afocal length shorter than 10 inches. Image (lens-to-

16

film) distance becomes the determining factor formagnification of the photographed image. A camerawith a 2-inch (50 mm) lens will record an image onlyabout one-fifth the size of the image seen in themicroscope. Magnification on film can then be deter-mined by the ratio between the camera-lens focallength and 10 inches (250 mm), multiplied by thevisual magnification of the microscope. Cameralenses, except those on inexpensive cameras, usuallyhave their focal lengths engraved on their mountings.Fixed-focus cameras normally have no designation foreither focal length or lens aperture.

To determine the actual magnification recorded onfilm, you can place a micrometer slide under themicroscope (see Figure 4, page 11). The lines of theslide should be focused sharply and the image record-ed. When the film is processed, you can measure theseparation of the recorded lines and compare themeasurement with the actual separation of the lineson the micrometer slide. For example, if two lineson the slide were 0.01 mm apart, and the samerecorded lines were 0.5 mm apart, the recordedmagnification would be x50 (0.5divided by 0.01).

There are other disadvantages to using a camerawith an integral lens in photomicrography. One isthat the entire microscope field may not fill the filmframe. The out-of-focus, peripheral area of the micro-scope field is then recorded. This condition can bealleviated to some extent by using high-magnificationeyepiecesso that only the central, best-corrected partof the microscope field will be photographed.

Complex camera lenses will sometimes create inter-nal reflections (due to multiple-element construc-tion). These reflections reduce image contrast inphotomicrography. Because simple, inexpensive cam-eras have fewer lens surfaces to reflect light, theywill often produce better photomicrographs thanexpensive, complex cameras. Of course, a simple cam-era must be used correctly.

Some camera firms make microscope "adapters"that can be used to place a camera with an integrallens in correct position over a microscope. Suchadapters are often made, however, for adjust-able-focus cameras, not for the fixed-focustype. Cam-era manufacturers should be consulted for this acces-sory to their cameras.

CAMERAS WITHOUT INTEGRAL LENSESReflex CamerasThe next type of camera that can be adapted foruse over a microscope is the single-lens reflex (SLR).Very often a camera of this type uses 35 mm film,

has a focal-plane shutter, and will accommodate arange of interchangeable lenses, from wide angle totelephoto. Many firms that manufacture reflex cam-eras also offer microscope adapters. Normally, whena reflex camera is to be used over a microscope, thelens is removed from the camera and one or moreextension tubes is placed on the camera in the lensposition. A microscope adapter ring, containing themicroscope eyepiece, is then fastened in the frontextension tube. The whole assembly of camera, tubes,and adapter can then be placed on the microscope,fitting the microscope eyepiece and the adapter ringdirectly into the drawtube of the microscope. Thisassembly, in some designs, can be attached to a rigidstand that is supported independently of the micro-scope. This will prevent transfer of vibration fromthe camera to the microscope.

The micro-image is focused by adjusting the focusknob on the microscope while viewing the image inthe camera's viewfinder. One disadvantage of thissystem is that the image is usually focused on aground glass within the viewfinder. Critical focus offine detail is difficult to achieve on a ground glassbecause of the coarseness of the ground surface. Ifa clear glass can be used in place of the ground glass,or if a clear area is present on the ground glass nearthe center, this disadvantage can be overcome.

Bellows Extension CameraSome firms that manufacture reflex cameras also offeran adjustable bellows, which is normally used inclose-up photography. This bellows (with no lensattached) can be used on a reflex camera over amicroscope and has the advantage of adjustability,so that magnification and the amount of recordedfield can be varied for control of image composition.When the camera is used in this manner, it is advisableto attach the bottom plate on the bellows track toa rigid vertical stand.

With a reflex camera, magnification can be varied,a wide range of shutter speeds is available (usually1 second to 111000second) for exposure control, andthe entire film frame in the camera can be filled. Also,many modern reflex cameras include a "behind-the-lens" metering system for monitoring the imagebrightness in exposure determination. There are onlytwo drawbacks, or factors, which can affect recordedimage quality. One, already mentioned, is thedifficulty of critically focusing the image on a groundglass.The other is the possibility of creating vibrationwhen a focal-plane shutter is actuated. This can beminimized by using a shock mounting pad under themicroscope. (See also "Camera Vibration" page 20.)

PHOTOMICROGRAPHIC CAMERASThere are several commercially made cameras de-signed specifically for photomicrography. The mostpopular are those called eyepiece cameras; a featuregenerally common to cameras in this group is abeam-splitter eyepiece. The microscopic image can beviewed, focused, and composed by means of thisauxiliary eyepiece.A central, rectangular area is oftenshown in the center of the field to indicate the portionof the field that will be recorded on film (Figure 9).

II /I /I /I /I /I /I /I /I /I /I /I / TELESCOPICI / EYEPIECE

~/-f12l~1- ---~U r::~;;;~~~~~==_\i~~\\l,~~'>r11 RETICLE

_ MICROSCOPEEYEPIECE

FILM HOLDER ORROLL FILM ADAPTER

FIGURE 9-Eyepiece Camera-fixed bellows. If bellows draw (thelens-to-film distance) is less than 10 inches (250 mm), a correctionlens is often included to adjust the focus to the film plane. Themagnification on the film is then the same as the visual magnifica-tion.

35 mm Eyepiece CamerasCameras of this type usually accommodate 35 mmfilm magazines, although some use sheet films (orplates). Some with more versatility are designed foruse with either roll or sheet film.

The film plane in a 35 mm eyepiece camera is ina fixed position, usually far enough from the micro-scope eyepiece so that only the central area of thefield is recorded. This feature avoids the out-of-focusperipheral area caused by curvature of field, whichis inherent in many microscope objectives and whichwould be recorded in a camera with integral lens.The distance from eyepoint to film may vary slightlyfor 35 mm eyepiece cameras of different manufacture,but is usually about 4 or 5 inches. Since the distance

17

is less than 10 inches, however, a simple correctinglens is incorporated so that the image seen sharplyfocused in the viewing eyepiece will also be in sharpfocus in the film plane of the camera. The use ofthis correcting lens also makes sure that the micro-scope will be used at the correct optical-tube length.

Focusing this eyepiece camera requires care. Thereticle (or crosshairs) in the telescopic eyepiece mustbe in sharp focus when the specimen is in focus.Persons under 40 years old may accommodate a focusdifference without realizing it. The best method isto focus the reticle first, and then focus the specimen.To check the focus, move your head from side to sidewhile viewing the specimen. If the reticle and a finestructure in the specimen appear to move in oppositedirections, a fine focus adjustment is needed. Whenthey seem to move in the same direction and staytogether, the focus is correct. This is often called"parallax" focusing.

The greatest advantages presented by an eyepiececamera are convenience and economy, especiallywhen 35mm slides are desired for projection purposes.This type of camera can be used with most micro-scopes. It requires little storage space when not inuse and can be set up quickly when needed. Anyefficient visual microscope setup can be used forphotomicrography with an eyepiece camera. Thisgreat convenience has made the eyepiece camera verypopular and useful for photomicrography.

Roll-film cameras, especially 35 mm cameras, havelimitations and disadvantages that should be consid-ered before equipment is purchased for specific use.For example:1.If 35 mm film is used, a small-size negative or

transparency isproduced. When prints are required,enlargement becomes necessary, and the extra costof an enlarger reduces the economy of a small filmsize. (Also, in enlargement the optical condition of"empty" magnification can occur. See page ll.)

2.Magnification is quite limited on 35 mm film. Somespecimens, such as large blood cells, must be pho-tographed at low to medium power because highmagnification might make them too large for thefilm size. This reduces best optical resolution possi-ble, and fine details cannot be well defined becauseonly objectives of lower numerical aperture can beused. Objectives of high numerical aperture arenecessary when the best resolution is desired.

Sheet-Film Eyepiece CamerasSheet-film cameras of the eyepiece type often repro-duce visual microscope magnification, since the eye-piece-to-film distance can be fixed at 10 inches (250

18

mm). In addition to an observing eyepiece, thesecameras often have a ground-glass back to facilitateboth composing and focusing of the image to berecorded. Film size is usually 4 by 5 inches. Becauseof this larger film size, greater magnifications can berecorded with these cameras than with roll-film cam-eras, especially 35 mm cameras. Also, the same fieldcan be easily exposed on several different emulsionsand then developed individually if desired. Adapterbacks are available, however, to convert sheet-filmcameras to the use of roll films when desired.

Trinocular Attachment CamerasAnother type of photomicrographic camera is shownin Figure 10.This type is especially designed by some

FIGURE 10- Typical trinocular microscope arrangement. A prismdeflects the light either to the film plane or to the eyepieces.

manufacturers for use on their "trinocular" micro-scopes. A binocular microscope has a binocular ar-rangement for visual work and a third tube forphotomicrography. The visual and the photomicro- .graphic optical systems are "parafocal"; that is, theimage seen visually in the microscope will also bein focus in the camera. A beam splitter is not used.A 35 mm back and a sheet-film back are interchange-able. Also, a special "field of view" eyepiece issupplied, containing a focusable eye lens and a reticle

to indicate the area of field which will be recorded.Although this is an excellent system for photomicrog-raphy, it has one disadvantage-it cannot be usedto photograph moving organisms. A prism is used tochange the light path from the visual system to thephotographic, so it is impossible to see the field whilea photograph is being made. If only stationary sub-jects are to be photographed, there is no problem.The eyepiece camera, on the other hand, can be usedto photograph both stationary and moving subjects.

Cameras with Adjustable BellowsA camera that has an adjustable bellows and acceptsa large film size is considered best for high-qualityphotomicrogra phy.

The well-equipped photomicrographer always usesa camera with adjustable bellows. Such a camerausually contains a shutter capable of a wide rangeof exposure times, a light-locking device to excludeall light from the film except that from the micro-scope, and a ground-glass screen for focusing andcomposition. No lens is used or needed in this camera.Sheet films or plates in appropriate holders are nor-mally used, but the camera will often accommodateroll films by means of suitable adapter backs. (SeeFigure 11.)

E~;=================;;3-- GROUNDGLASS INFILM PLANE\

\\\\\\\\\\

//

//

//

//

//

/

___ ADJUSTABLE

BELLOWS

/ \ '-r------ SHUTTER

EYEPIECE ---IH ..••.

1----- LIGHT LOCK

DRAW- TUBE -----+

FIGURE 11-Adjustable bellows camera. Focusing the camera isaccomplished by adjusting the microscope focus until the imageis sharp on the ground glass. The eyepoint is placed at the shutterof the bellows.

Film size is commonly 4 x 5 inches or larger toallow highest magnifications and greatest field sizes.This also allows complete versatility in the selectionof objectives and eyepieces.

A camera with adjustable bellows is capable of acomplete range of magnification, since eyepiece-to-film distance is adjustable. Microscope magnificationis reproduced at the stated Hl-inchdistance.

Commercially made cameras of this type are usual-ly mounted vertically on a rigid stand (Figure 12 .

FIGURE 12-Professional photomicrography setup. Light source,optics, and camera are in the same vertical plane.

Actually, a heavy, rigid stand is a necessity in orderto avoid vibration and consequent unsharpness in theimage. In addition, a separate means of absorbingvibration and shock, such as a vibration isolationtable, is recommended for consistently high quality.particularly when long exposure times are indicated.

Aprecaution that should be observedwith this t -peof camera is correct positioning ofthe shutter. Ideallythe shutter should have a reasonably large openingand small, light, fast-moving blades. It should bepositioned so that the blades are at, or very near,the eyepoint of the microscope eyepiece. If the bladesare too far behind the eyepoint and short exposuretimes are used, a light silhouette-image of the shutterblades may be recorded. (See Plate Ill-A, page 39.)

19

Light LockCommercial photomicrographic cameras of the ad-

. justable-bellows type usually contain an efficientsystem for excluding extraneous light from the cam-era. This is the "light-lock," the connection betweenmicroscope and camera. When an available viewcamera or an enlarger is adapted for this work, alight-lock system must be devised. A very efficientand convenient light lock can be made by using twodifferent-size lens hoods-the smaller one is attachedto the camera or shutter and the larger to the tubeof the microscope. The two hoods will nest togetherto exclude all light from the camera except thatcoming through the microscope.

In practice, the camera is usually moved out ofposition so that the microscope can be used visuallyto locate an appropriate field. The camera is thenmoved into position so that its lens hood fits intothe lens hood on the microscope. The camera, ofcourse, should be centered with respect to the micro-scope so that there is no physical contact betweenthe two lens hoods. Enough space exists to allowmovement of the microscope barrel for critical focus-ing of the image on the ground glass.

Lens hoods in various sizes are standard cameraaccessories and can be bought in most camera stores.

THE GROUND GLASSFrequently in photomicrography the image to berecorded consists of extremely fine detail. The surfacestructure of a ground-glass screen, however, is oftenso coarse that it interferes with this detail; criticalfocus of the image is difficult, if not impossible. Somemanufacturers of photomicrographic equipment pro-vide screens with clear centers for critical focus ofthe image on the plane of the ground surface. Aground-glass screen with clear center can also beprepared in the following manner:

With a soft pencil, draw two diagonals on theground glass surface, which should be facing themicroscope. Place a small drop of warm Canadabalsam (or other mounting medium) where thesediagonals cross. Drop a small, round, micro cover glassonto the balsam and press it into place gently. Allowthe balsam to set; a permanent transparent center

20

on the ground glass will result. This procedure pro-vides crosshairs for parallax focusing.

For photomicrography, place a mounted handmagnifier of at least x 5 over the clear center of thescreen. Put your eye to the magnifier and glanceslowly back and forth over the crossed lines. Whenthe image is critically focused in the plane of thelines, there will be no movement of the image inrelation to the crossed lines.

CAMERA VIBRATIONVibration in a camera setup can result from micro-scope manipulation, from drawing and replacingdark-slides in film and plateholders, from setting themechanical shutter and from vibration within thebuilding. It is always good practice to wait a fewseconds for vibration to cease before making theactual exposure. Even then, if the shutter is actuatedmanually, additional vibration can occur and maycause some unsharpness in the recorded image. Forthis reason, if the shutter is part of the camera, theuse of a suitable cable release is strongly recom-mended. A self-timer can also be used to provide ameans of automatic delay before the shutter isoperated.

Some cameras contain focal-plane shutters; othershave leaf-type, or "between-the-lens," shutters. Thelatter type is much more satisfactory for photomi-crography, since a focal-plane shutter may imparta certain amount of vibration when an exposure ismade. This is caused by the shock of the focal-planeshutter striking the side of the camera as the shutteropens and closes.This shock can be alleviated by usinglonger exposure times to minimize the vibration effectcaused by the shutter or by using special absorbingpads or an anti-vibration table under the microscope.These will dampen the vibrations; tests will determinetheir efficacy.Such units are available from scientificequipment supply firms.

In general, heavy shutters and focal plane shutterson the camera should not be used for timing theexposures. It is better to set them to the "open" po-sition just prior to making the exposure and then totime the exposure with an auxiliary shutter placed inthe light beam between the lamp and the condenser.

MICROSLIDESHOW MICROSLlDES ARE PREPAREDBasically, any specimen to be examined or pho-tographed through a microscope is mounted in asuitable medium on a microscope slide and coveredwith a cover glass. The specimen is usually coloredby means of various biological stains to producecontrast and visualization ofstructural details. Tissuesections and smears are the most common types ofspecimens prepared for the microscope, althoughother subjects can also be suitably prepared.

The actual techniques used in slide preparation canbe quite involved and complex, and will not beexplained in detail in this book. However, the funda-mentals of slide preparation will be reviewedbriefly. More detailed explanations can be found inbooks on microscopy and microtechniques.

A wide selection of prepared microslides is availableby mail order from several scientific and biologicalsupply firms. These slides include tissue sections,smears, and many other types of specimens. Preparedslides are used extensively in classroom instructionin biology,botany, medicine, etc.

Slides and Cover GlassesNormally, a specimen is mounted on a glass slidewhich is usually 3 inches long and 1 inch wide. Allbrightfield microscopes are equipped to handle slidesof these dimensions. Larger slides are also used,although less often than the 1x 3-inch size.The largersizes (Ph x 3-inch and 2 x 3-inch) will accommodateeither large sections or a long series of sections.

The surface of the slide should be flat. Also, theglass should be of high quality and chemically stable(noncorrosive), since it may be subjected to manyreagents. All commercially available microscopeslides are produced with these features and are cur-rently available from a number ofsupply houses.

The thickness of the microscope slide is of definiteimportance when high-power photomicrography isconsidered. Substage condensers are designed for usewith slides of specificthickness, within a narrow rangeof tolerance. Unfortunately, the specifications forslide thickness vary with different microscope manu-facturers. One condenser, for example, may requirea slide thickness of 1.2 mm, whereas another mayspecify 1.6 mm. The specification for slide thicknessis related to the working distance of the condenser.Slide thickness is less critical for visual microscopyor for photomicrography at lower powers when thetop of the condenser is removed. In this case, the

condenser has a much longer working distance andthe actual slide thickness is unimportant.

If a specimen is to be photographed at highmagnification, however, it may be necessary to makea special preparation on a slide of the correctthickness, as specified for the condenser in use. Ingeneral, however, a slide thickness of 1.2 mm or lesswill suffice for most subjects to be examined orphotographed at low magnification (up to about

. 150X).The cover glass is usually a circle, square, or

rectangle of very thin optical glass. Various sizes areavailable for the different shapes, but if a 1 x 3-inchslide is used, the cover glass should be of suitablesize to fit the slide. The most common size for a1 x 3-inch slide is 22 mm, and can be either roundor square. Thus, rectangular cover glasses should beless than 1 inch (25 mm) wide. Smaller sizes are usedoccasionally for small specimens. Recently, plasticcover glasses have been introduced which should notbe used for photomicrography.

Ideally, the thickness of the cover glass should beas specified by all microscope manufacturers, whichis either 0.17 mm or 0.18 mm. Cover glasses areavailable, however, in different thickness ranges asNo. 0, No. 1, No. Ph, No. 2, and No. 3. Each numberindicates a narrow range of thicknesses, with No. 0being the thinnest and No. 3 the thickest. The com-plete range is from less than 0.1 mm to slightly over0.3 mm. A box of No. Ph cover glasses includes theideal range of 0.16mm to 0.19mm, with the majoritybeing 0.17 mm. It is suggested, therefore, that when-ever a microslide is prepared, the No. 1Vz cover glassbe used. The only exception would be for preparationof some whole mounts, where a thick cover glass (No.2 or No. 3) would be of advantage. (For a descriptionof whole mounts, see page 23.) When the rightthickness of cover glass is used, the slide. can beexamined visually and is correct for most photomi-crography, particularly with a high, dry objective.

Tissue SectionsA large majority of specimens prepared for the micro-scope are very thin slices, or "sections," of biologicalmaterial, usually either animal or plant tissue. Sec-tions are cut on an instrument known as a "micro-tome," with the rotary type being the most common.Ablock of tissue is held firmly in a clamp which movesup and down as a hand-actuated wheel is rotated.As the block moves downward, it passes over the edge

21

of a very sharp, stationary knife blade and a thinslice of the material is produced. The thickness ofthe slice is controlled by a setting on the microtome.Serial sections, one after the other, of the samethickness are cut in this way.

Another type of microtome is used for larger piecesof tissue, and makes large sections. This is the "slid-ing" microtome, where the block remains stationaryand the knife movesback and forth to cut thin slices.

Most tissue specimens, however, are not hardenough to be placed in the microtome and be cutwithout preliminary preparation. First of all, thetissue is fixed and hardened with a chemical reagent.Then it is dehydrated, cleared, and placed in a bathof molten paraffin wax, where it is left long enoughfor the wax to impregnate the tissue. The tissue mustthen be embedded in a block of paraffin. This isaccomplished by placing the impregnated tissue ina box-likemold containing melted paraffin. The moldis cooled rapidly by immersing it in cold water toharden the paraffin. The result is a block of waxcontaining the tissue specimen. This block is trimmedto a convenient size and attached to a holder, whichis inserted in the microtome clamp.

The actual techniques of specimen preparation andsectioning are much more involved than are describedhere. Complete details are available in pertinentliterature on the subject.

Sections are cut on a microtome either individuallyor as "ribbons." A ribbon is a long chain of sectionswhich cling to each other at the edges. Either onesection or a short ribbon is placed on a microscopeslide that has been treated with an adhesive material.The paraffin is then removed by bathing the slidein a suitable solvent. The sections fastened to theslideare usually colorlessand practically transparent,unless stained (see below) before embedding. Littleor no detail would be visible if they were examinedin a microscope. In order to render the details visible,the sections must be colored, or "stained"; manydifferent biological stains are used for the purpose.Double staining, that is, the use of two stains, iscommon in order to differentiate between specificparts of a specimen. For example, with animal tissuethe nucleus of a cell might be stained with one colorand a stain of different color used for the cytoplasm,the area of the cell around the nucleus. The mostcommon stains used for animal tissue sections are"hematoxylin" and "eosin." (See Table 5, page 33.)These stains appear as blue and light red, respectively.

Many other colored stains are also used in bothplant and animal histology. Some techniques utilizetriple, quadruple, and even quintuple staining. Singlestains are used rarely.

When tissue sections are to be prepared, it wouldbe best for photomicrography if they were as thinand as well stained as possible. As discussed previous-ly, thick sections present a problem when photomi-crographs are to be made at medium to highmagnification. There are understandable limits, how-ever, in the preparation of thin specimens by theparaffin technique. Sections thinner than 4 or 5 urnare difficult to make. However, sections as thin as1 or 2 p,m are needed for high-magnification work,both visually and photographically.

In order to make these ultra-thin tissue sections,it is necessary to resort to a different impregnatingand embedding substance than paraffin, as well asto use a different technique for preliminary prepara-tion of the tissue. The media most commonly em-ployed in place of paraffin are the methacrylate andepoxy resins. Sections are cut on an "ultra-micro-tome" by either a glass or a diamond cutting edge.This technique is commonly used in preparing sec-tions for electron microscopes.

Another technique for sectioning tissue for the lightmicroscope involves quick-freezing. Tissues may behardened sufficiently by freezing so that the usualinfiltration and embedding techniques are eliminated.The material to be sectioned is fresh and can be eitherfixed or left unfixed. The principal advantage, ofcourse, is speed, since the steps of freezing and sec-tioning can be carried out in minutes=compared withhours for the paraffin technique. The disadvantagesare relatively thick sections, less clarity of detail, and

FIGURE 13-Same as Plate I (page 37) on KODAK EKTAPAN Film.A yellow filter (KODAK WRATIEN Filter, No. 12) was used to lightenstain calor and to emphasize taste buds.

22

the inability to handle large pieces of tissue becausethey do not freeze entirely throughout. In pathology,however, the ability to make stained, mounted speci-mens ofhuman tissue in a very short time is a definiteadvantage.

No matter how a section is made it is mountedon a microscope slide, stained; a drop of suitablemounting medium is placed on it; and then thepreparation is covered with a cover glass. The micro-slide is then ready for viewing or for photographythrough the microscope.

SmearsThe technique ofpreparing certain types of specimensby "smearing" is relatively simple compared withmaking tissue sections. A sample of fluid material-such as blood, a bacterial culture, or an exudate-isspread to a thin layer on a clean microscope slide.The smear is then dried and fixed (made permanent).A suitable mounting medium is applied and thespecimen is coveredwith a cover glass.

The methods of smearing will vary slightly fordifferent types of specimens. Blood smears are themost common and are made routinely in hematologylaboratories. Two methods of making blood smearsare practiced. In the first method, a drop of bloodis placed on a clean slide about 1 inch from the end.A second slide, held at about 45° and in contact withthe first slide, is allowed to touch the blood so thatcapillary action distributes the blood along the trail-ing edge of the slide. The slide is then pushed forwardquickly to produce a thin smear on the bottom slide.The smear is allowed to dry in air and it is readyfor fixing.

The second method for blood smears involves plac-ing a drop on a clean cover glass. A second cleancover glass is touched to the drop of blood on thefirst and then dropped diagonally across the first glass.The drop spreads quickly between the two glasses.The two cover glasses are separated immediately, bygrasping two projecting corners and pulling with asmooth motion. Each cover glass then contains asmear which can be dried in air, then fixed andmounted on a clean slide.

Wright's Stain, a combination of methylene blueand eosin, is dissolved in methyl alcohol and com-monly used for fixingsmears in the United States.

Preparation of bacterial smears and smears fromexudates is even simpler. A drop of dilute liquidmaterial is picked up with a small wire loop. Thedrop is then smeared on a clean slide by moving theloop in contact on the slide, either back and forthor in a circular motion. The smear is then dried,stained, and a mounting medium and cover glassapplied.

Whole MountsThere are many specimens which are mounted direct-ly onto microscope slides in specific media withoutresort to either sectioning or smearing. These includesmall insects, protozoa, crustacea, pollen grains, andfibers. The techniques of preparation and mountingare quite diverse. The reader is therefore directed toliterature on microtechniques, found in most scientif-ic and biological Iibraries. Many types of wholemounts can also be purchased as prepared slides fromsupply firms.

The Mounting MediaMicro-specimens for a brightfield microscope arenearly always mounted in some kind of medium, ona slide and under a cover glass. The purpose andcharacteristics of the medium are variables, depend-ing on the type of specimen.Mounting-media basicallyare divided into two classes: those suitable for perma-nent mounts and those suitable for temporarymounts.

With regard to stained tissue sections and smears,the mountant is usually permanent; it serves toprotect and preserve the specimen for future studyor photography. A medium for this purpose must bereasonably colorless, so as to produce a neutral back-ground and not degrade the specimen colors. It shouldhave a refractive index fairly close to that of thespecimen in order to produce the highest degree oftransparency. It should be chemically inert both tothe glass and the specimen, and should not causestains to fade. Also,it should adhere to glass.

A permanent mounting medium is usually eithera natural or a synthetic resin. Canada balsam isprobably the most well-known example of a naturalresin. It has been used as a mounting medium for overa hunched years, and is still commercially availablefrom many sources. Slidesprepared with this medium,however, tend to become slightly yellowish with age,or to appear slightly yellowish when an excess of themedium is used. Some fading of stain colors alsooccurs after a long period of time.

Synthetic media are employed quite extensivelytoday, and are available under a variety of tradenames. They are considered in some ways to besuperior to balsam and are often used as substitutes.Table 3 lists some of the common mounting media.

Occasionally, a permanent medium is selectedwhich has a higher refractive index than either theglass or the specimen. In this case, the aim is to makethe specimen more visible, since it may be colorless(unstained) and practically transparent. The greater

23

the difference in refractive index between the mediumand the specimen, the higher will be the visual con-trast. One application is in fiber microscopy, especiallywith animal hairs. The surface texture of wool fibers,for example, is much more visible in a medium ofhigh refractive index than in one where the indexis close to that of the specimen. Some media of thistype are Aroclor resins, Naphrax, and polystyrene.(SeeTable 3.)

When permanent mounts are prepared, a quantityof medium is placed on the specimen and coveredwith a cover glass. Sufficient pressure must be applied,either by a weight or by a spring-loaded press, to pushthe cover into place. The cover-glass surface mustbe parallel to the glass slide, and a minimum amountof mounting medium should exist between the coverand the specimen. The slide must be kept underpressure until the mounting medium has hardened.The slide is then ready to examine and photograph.

Temporary mounting media are often used withspecific subjects for speed and convenience. A slidemade with a temporary mount is often discarded afteruse. Some of the temporary fluid media include water,glycerine, certain oils, Karo SYTUP, and many organicliquids. One precaution which must be observed isthat the specimen should not be soluble in the medi-um 01' be reacted upon by it. The cover glass is usuallytaped down to prevent movement.

TABLE 3

Chemical CrystalsThe formation and photomicrography of chemicalcrystals is fascinating, particularly when you useeither a polarizing microscope or a conventionalbrightfield microscope equipped with polarizingfilters. Some crystals are "birefringent," or "aniso-tropic," and appear brightly colored when viewedbetween crossed polarizers. Very striking color photo-micrographs can be made of crystals or crystal pat-terns by using polarized light.

The preparation of chemical crystals on a micro-scope slide is relatively easy. The simplest methodis by evaporation. Dissolve a small amount of chemi-cal in distilled water (or other solvent) in a test tubeOT small vial. Place a drop of the solution on a cleanmicroscope slide and allow the solvent to evaporate.Crystals will begin to form in a short time. (Applica-tion of low heat will hasten crystallization.) Thegrowth can then be studied under a low-powermicro-scope with crossed polarizers. When all of the solventhas evaporated, crystals can be photographed dry,or a mounting medium of high refractive index canbe applied and a cover glass used. The mountingmedium can beapermanent type, butitssolventshouldnot attack the crystals. This might happen with someorganic chemicals but is unlikely with inorganic ma-terials. (SeeTable 3.)

Fusion is another technique which produces color-

Common Mounting Media

Mounting RefractiveMedia Index Use Available From

Canada Balsam 1.53 Bioloqical, general Scientific supply firms

Caedax 1.58 General Ward's Natural ScienceEstablishment, Inc.

Diaphane Green 1.54 Especially recommended VWR Scientific, Inc.for hematoxylin-stainedspecimens

Har/eco Synthetic 1.52 General Hartman LeddonResin Company

Permount 1.54 Biological, general Fisher Scientific Co.

Aroclor Resins 1.63 High refractive index work Monsanto Chemical Co.

Carmount 165 1.65 High refractive index work R.P. CargilleLaboratories, Inc.

Castor Oil 1.47 Temporary mounts, VWR Scientific, Inc.general

Cedar Oil 1.52 Temporary mounts, VWR Scientific, Inc.general

Immersion Oil 1.51 Temporary mounts, R. P. Cargille Lab.general

White Karo 1.42 Semipermanent mounts, Grocery storesSyrup ultraviolet

24

ful crystal patterns under polarized light. Avery smallamount of an organic chemical is placed on a micro-scope slide and a cover glass placed on top. Then,if heat is applied to the bottom of the slide, thechemical melts and spreads evenly under the coverglass. The heat is removed, and as the melt cools,crystal growth begins when the temperature dropsbelow the melting point. All of the crystals formedwill "fuse" together, very often producing beautifullycolored patterns.

Only a low-power microscope is necessary for thisphotography; either a 5 X or 10X objective willusually suffice. Crystal patterns are often too thickfor higher power.

HOW MICROSLlDES ARE ILLUMINATEDA complete understanding of the principles and prac-tices of obtaining efficient illumination is just asimportant to the photomicrographer as knowing thecapabilities and limitations of the compound micro-scope. Correct adjustment of the optical system ofthe microscope is, in fact, dependent upon an efficientsystem of illumination. An objective, for example,cannot be used effectively unless the substage con-denser is properly adjusted and the substage dia-phragm is set at the correct aperture. Theseadjustments are made by following the system knownas Kohler illumination. (See page 29.)

The light source itself should provide sufficientintensity to allow reasonably short exposure times.The lamp housing should be suitably designed toallow easy access to the light source and shouldcontain those elements necessary for proper adjust-ment of the illumination furnished to the microscope.

When a color film is to be exposed, the light sourceshould conform to, or allow suitable filtration to meet,the requirements of the film. A source with a continu-ous visible spectrum is necessary. Most common lightsources meet this requirement. The only exceptionis the mercury-vapor lamp, which emits line spectra.This source, however, is of special interest for ultra-violet and fluorescence photomicrography. It can alsobe used in regular black-and-white work.

Efficient illumination is not only dependent uponcorrect, suitable adjustments of the microscope andthe illuminator, but is very much dependent oncorrect alignment of all components of the system-from the light source to the film plane. Many effectsof uneven illumination in the image, especially colorfringes, can be traced to improper alignment. If cen-tering devices are not provided for the condenser andthe light source, alignment already has been estab-lished by the manufacturer. The microscope manualor manufacturer should be consulted if alignmentproblems persist.

Light SourcesThe most common light source in general brightfieldphotomicrography is the incandescent tungsten fila-ment lamp, available in a wide selection of voltagesand wattages. Most microscopes having built-in illu-mination utilize either a 6- or l2-volt coil filamentlamp, which varies in color temperature from about2700 K to 3200 K, depending on manufacture andelectrical conditions at the time of usage. Operationof this low-voltage lamp is through a transformerhaving several settings. The highest setting is usuallysuggested when color film is to be exposed in orderto provide the highest color temperature. Even whena color film balanced for tungsten illumination is tobe exposed, it is usually necessary to use appropriatelight-balancing filters to adjust the illumination sothat it will be right for the film. These filters willvary with different lamps.

When a microscope does not have built-in illumina-tion, an external illuminator must be used. Separateilluminators are available from microscope manufac-turers or dealers. These illuminators contain eithera nO-volt, lOO-watt, coil-filament lamp or a 6-volt,1.8 amp. ribbon-filament lamp. Here again, however,the illumination must be adjusted with appropriatefilters to suit the color film in use. (Plate I-B, page 37.)

One of the problems involved with a tungsten lampis that the glass envelope becomes blackened withage, due to the deposit of tungsten resulting fromvaporization. This effect causes a reduction in izhtintensity and a drop in color temperature. When alamp has become visibly blackened, it should bediscarded and replaced with a new lamp.

This problem does not exist with the tung en-halogen lamp. Although it is also a tungsten coi -filament lamp, halogen gas is contained within theenvelope. When the lamp is ignited, tungsten evapo-rates from the filament and is intercepted by thehalogen gas particles. These combine because of theheat involved, and the tungsten is redeposited uponthe filament. The halogen gas is released and the cyclebegins again. Deposition of tungsten (on the glassenvelope) is thereby eliminated, and the lamp retainsits initial brightness and color temperature (3200 Kthroughout its life. Tungsten-halogen lamps areavailable in suitable housings from some microscopemanufacturers. New microscopes having built-in illu-mination from a tungsten-halogen lamp have beendeveloped.

The tungsten-halogen lamp-available at presentin a l2-volt, lOO-watt size-is an excellent source forphotomicrography. It emits high-intensity, efficientillumination because the coil filament is small andcompact. Lamp life is about fifty hours, and it can

25

be replaced easily and inexpensively.The xenon arc is another light source that can be

used in photomicrography. This lamp produces illu-mination of high intensity and of daylight quality.The latter feature is especially important, becauseit allows the use of daylight-type color films withlittle or no filtering. The lUC is produced acrosstungsten electrodes in a clear envelope which containshighly pressurized xenon gas. The emission of thexenon arc is continuous not only in the visible spec-trum but also in the long-wave ultraviolet and in-frared spectral regions.Xenon-arc lamps operate froma special power supply connected to a 110-to l20-voltsource.

The zirconium arc is another excellent light source.It is very small, being almost a point source. Its colortemperature is 3200 K, and therefore it is quitesuitable for color films balanced for 3200K illumina-tion. The light intensity of a zirconium arc is notas high as tungsten-halogen or the xenon-arc lamps,but is high enough for efficient photomicrography.The lOO-wattsizeis usually suggested, although lowerwatt ages are also available. Like the xenon arc, itoperates from its own power supply.

Electronic flash lamps can also be used in photomi-crography, specifically in photographing moving or-ganisms. The illumination is of daylight quality andtherefore is satisfactory for use with daylight-typecolor films. Since the flash is instantaneous, an auxil-iary tungsten lamp of low brightness is usually neces-sary for purposes of alignment, for producing Kohlerillumination, for focusing and composing the speci-men image, or for general viewing of the specimenprior to photography. The flash should be synchro-nized with the operation of the camera shutter.

Mercury-uapor lamps serve as excellent monochro-matic-light sources. With appropriate filters, the mer-cury green line at 546 nm, the blue line at 436 nm,or the 365 nm line in the ultraviolet can be used formonochromatic black-and-white photomicrography.A mercury-vapor lamp should not be used in colorphotomicrography with a brightfield microscope,since its illumination only contains line spectra, andthus, will not givea true rendition of the subject.

IlluminatorsIlluminators for visual microscopy and for photomi-crography can be obtained from all major opticalcompanies, particularly from those that manufacturemicroscopes. Some requirements for a photomicro-graphic illuminator are not absolutely necessarywhen only visual microscopy is intended. Generally,however, a brighter light source is required for photo-micrography than for visual work.

26

MICROSCOPE LAMP

LAMP HOUSE

VARIABLE -_FIELD DIAPHRAGM .....L.-----t---,

-+-+-- FIlAMENT LAMPIN CLEAR ENVElOPE

FIGURE 14- Typical lamp used in photomicrography.

Because complete control of illumination is neces-sary, photomicrographic illuminators should containboth a lens to project an image of the lamp filamentand a diaphragm to control the size of the illuminatedfield in the microscope. The lens is usually called a"field condenser"; the diaphragm, a "field dia-phragm." Also, the terms "lamp condenser" and"lamp diaphragm" are used. (See Figure 14.)

Another refinement, not always included on anilluminator, is a facility for centering the light sourcewith respect to the field condenser lens. We oftenassume that a light source in a lamphouse is centered,but this is not always true. Lamps, themselves, varyin regard to the position of the filament in theenvelope. A centering facility corrects any discrep-ancy.

Since filters are almost always used in photomi-crography, a filter holder should also be included onthe illuminator, in front of the field condenser lens.

OBJECTIVE

'"-'<-_~ LIGHT CONE\ / COVER GLASS

I ; \ ! SPECIMEN SLIDE/ \

'\I--v----- SUBSTAGECONDENSER

SUBSTAGE VARIABLEDIAPHRAGM

1-------

FIGURE 1S-Cone of light from substage condenser with apexin specimen plane. Inverted cone fills aperture of Objective.Critical focus of condenser is necessary.

Methods of IlluminationThere are two ways to illuminate specimens-bytransillumination and by reflected light. The secondis used for photographing the surfaces of thick oropaque subjects. It is accomplished with microscopelamps that project a small spot of light or by specialbuilt-in ring or axial systems. Metallography utilizesthe latter and this field is discussed later in this book.The majority of subjects encountered in generalphotomicrographic work are examined and pho-tographed by transmitted illumination.

The simplest setup involves the use of axial trans-mitted light. Rays of light are reflected from the planesurface of the substage mirror through the substagecondenser, through the subject and the field aroundit, and into the microscope objective. (See Figure 15.)

A movable substage condenser permits accuratecontrol and concentration of the light and the useof high-power objectives. The illumination should fillcompletely the diaphragm opening of the condenserwith light of even intensity. The condenser must beproperly adjusted to insure that the back lens of theobjective is filled evenly with light. (See Figure 16.)The plane, or flat, side of the substage mirror shouldalways be used when a condenser is employed. Thespherical mirror is used only when a 48 mm objectiveis used without a condenser.

If the illumination system has not been properlyadjusted, a photomicrograph may be disappointing-even if the highest quality optics have been used theimage has been focused critically, and the expo ureof the film has been correctly determined. The twobasic requirements are (1) that the whole illuminationsystem be centered, and (2) that the cone of lightfrom the illumination system completely fills theaperture of the microscope objective, providing uni-form illumination over the whole subject field.

Kohler illumination is the most common systemof illuminating a microscope specimen in photomi-crography. It can be used in visual work, too, becauseit provides best image quality and highest resolution.

Basically, Kohler illumination consists of using thefield or lamp condenser to focus an image of the lampfilament on the substage condenser, which in turnfocuses an image of the lamp condenser in the planeof the specimen. Thus, in effect, the lamp collectorbecomes the source of illumination. (See Figure 17.)

The chief practical advantage of this technique '-that, when the elements are properly aligned, a uni-formly illuminated field is provided, with practicallyno restriction as to the structure of the light source.Hence, it is possible to employ a nonuniform source(such as a coil-filament tungsten lamp) or a high-in-tensity source of small area (such as a zirconium arc

FIGURE 16-Adjusting the aperture diaphragm for optimum re-solving power of the objective (a) wide open, (b) stopped down,Cc)correct aperture. The bright area should be 4/5 the diameterof the distant circle.

27

or a xenon arc).Essentially, the method of producing Kohler illu-

'ruination in photomicrography consists of focusingan image of the lamp collector in the plane of theobject and an image of the lamp filament in the planeof the aperture diaphragm. Refer to Figure 17. Inactual practice, the steps in establishing Kohler illu-mination are listed below and in the followingsection:

1.Check alignment of the lamp with respect to thelamp collector. A quick check of alignment can bemade by placing a piece of thin white paper overthe field diaphragm and focusing the lamp collectorto produce an image of the lamp on the paper. Theimage of the lamp should be centered within theoutline of the diaphragm. The lamphouse-containing the lamp, collector lens, and dia-phragm-should be placed about 10inches from themicroscope. The actual distance should be suchthat the image of the lamp filament projected ontothe mirror is slightly larger than the maximumopening of the aperture diaphragm. The alignmentof the lamp with respect to the lamp collector isnecessary only once. When the lamp is changed,realignment is required.

2.Adjust the lamphouse so that the image ofthe lampis centered on the mirror. Adjust the mirror so thatthe image reflected by it onto the aperture dia-phragm is centered on the diaphragm. Focus thelamp collector to produce a sharp image ofthe lampfilament on the aperture diaphragm.

3. Stop down the field diaphragm to a small aperture.While looking into the microscope or at the groundglass of the camera, focus the condenser to obtaina sharp image of the field diaphragm in the speci-men plane. For this step, the microscope must bein focus on a specimen. (See "Chromatic Aberra-tion," page 43.)

4.The image of the field diaphragm should be cen-tered in the observed field. If it is not centered,alignment of the lamp to the mirror or lamphouseadjustments were not properly made and shouldbe corrected.

5.While still observing the diaphragm image, openthe field diaphragm until its diameter is equal to,or just slightly greater than, the entire microscopefield as seen.

Having accomplished the requirements of Kohlerillumination, it is necessary to take full advantageof this illumination. The back aperture of the objec-tive lens must be filled to obtain maximum resolvingpower by adjusting the aperture diaphragm.

28

Adjusting the Aperture DiaphragmThe aperture diaphragm is located beneath the con-denser and is adjustable. Its purpose is to controlthe diameter ofthe light beam entering the condenser,thereby controlling the angle of the cone of lightentering the objective lens so that the full apertureis utilized. The aperture diaphragm is adjusted in thefollowing manner (step numbers are continued fromprevious section):6.Focus on the specimen and remove the eyepiece

from the drawtube of the microscope.7.Look down the tube. The back lens of the objective

will be visible as a bright circle. (This step is aidedby the use of a pinhole eyepiece.) The centeredlamp-filament image (Step 4, above) will also bevisible.

8. Reach below the condenser and adjust the openingof the aperture diaphragm until its edge is seenwithin the bright circle. The correct setting occurswhen the diameter of the diaphragm image is about0.8 that of the back lens image, as seen in Figure 16.If the edge of the diaphragm image is not visible,

the aperture diaphragm is open too far. If it is leftthis way, the effect will be a loss of contrast in theimage of the specimen. Poor image quality will result.

If only a very small diaphragm image is visible,the diaphragm opening is reduced too far. The effec-tive numerical apertures of the objective and con-denser are thus reduced, and poor resolution results.Also, pronounced interference fringes appear in thespecimen image. Such fringes produce low imagequality.

The effect on image quality for three settings ofthe aperture diaphragm are shown in Figure 16.Proper adjustment of this diaphragm cannot be over-emphasized if the best image quality is desired.

Some microscopes have numbered settings markedon the edge of the aperture diaphragm. Since eachobjective will require a different setting, this facilityenables one to adjust the diaphragm accurately with-out looking down the tube of the microscope. Eachtime the objective is changed, the correct, predeter-mined setting can be made easily. It is also possibleto mark the diaphragm with the correct setting foreach objective.

Adjusting the Field DiaphragmThe principal functions of the field diaphragm arecontrol of image contrast by minimizing flare andcontrol of the size of the illuminated field. Thisdiaphragm has no effect on image brightness. Ifbrightness is affected, the microscope is not properlyadjusted for Kohler illumination.

1 lull 1 111111I11

!\ I 1111111111110,"" 1101111111'I ,Ill 1111"111'1111111'1111111'1111111111111111101PI"1.11I" 11111111111111111',111111111111I11I1I1J1111111111/"11111flllld tllilplllllllllllllltlloollllOlllt 011111111111111110dllljllllll[J1l1. I\ltol UH.l IIgl1t beam passesthrough the condenser, the uniform bundle oflight passes through the specimen. Theoretically,an image of the filament is also formed at the exitpupil of the Objective. As the light beam continuesthrough the microscope, it again forms an imageof the filament, this time at the shutter or cameraaperture. This diagram shows the path throughthe microscope of just two light rays originatingat the same point on the filament. In actuality, thecollector lens focuses an infinite number of rayson the aperture diaphragm.

hll ",,"111 III

I IIIill lilY" 11111dllllvllI 1111III 11111IIIIld IIll1pl 111111"1by 1I1ucoltoctot IUIIII. 1110COlldol1S01 .ocotvos th

rays from the field diaphragm and brings theminto sharp focus in the plane of the specimen. By

focusing an image of the field diaphragm in thespecimen plane, the focused light picks up animage of the specimen and projects it into the

Objective. As these light rays travel through themicroscope toward the eyepiece, an aerial image

of the specimen is formed at the entrance pupilof the eyepiece. The eyepiece receives the image

rays and focuses an image of the subject on thefilm plane for photography or on a viewing screenfor visual purposes. This diagram shows the path

of just two image-forming light rays through themicroscope. Actually, the image of the specimen

is formed by an infinite number of focused lightrays passing through the specimen.

FILM PLANE- I§£::")\)' c.:- tIII(~I---

£1. CAMERA ORFILM HOLDER .\\

~ SHUTTER ~=-- EYEPIECE EYE LENS:w: t=EYEPIECE I "I 1\ 11EYEPIECE .lrlUFIELD LENS

ENTRANCE PUPILOF EYEPIECE

BODY TUBE I

EXIT PUPILOF OBJECTIVE

I • OBJECTIVE • I

SPECIMEN .~

~~

~~. CONDENSER .~

'/'*1---- BASE ~(~

//-. G5'~~s\ MIRROR I<,(,(~ •

I COLLECTOR LENS I

,-I - LAMP I

FIGURE 17-Kohler illumination is a method of brightfield illumination necessary to successful photomicrography.Establishing Kohler illumination provides a uniformly i'lluminated field from a nonuniform source such asa filament lamp. The paths of the light beam and il'llilge-forming beam are diagrammed here.

In photomicrography, the field diaphragm is ad-justed until the diameter of its image is equal to,or just larger than, the diagonal of the film size. Itsimage can be seen and adjusted in size while lookingeither through a beam splitter or at the film planeofthe camera (as on a ground glass).

If the micrsocope is only used for visual work, thefielddiaphragm can be opened until the entire circularfield is seen in the microscope.

CondenserAs stated previously, an achromatic condenser ishighly recommended for all brightfield photomi-crography, but particularly for use with color films.It is essential, however, that the condenser, whateverthe type, be accurately centered with respect to theobjective for good photomicrography. Loss of lightand uneven illumination can result when the con-denser is out of alignment.

Since specimen slides may vary in thickness, it isusually necessary to readjust the position of thecondenser correctly each time the slide is changed.

The best slide thickness is usually specified by themanufacturer for each condenser. Slide thicknessbecomes quite critical when the use of immersion oilis required between the bottom of the slide and thetop lens of the condenser. If the slide is too thin,oil contact is lost when an attempt is made to focusthe condenser according to the Kohler system. If theslide is too thick, it may be impossible to focus thecondenser correctly.

Built-In IlluminationThe present tendency in the production of modernmicroscope is toward more convenient and compactillumination systems. Many microscopes (American,European, and Japanese) include illuminators builtinto the base. The illumination provided to the speci-men is usually a modified type of Kohler illumination,in that either a diffusing disk or a lens with a diffusesurface is placed in front of the light source. Noadjustment is provided for either the lamp positionor the field condenser. As a result, the lamp-filamentimage cannot be projected to the plane of the aperturediaphragm. Centering screws, however, are usuallyincluded for the lamp itself so that its position canbe adjusted for even illumination.

The entire illumination system is fixed in place,and the lamp and the collector lens are so positionedthat an image of the diffuser is formed on the dia-

30

phragm, A field diaphragm is provided so that itsimage can be formed in the specimen plane by adjust-ing the condenser. An aperture diaphragm is alsoincluded; its setting should still be established cor-rectly, as discussed on page 28.

Low-wattage tungsten lamps are generally used inbuilt-in systems. The illumination provided to thespecimen must always be corrected with appropriatelight-balancing filters if color films are to be exposed.It should be noted that built-in or separate systemsintended only for visual observation are not suitablefor photomicrography. An example is a form of opalglass illuminator used for making blood-cell counts.

Image Brightness and Neutral FiltersVery often when the microscope and the illuminationare correctly adjusted, the image in the microscopewill be extremely bright-too bright, in fact, for corn-fortable visual observation. The brightness of theimage can be reduced by placing a neutral densityfilter in the light beam. The degree of brightness willindicate how dense the neutral filter should be inorder to provide a comfortable level of brightness.Usually, a density of 1.00 or 1.20 will suffice; manybuilt-in illumination systems incorporate such a neu-tral filter for this purpose. Generally, it should beremoved for photography, since it will prolong expo-sure time.

When a separate microscope illuminator is used,it is good practice to have a neutral filter availableto reduce image brightness for visual work. In thiscase, the neutral filter is placed in a filter holder onthe front of the illuminator, and can easily be removedwhen a photomicrograph is to be made.

Although closing the aperture diaphragm decreasesimage brightness, image quality suffers for both visualand photographic purposes.

Neutral filters of several densities are actually usedin photomicrography to control exposure time so thatit will be within the range of available shutter speeds.

A method of reducing image brightness with tung-sten filament lamps is to use a variable transformer,such as a Powerstat or Variac unit. This can reducelamp intensity to a comfortable level for visual useor for intensity control when black-and-white filmsare used. Lamp life will be considerably prolongedalso. If color films are to be exposed, the lamp mustbe used at the normal rated voltage for photography.Newer tungsten-halogen lamps must not be usedbelow their rated voltage.

daylight illumination and for short exposure timesof 1110second or less.

When a particular film is to be exposed in photomi-crography, and the illumination differs from that forwhich the film was balanced, specific filters must beplaced in the light beam to adjust the illumination.KODAKLight Balancing Filters were made for thispurpose. Failure to make this adjustment may resultin erroneous color balance in the recorded image. ( eeKODAKLight Balancing Filters, page 35.)

All color films are rated according to their "speeds ,.or sensitivity to light. Film speeds are assigned bya standard rating system as numbers which aredirectly proportional to sensitivity-the larger thenumber, the faster the speed. A film rated at a speedof 160is twice as fast in its reaction to light as anotherfilm rated at 80, four times as fast as a film at 40.etc. The film speeds discussed in this book are thospecified by the American National Standards In ti-tute. The speed number given to a film is commonlycalled the "ASA speed."

Table 4 contains a listing of Kodak color films forphotomicrography, their speeds, how they are bal-anced, the various available sizes, and the processinvolved.

COlOR PHOTOMICROGRAPHY

FILMSThe ultimate function of the microscope and theillumination is to produce the best possible image ofthe specimen. If the specimen is colored, as withbiological stains, then the image will also be colored,and can be recorded to best advantage on a colorfilm. Filters are almost always necessary when a colorfilm is to be exposed, principally for control of colorbalance.

A working knowledge of color film characteristics,as well as of the action of specific filters, is essentialin color photomicrography if the micro-image is tobe recorded efficiently.

KODAK Color Films for Use InPhotomicrographyEastman Kodak Company manufactures two generaltypes of color films for exposure in a camera. Theyare designated as reversal color films and negativecolor films. The reversal films yield direct-positivecolor transparencies after reversal processing; theresultant colors are comparable to those seen in theoriginal subject. Color transparencies are viewed ei-ther by projection or on a suitable illuminator. Thenegative films yield color negatives after processing;the colors in the recorded image are complementaryto the corresponding colors in the subject. Colornegatives must be printed onto a positive color mate-rial in order to obtain a reproduction of the originalsubject colors.

KODAKEKTACHROMEFilms, KODACHROMEFilms,and KODAKPhotomicrography Color Film 2483 arereversal color films, whereas KODACOLOR-IIFilm andKODAKEKTACOLORProfessional Films are of thecolor negative type.

Color films are further classified with regard to thekind of illumination for which they are balanced inmanufacture. This is either artificial light (tungsten)or daylight. A film intended for use with artificial

~ light is usually specified as Type A, Tungsten, or TypeB, indicating that the light source for which the filmwas balanced had a color temperature of 3400K (TypeA) or 3200K (Tungsten and Type B). A film specifiedas "Daylight Type" is balanced for average sunlightat 5500 K. In addition, KODAKEKTACOLORProfes-sional Films are designated either Type L or TypeS. The Type L film is balanced for tungsten lightat 3200 K and for long exposure times (1110 secondto 60 seconds). The Type S film is balanced for

Selection of Color FilmsThe film characteristics to consider in selecting a colorfilm for photomicrography are size, speed, and theability of the film to record specimen colors as accu-rately as possible. Other factors are contrast, granu-larity, illumination, and color balance.

The film size, of course, is governed by the camerato be used. Most photomicrographic cameras accom-modate 35 mm film with a 24 x 36 mm frame size.Others accommodate sheet films of specific size. Thewidest selection of color films is in 35 mm size, whilethe narrowest is in sheet films.

In photomicrography, 35 mm reversal color filmsare most often used. These films yield color slidescommonly used in 2 x 2-inch slide projectors, suchas KODAKCAROUSELProjectors. When these filmsare processed, the individual frames are usuallymounted either in cardboard or in glass mounts fordirect insertion in the projector. The convenience of35mm slides is unquestioned-for handling, filing, andtransporting.

The selection of a Kodak reversal color sheet filmis as follows: Photomicrography Color Film 24

31

(4 x 5-inch size only), intended for Process E-4; KODAKEKTACHROMEFilm 6116, Type B (Process E-3), and

. KODAK EKTACHROMEFilm 6115, Daylight Type(Process E-3). The latter two films are available inall standard sheet sizes. Projectors for sheet-filmtransparencies are not available from Eastman KodakCompany, but can be obtained from photographicdealers. A projector of this type usually handles a3~ x 4-inch glass slide, so any film larger than thissize must be cut down to fit masks and cover glass.

Negative calor films offer versatility with regardto the kinds of prints which can be made from calornegatives. Positive calor transparencies can be madefrom a calor negative-either on KODAKEKTACOLORSlide Film 5028 for 35 mm slides or on KODAKEKTACOLORPrint Film 4109 (ESTARThick Base) tomake large transparencies in a variety of sizes. Calor

prints ofmany sizes can be made from a color negativeby using KODAKEKTACOLOR37RC Paper. This paperproduces excellent calor prints from Kodacolor II andEktacolor negatives. Black-and-white prints can bemade readily from calor negatives by using KODAKPANALUREPaper or other panchromatic paper.PANALUREPaper is a fast panchromatic enlargingpaper which renders colors in their correct gray tone.KODACOLORII Film can be obtained in 35 mm aswell as in other roll sizes.

The high film speed is important when a specimenis in motion, or possibly when the illumination inten-sity is very low. In either case, the use of a high-speedfilm may be of advantage. However, micro-subjectsnormally are stationary and film speed is unimpor-tant because exposure time can be controlled overa wide range.

TABLE 4 Kodak Color Films for Photomicrography

Commercial Oo-it-Yourself KitFilm Balance ASA Speed* Sizes Process Processing Processing Sizes

t\ODAK PCF135 (36 exp)Photomicrography Daylight 16 PCF415 (35mm x 125-ft) E-4 x l-qtColor 2483 4 x 5-inch sheet-film size

~ODAKHigh Speed Daylight 160 EH135 (20 & 36 exp) E-4 x x l-qtEKTACHROME(Daylight) EH120, EH126 (20 exp)

KODAKHigh Speed 3200 K 125 EHB135 (20 & 36 exp) E-4 x x l-qtEKTACHROME(Tungsten) EMB120

KODACHROME25 Daylight 25KM828

t No KitsKM135 (20 & 36 exp) x -

KODACHROME11 3400 K 40 KPA 135 (36 exp only) t x No KitsProfessional, Type A -

kODACHROME64 Daylight 64 KR135 (20 & 36 exp)t x No Kits -KR1~6

EX135 (20 & 36 exp)KODAKEKTACHROME-X Daylight 64 EX126 (20 exp), EX127, EX120 E-4 x x l-qt

EX620, EX828

CXl16, CX120, CX620 l-pintKODACOLOR11 Daylight 80 CX126, CX127, CX135-20 C-41 x x l-gal ~CX616, CX828

KODAKEKTACHROME6115,%-9alDaylight Type Daylight 50 All standard sheet-film sizes. E-3 x x

(Process E-3) l-gal

KODAKEKTACHROMEProfessional, Daylight 50 EP120 E-3 x x %-galDaylight Type l-gal(Process E-3)

KODAKEKTACHROME%-gal6116, Type B 3200 K 32 All standard sheet-film sizes. E-3 x x

(Process E-3) 1-gal

KODAKEKTACOLOR 64Professional 3200 K (5-second All standard sheet-film sizes. C-22 l-pint6102, Type L x x l-gal(Long Exposure) exposure)

KODAKEKTACOLOR CPS135 (36 exp)Professional, Daylight 100 CPS120, CPS220 C-22 x x l-pintTypeS CPS620 and all standard 1-9al(Short Exposure) sheet-film sizes.

°Film speeds quoted depend upon illumination level and exposure time, and refer to use of films under recommended conditions.tProcessed by Kodak or laboratory offering such service.

32

Normally, color films are not produced with a wideselection of either contrast or granularity. Recently,a new film, KODAKPhotomicrography Color Film2483,was introduced. It features the highest contrastand highest resolving power of any Kodak colorreversal film used for photomicrography. (Data onthis film is available on request. See page 3 for details.)Of the other Kodak color reversal films, KODACHROME64 and EKTACHROME-XFilms exhibit the most con-trast, although their contrasts are only slightly higherthan the other color films. KODACHROME25 Filmexhibits finer granularity than either KODACHROME64 Film or EKTACHROME-XFilm. These charac-teristics have been established in manufacture. Anyattempts to change contrast, granularity, or speedby departures from recommended processing mayresult in an undesirable change in calor balance.KODAKEKTACHROMEFilms designed for Process E-4,however, do have some leeway in this procedure.

As previously stated, color films are made for usewith either daylight or artificial light. The choice hereis optional, since either type can be used successfullyin photomicrography if the illumination is properlyadjusted with light-balancing filters. Normally, how-ever, if tungsten illumination is used with the micro-scope, a calor film balanced for artificial light shouldalso be used. Daylight-type films are suggested whenthe light source has daylight quality. Such sourcesas the xenon arc or electronic flash give this illumina-tion. Calor films balanced for daylight can also beused with tungsten sources if the illumination iscorrectly adjusted. The use of daylight films extendsthe selection of films available when one is lookingfor a film which will record and reproduce specimencolors most accurately. The rendition of specimencolor is related to the sensitivity and spectral responseof the calor film.

Rendition of Stain ColorThe many biological stains used in specimen prepara-tion, such as tissue sections and smears, representall of the colors of the visible spectrum. Color films,however, show considerable difference in sensitivityand response to various colors. For this reason, thecolor of one stain may record better on one colorfilm than on another. It depends on where that colorappears in the spectrum and how sensitive the colorfilm is to that region of the spectrum. The renditionof a particular calor also depends on the particulardyes formed in the emulsion layers of the film;different color films utilize different dye systems. So,in photomicrography, specimen dye-colors are record-ed on a color film containing different dyes. Unfortu-

TABLE 5 Field of Use>. e-,

Q.Cl 00 ":§ Ul

>. lii eEASTMAN Biological Stains* Cl o

0 >. (5 ~Cl"0 0 to1ii "0 '9 E:i: 1ii >. Cl>>. Cl c0; :i: Cl 0 Ul

0 "0 Cl>E E "0 s: (;

Calalog C ~ >. iO j

Number Chemical Name « Il. U Il. u::;..

C1740 Acid Fuchsln ,/ V- V- y' y'

IC1051 Alizarin Red S y' y' y'

C1741 Aniline Blue W.S. y' ,I V-C8688 Auramine 0 ,/ y' V-C8680 Azocarmine G y'

C8689 Azure A y' y'

C8683 Azure B y'

C8693 Azure C ,I y'

C1762 Basic Fuchsin y' y' y' ,/C1742 Bismarck Brown Y ,/ y' y'

C1743 Brilliant Cresyl Blue ,/ y'

j C624 Brilliant Green ,/C1745 Carmine Alum Lake y' y' y' y'

C8684 Chlorazol Black E ,I iC770 Congo Red ,I y'

C8687 Cresyl Violet Acetate y' y'

C1350 Crystal Violet ,I y' ,I y'

C5322 Eosin B y' ,IC1746 Eosin Yellowish y' V- y' V-C110 Erythrosin B ,/ V- y'

C8702 Ethyl Eosin y' ,IC868S Fast Green FCF V- y' ,IC8685 Giemsa Stain ,I IvC309 Hematoxylin ,/ y' y' ,/

C1009 Indigo Carmine y'

C4444 Janus Green B ,I ,IC8705 Jenner's Stain V-C5911 Light Green SF, Yellowish y' y' y' ,IC1264 Malachite Green ,I ,/ ,; ,;

C1979 Martius Yellow y' y'

C573 Methylene Blue y' y' ,/ y'

C8913 Methylene Violet (Bernthsen) y'

C1767 Methyl Green y' y' ,/C432 Methyl Orange y' ,I ,/ y'

C1309 Methyl Violet 2B V- y' y' ,;

C725 Neutral Red y' ,/C3536 Nigrosine W.S. y' ,I ,/C8679 Nile Blue A y' y'

C1751 Orange G y' y' y' ,IC196 Orange 11 y' y'

C1294 Orcein ,I V- ,;C8681 Phloxine B y' V- ,/C1752 Pyronin B V-C8707 Pyronin Y y'

C2245 Rose Bengal ,/ ,;C1753 Safranin 0 (Reddish) y' v v V-C1754 Sudan III vC1273 Sudan IV y' v V-C8690 Sudan Black B y' y'

C8709 Tetrachrome Stain (MacNeal) ,/ v'C1755 Thionin y' ,; v ,IC1756 Toluidine Blue 0 ,/ ~/ y'

C8682 Wright's Stain ,( ,/

*Availablein small quantitiesfrom suppliersof EASTMANOrganicChemicalsor directlyfrom EastmanKodakCompany.

33

nately, the results are not always satisfactory.. There is no one color film which will record allspecimen colors accurately. If specimen colors mustbe reproduced with reasonable fidelity, it may benecessary to experiment with different films to findone which is most suitable.

Although a comprehensive survey has not beenmade on the rendition of all biological-stain colorson the different col or films, there are some commonstains whose color rendition is known. Probably themost common stain is one called "eosin." This reddishstain is widely used with tissue sections in animalhistology in combination with another stain knownas "hematoxylin," and in combination with "methy-lene blue" stain for blood smears (Wright's Stain).This stain color reproduces well on KODAK Photomi-crography Color Film 2483 but not on KODACHROMEFilms, especially when the stain is very light in color.The use of a didymium glass filter enhances itsportrayal with KODACHROME Films. KODAKEKTACHROME-X Film does show excellent renditionof eosin color without this special filter. Filtering (seenext section) can often yield an improvement in therepresentation of many other stains.

In some neurological techniques a silver prepara-tion is often used. Tissue and nerve cells appear deepyellow as shown on EKTACHROME-X Film in PlateIV, C, page 40.

Prccesalnq Color FilmsAfter color films have been exposed in a camera, theymust be properly processed, either by the user or bya commercial processing laboratory. Eastman KodakCompany provides processing service forKODACHROME Films and for KODAK EKTACHROMEFilms in rolls. KODACOLOR II Film, also in rolls, isboth processed and printed by Kodak. Sheet colorfilms, however, are not processed by Kodak.

Many commercial laboratories are equipped toprocess and print all Kodak color films. Some of theseprocessing laboratories offer specialized services, suchas fast service enlargements with cropping, etc., whichwill be helpful to the photomicrographer.

Color films to be processed by Kodak laboratoriesshould be sent either through photographic dealersor in special mailers directed to Kodak. KODAKPrepaid Processing Mailers can be purchased fromdealers. When mailers are used, processed films willbe returned directly to the customer.

Some color films can be processed by the user. Theyinclude KODAK Photomicrography Color Film 2483,EKTACHROME Films (roll or sheet), KODACOLOR IIFilm, and EKTACOLOR Professional Films. (See Table4, page 32.) Chemicals are available in kit form for

34

each color process. Complete information is given indata sheets about mixing chemicals, processing times,agitation, and temperature. Further information isavailable in Kodak Publication No. R-19, KODAKColor DATAGUIDE, available from your photo dealer.

KODACHROME Films cannot be processed by theuser. They must be processed by a facility that hassuitable equipment for this purpose.

Increasing Color-Film SpeedIt is possible to use either KODAK EKTACHROME-Xor KODAK High Speed EKTACHROME Films at film-speed ratings higher than normal by increasing thefirst-developer time a specific amount. These filmscan be processed with KODAK EKTACHROME FillnChemicals, Process E-4. Increasing film speed byaltering processing is not possible with any otherKodak color films.

Best color quality will be obtained when either ofthe above films is processed normally. If a compromisein color quality is acceptable, however, the increasedspeed may be an advantage in some applications.

Processing for increased speed can easily be accom-plished when the user develops his own col or film.If film is usually sent to a commercial laboratory,the user should check with the laboratory to findout if this service is available.

Table 6 gives the suggested changes in first-developer times, Process E-4, and the resultant speedratings for various EKTACHROME Films.

TABLE 6Increasing Color Film Speed

First-DeveloperKOOAK High Speed KooAK High Speed KODAK

EKTACHROME Film EKTACHROME Film EKTACHROME-XTime(Daylight) (Tungsten) Film

Normal' 160 125 64

Normal plus35% increase 320 250 125

Normal plus75% increase 640 500 250

• Refer to instruction sheet packaged with Process E-4 chemicals for normal first-developer time.

Kodak Processing Service for Increased SpeedA KODAK Special Processing Envelope, ESP-l (avail-able from photographic dealers), enables a customerto send KODAK High Speed EKTACHROME Film (Day-light or Tungsten) to a Kodak laboratory in theUnited States for special processing for increasedspeed. The Daylight film will be processed for an ASAspeed of 400; the Tungsten film, for an ASA speedof 320. The cost of the special envelope prepays theextra charge for this service. This special service is

available only for the 135-20, 135-36, and 120 sizesof KODAKHigh Speed EKTAcHRoMEFilm.

FILTERSThe ultimate function of both the microscope andthe illumination is to produce the best possible imageof a specimen. A photomicrograph is a record of thisimage on a photographic, or light-sensitive, material.If the specimen is colored, as with biological stains,then the image will also be colored and will berecorded ideally on a color film. If a colored imageis to be recorded on a black-and-white material, thecolors ' must be reproduced as tones of gray thatsatisfactorily represent the color brightnesses in thespecimen. It is usually necessary to use specific lightfilters either to provide correct rendition of colors oncolor film or to record the calms as appropriate graytones with suitable contrast on a regular film or plate.

KODAK Light Balancing FiltersThese filters are intended for the adjustment ofillumination when it differs from that for which acalor film is balanced. Illumination calor quality isusually expressed as "color temperature" in degreeson the Kelvin scale. KODACHROMEII ProfessionalFilm (Type A) is balanced in manufacture for a lightsource having a color temperature of 3400 K. If thelight source to be used in exposure of this film hasa lower color temperature than 3400K, its illumina-tion can be adjusted with one or more KODAKLightBalancing Filters of the 82 series to approximate a3400 K color temperature. There are four filters inthis series: No. 82, 82A, 82B, and 82C. Each willeffectively raise color temperature by definite incre-ments. Of course, these filters don't actually changethe color temperature of the light source, but theydo modify the illumination to simulate a higher calortemperature. The No. 82 filter will effectively increasethe color temperature by 100K; the No. 82A,by 200K; the No. 82B, by 300 K; and the No. 82C, by 400K. If the light source had a color temperature of 3000K, therefore, its illumination could be adjusted to3400 K with a No. 82C filter. The above filters canbe used in combination to adjust illumination bygreater amounts than 400K. For example, if the lightsource were 2800K, its illumination could be adjustedto 3400 K with a No. 82C plus a No. 82A filter. Allofthe 82 series filters are light blue in color.

Another group of KODAKLight Balancing Filtersapplies when color temperature is too high for a film.This is the No. 81 series, which includes No. 81, 81A,81B, 81C, 81D, and 81EF. These filters are yellowishin color and will effectively decrease color tempera-ture by 100 K, 200 K, 300 K, 400 K, 500 K, and 600

K. These figures are only approximate, however incethe actual amount depends upon the initial colortemperature. The No. 81 series filters are not oftenused in photomicrography, however, since too higha color temperature seldom occurs.

An important fact to be considered is that theactual color temperature of a light source may beunknown. Then, too, the illumination quality at thefilm plane may be different than that emitted frothe source because of optical absorptions within themicroscope and the illumination system. Mo t b . -in illumination systems, for example, contain a diffussurface, which will effectively decrease color temper-ature by about 200 K. So, even if the somce ereknown to have a specific color temperature, - eillumination color-quality supplied to the filmbe different. Most often, it is lower in effective co ortemperature than is indicated by the source.

Tungsten-filament lamps are often used in p 0-0-

micrography, particularly in the buil -in illumina '0

systems, where 6-volt coil-filament lamps are co -mono These lamps, manufactured by several 'differ in color temperature through a range of a2800 K to 3200 K, even when used at the specifiedvoltage. A lamp in one microscope system, then, maydiffer in color temperature from the lamp in anothersystem. It is difficult to say, then, which light-balanc-ing filter, or filters, should be used.

The most practical method for determining thecorrect filters is to make color-balance tests, usingdifferent filters or combinations of filters. The 82series of light-balancing filters usually applies withtungsten illumination, since the color temperatureis almost invariably too lowfor an artificial-light film.If the initial color temperature of the source is un-known, a series of exposures might be made usingthe No. 82, 82A, 82B, 82C, 82C + 82, and 82C +82A filters and a reversal color film balanced forartificial ·illumination. After the color film is pro-cessed, the different exposures could be examined onan illuminator to determine which one showed themost pleasing rendition of specimen colors and aclean, almost-white background. The filter or filtersused to make that exposure would then be correctfor future exposures. If a transformer was used toprovide the 6 volts to the lamp, the same settingshould always apply for making future photomicro-graphs. Several voltage settings aloeoften availableon a transformer. The highest setting should normallybe utilized, since the highest color temperature forthe lamp is obtained at this setting.

If the color temperature of the source is known,even approximately, the color-balance test series canbe much shorter; very often, only about two filters

35

or combinations need to be applied.Light-balancing filters should be placed between

"the illuminator and the condenser of the microscope.Separate illuminators usually contain filter recepta-cles for the purpose.

TABLE 7

KOOAK Filters' for KODAK Color Films

Tungsten. Type B. or Type A Daylight-TypeType L Film (3200 KI Film (3400 KI FilmLight Source

Filter No. Filter No.Filter No.

6-Volt Ribbon-Filament(Average C.T.=3000 K) 82A 82C 80A+82A

6-Volt Coil-Filament(Average C.T.=3100 K) 82 82B 80A+82

lOO-Watt Coil-Filament(Average C.T.=3l00 K) 82 82B 80A+82

300- to 750-Watt Coil-Filament (3200 K) None 82A 80A

I Zirconium Arc (3200 K) Nonet 82A 80A

Photoflood lamp (3400 K) 8lA None 80B

I CarbonArc (3700 K) 81Dt 818 aocXenonArc (5500 K) 858 85 Nonet

"The filters suggested are considered to give approximate calor-temperature compensation. Calortest exposures are often necessary to obtain best color balance.

tA No. 2B lilter is olten used to absorb unwanted ultraviolet Irom arc illumination.

Table 7 contains average filter recommendationsfor different light sources and various color films. (Seealso the nomograph on the inside front cover.) Theserecommendations can only be considered as approxi-mate, because ofthe variance in the color temperatureof light sources and the electrical conditions at thetime of exposure. Voltage regulators are desirable forcritical work.

KODAKLight Balancing Filters can be obtainedfrom photo dealers. They are supplied as gelatin filmsquares (50, 75, 100, or 125 mm). These gelatin filmsquares can be inserted into two-part pressed metalframes called KODAKGelatin Filter Frames, whichmake the filters convenient to handle and store. Theseframes are also available through photo dealers. Ifglass-mounted filters are needed, they can be obtainedfrom suppliers such as Tiffen Optical Company, 71Jane Street, Roslyn Heights, New York 11577.

All daylight-type color films can be used in photo-micrography, regardless of the type of illumination,if the correct filters are placed in the light beam toadjust the illumination to daylight quality. Thesefilms can also be used, of course, with light sourcesof daylight quality-such as the xenon arc or elec-tronic flash-and probably without any light-balanc-ing filters. With tungsten lamps and the zirconium

36

arc, however, the use of specific light-balancing filtersis necessary if daylight-type films are to be exposed.

KODAKWRATTENGelatin Filter, No. 80A willadjust illumination color-quality from 3200 K todaylight. If color temperature is below 3200 K, asit often is, one or more of the 82 series filters willalso be needed. In order to establish which filter ofthe 82 series should be used with the No. 80A filter,a color test-exposure series can be made. Five expo-sures will probably suffice; one with the No. 80Afilteralone and four with the combinations of No. 80A +No. 82, No. 80A + No. 82A, No. 80A + 82B, andNo. 80A + No. 82C. One of the resultant exposureswill show the best color balance. The correct combi-nation should be used in subsequent photomicro-graphy when a daylight-type film is exposed.

KODAK Color Compensating FiltersUndesirable color effects are caused by several factorsin color photomicrography in addition to the onealready mentioned-exposing a color film to illumina-tion other than that for which the film was designed.These effects can usually be corrected with KODAKColor Compensating Filters-available in variousdensities in red, green, blue, cyan, magenta, andyellow. These filters are commonly called "CCFilters" and can be obtained through photographicdealers. They are supplied as gelatin film squares andcan be inserted in KODAKGelatin Filter Frames.

The various general effects that can be correctedwith CC Filters are discussed in "Factors AffectingColor Balance," page 41. Pale color compensatingfilters can sometimes be used to enhance the renditionof certain stains. However, the benefits gained by useof a filter of the approximate color of the stain mustbe balanced against a possible degradation of compli-mentary stain color.

Neutral Density FiltersNeutral density filters can be used in photomicro-graphy to reduce image brightness as a means ofcontrolling exposure time. A neutral filter will absorba specific amount of light, depending on its density,without affecting the color quality of the illumina-tion. The principal application of such filters is inthe exposure of color films. They can also be usedwith black-and-white films to prevent overexposure.

If a very intense light source is used to provideillumination, the correct exposure time (as deter-mined by a test-exposure series or by light measure-ment) may be shorter than the fastest availableshutter speed. A neutral density filter can be placedin the light beam to reduce image brightness so thatthe exposure time will be within the shutter range.

Plate I, A-Taste buds in rabbit tongue. x 100. Iron hematoxylin stain.KODAK EKTACHROME Film, Type B.

Plate I, B-Photomicrographs on KODACHROME 11 Film,Daylight TypeA. Uncorrected tungsten illuminationB. Illumination adjusted with KODAK Light Balancing

Filters (NO. 80A and 82A)

Plate I, C-Highlight dots on printed material- x 100.KODAK EKTACHROME Film, Type B.

a

b

Plate I, D-Diatom (Phase)- x 1000. KOOA

EKTACHROME Film, Type B.

Plate 11, A-Amoeba proteus (interference microscope)-x 150.

Plate 11,C-Micro-artery- x 125. Aldehyde fuchsin and lightgreen stains. KODAK EKTACHROME Film 6116, Type B (ProcessE-3.)

Plate 11,D-Fluorescence photomicrograph. Acridine orangestain. KODAK High Speed EKTACHROME Film (Daylight).

Plate 11,B-Human pancreas, x 150. Comparison ofimage quality with (a) apochromat, (b) achromat.

Plate 11,E-Unpolished surface of an industrial dia-mond photographed by the Francon system. Thismethod is similar to the Nomarski system (discussedunder "Specialized Techniques and Applications"),but it produces interference from surface light re-flected by thick or opaque specimens instead of fromtransmitted light.

Plate Ill, A-Image of shutter blades-shutter too far from eyepoint.

Plate Ill, 8-(a) Image of field diaphragm; (b)Image removed by opening field diaphragm.

Plate Ill, C-Printed micro-circuit- x 40. Reflected light.

a

Plate Ill, O-Black stem rust of wheat.

a b

Plate IV, A-Effect of eosin stain (a) without didymium filter; (b) with didymium filter.

Plate IV, B-Zinc-silver alloy, chemical stain. Plate IV, C-Nerve cells in human cerebellum- x 200, Silverimpregnation. KODAKEKTACHROMEFilm, Type B.

Plate IV, D-Human hair, brown-x 150. KODAK EKTACHROMEFilm,Type B.

The absorption of light by a neutral filter is directlyproportional to the filter's density. The greater thedensity, the greater the amount of light absorbed.Also, the greater the density, the lesser the amountof light transmitted. Density is defined mathemat-ically as the logarithm of the reciprocal of transmit-tance (D = log+). KODAKWRATTENNeutral DensityFilter, No. 96, is available in a wide range of densitiesfrom 0.10 to 4.0. The amount of light transmittedby these neutral density filters varies from 80 percentto 0.01 percent, respectively. The following tableincludes various filter densities available and theircorresponding transmittances.

Density PercentDensity Percent

Transmi1tance Transmi1tance

0.10 80.0 0.80 16.0

0.20 63.0 *0.90 13.0

*0.30 50.0 1.00 10.0

0.40 40.0 2.00 1.0

0.50 32.0 3.00 0.10

*0.60 25.0 4.00 0.010

0.70 20.0

*Most often used in photography.

The netural density filters most often used inphotography to control exposure time have densitiesof 0.30, 0.60, and 0.90. As shown in the table, thesefilters have transmittances of 50, 25, and 13 percent.Since a density of 0.30 has a transmittance of 50percent, it can be used to reduce brightness by a factorof 2. A density of 0.60 has a transmittance of 25percent and a reduction factor of 4. These densitiescan be used in combination because the total densityequals the sum of the individual densities.

Here is a sample exposure calculation involvingneutral density filters: Suppose that a very brightlight source provides illumination, and that a reason-ably fast film is to be exposed. The correct exposuretime is determined to be 11125second, but the fastestavailable shutter speed is only 1160 second. Thisshutter speed would cause overexposure. A densityof 0.30 could be placed in the light beam to reduceimage brightness by 50 percent; 1/60 second wouldthen be the correct exposure. If a density of 0.60wereplaced in the light beam, the correct exposure settingwould then be 1/30 second.

Because reversal color films have a very shortexposure latitude, it sometimes happens that the bestexposure is considered to be between two shutterspeeds. For example, 1/60 second may be too short,

causing slight underexposure; but 1130 second maybe too long, causing slight overexposure. In this case,a neutral density of either 0.10 or 0.20 could be usedto reduce the light level by less than a factor of 2;1130second would then be the correct shutter speed.

Neutral density filters are also used to reduce visualimage brightness. A very dense filter is placed in thelight path when image brightness is too high forcomfortable viewing. A density of 1.00 or more willusually suffice for this purpose, but it should beremoved for photography because it may prolongexposure time too much. Long exposures should beavoided, when possible, because of "reciprocity ef-fects" with color films and the resultant possibilityof color shift and decreased film speed. (See "FactorsAffecting Color Balance," below.)

KODAKWRATTENNeutral Density Filter, No. 96,can be obtained from photographic dealers as gelatinfilm squares in 50, 75, or 100 mm. sizes on 0.1 mmgelatin with protective lacquer coating. Larger specialsizes can be supplied also.

FACTORS AFFECTING COlOR BALANCEAt one time or another probably everyone who hasmade color photomicrographs has encountered unde-sirable color effects. Sometimes the reason is immedi-ately obviousor can be easily determined by reviewingthe conditions that affect exposure. Occasionally,however, the cause of erroneous color balance baffleseven the most careful photomicrographer. If the causeis known, suitable correction can often be made insubsequent exposures; if it remains an unsolvablepuzzle, a complete review ofpossible causes is indicat-ed. Here are some suggestions.

Many undesirable effects can be neutralized byplacing appropriate KODAKColor Compensating (CC)Filters in the microscope light beam. Some effectson color balance, however, are due either to incorrectuse of optics or to problems related to film storageand film processing. These effects cannot be compen-sated with filters. The only remedy is to followrecommended procedures.

The most common reasons for a color-balance shiftare listed in this section. Some are peculiar to photo-micrography, but many can be encountered in anytype of photography in which color film is used. Onlycolor reversal films are considered here, since colornegatives may not show a color change until theyare printed. When color negatives are slightly off-balance, the effects can usually be corrected in print-ing if they are not too pronounced.

Table 9, page 46,summarizes some of the difficultieswhich may be encountered, their possible causes, andtheir remedies.

41

Color- Temperature VarianceVariation in color temperature is probably the mostcommon reason for unexpected color shifts in colorphotomicrography. Whenever the illumination differsfrom that for which a particular film is balanced, thephotomicrograph will have shifted color. If the colortemperature of the light source is too high, an overallcold, bluish effect will be noticed in the photomicro-graph. If the color temperature is too low, the photo-micrograph will be too "warm" and will be eitheryellowish or reddish-yellow in appearance. The degreeof color shift will vary according to the amount ofcolor-temperature difference between the actual lightsource and that for which the film was balanced. Anexample of a large color shift is a micrograph madeon daylight color film with uncorrected tungstenillumination. (See Plate I-B, page 37.)Color-tempera-ture variance can be corrected by placing appropriateKODAKLight Balancing Filters in the illuminationbeam.

Heat-Absorbing FiltersMany light sources-such as high-wattage tungstenlamps and xenon arcs=emit a considerable amountof infrared in their illumination. This infrared isevidenced as heat and should be removed by anappropriate "heat-absorbing" filter to protect themicroscope optics, the specimen, and any filters inthe light beam. Some microscope illuminators haveheat-absorbing glass filters built in. Unfortunately,the owner of such an illuminator may be unawareof the presence of the filter, which is usually coloredgreen 01' blue-green. This coloration can affect thecolor balance of a photomicrograph. The transpar-ency may appear too green or blue-green. This effectcan be corrected in subsequent exposures by placinga "neutralizing" KODAKColor Compensating Filterin the light beam. A CC Filter of a complementarycolor is necessary. If the heat filter is greenish, a palemagenta CC Filter will absorb the green and producea neutral effect. If the filter is blue-green, a pale redCC Filter is indicated. Heat-absorbing glass filtersvary in their degree of coloration; it is not possible,therefore, to assign one specific CC Filter for correc-tion. The correct filter must usually be determinedby test exposures. If the heat filter can be removedfrom the lamp temporarily, you can place it on atransparency illuminator and view it through CCFilters of an appropriate complementary color untilone is found which best neutralizes the heat-filtercolor. Of course, permanent removal of the heat filteralso solves the problem, but this may not be wise,since permanent damage to the optics, filters, andspecimen may result.

42

In some types of photomicrography, colored-liquidfilters are used in the light beam to remove heat.In black-and-white metallography, for example, adilute solution of copper sulfate in an absorption cellis often used. The blue-green color of the solutionwould dominate the color of a photomicrograph madeon color film. During photography, therefore, thesolution should be replaced by a cell containingdistilled water.

Ultraviolet RadiationColor film is very sensitive to ultraviolet radiation,which can be recorded as "blue" by the blue-sensitiveemulsion layer. If ultraviolet is present, as in xenon-arc illumination, it may cause a color photomicro-graph to appeal' too blue. An ultraviolet-absorbingfilter, such as KODAKWRATl'ENFilter No. 2B, shouldbe used. The No. 2B Filter will absorb ultravioletbut will transmit all visible colors. The bluish effectof ultraviolet radiation on color film is often encoun-tered in the photomicrography of metals. Most elec-tronic fl.ashtubesalso emit ultraviolet. If a light sourcewhich emits ultraviolet is used in photomicrographywith color films, the No. 2B Filter is recommended.

Neutral Density FiltersSome "neutral" filters normally used to reducebrightness may have a slight yellowish color, eitherinherently or as a result of aging.This yellowish colorcan be neutralized with a pale-blue color-compensat-ing filter, either a CC05B or a CC10B. The effectgenerally isn't very great and can often be ignored.

Biological StainsThe stains used in coloring a section or smear toproduce contrast between the elements of the speci-men have individual properties of absorption andtransmittance. Some stain colors reproduce very wellwhen recorded on a specificcolor film,but others oftenappear quite different on film than when seen in themicroscope. Eosin and fuchsin, for example, do notrecord well on KODACHROMEFilm. Then: rendition,however, can be noticeably enhanced when a glassdidymium filter is placed in the illumination beam.The thickness of the didymium filter should not begreater than 2 mm. If it is greater, a background colormay appeal' or other stain colors may be degraded.Didymium glass filters are not available from East-man Kodak Company but are available from theComing Glass Works in Coming, New York 14830.If you order from Coming, specify half-standardthickness.

Both eosin and fuchsin stains record very well on

cyan, yellow, or magenta.When a photomicrographer changes from one film

emulsion to another, the resulting photomicrographmay show an undesirable background color. Thebackground should be white or very light gray. If itis not, this can be corrected in subsequent exposuresby placing a CC10 Filter of a complementary colorin the light beam. (SeeTable 9, page 46.)

When many rolls of a particular color film will beused over an extended period of time, several rollsof film with the same emulsion number can be pur-chased and stored in a refrigerator or freezer. If adeviation from normal color occurs in a film due tomanufacturing difference, the amount of deviationcan be determined by a filter-balance test with oneroll. The test consists of making one exposure withno color-compensating filter and six exposures witha CC10R, CC10G, CClOB, CC10Y, CC10C, andCC10M filter, respectively. When all seven photomi-crographs are placed on a suitable illuminator, oneof them will probably show a clean white or verylight gray background. In this way, the correct com-pensating filter can be determined.

Reciprocity EffectReversal color films of daylight type are normallydesigned for short exposure times-such as 1/30 sec-ond, 1/60 second, and 1/125 second. When the expos-ing illumination is correct, one of these short exposuretimes will usually produce normal color balance inthe photomicrograph. Whenever the exposure timefor a color reversal film is 1second or longer, however,an undesirable color effect may be noticeable in thecolor photomicrograph. With certain color films, evenan exposure of 1/8 or 1/4 second may produce anoticeable effect on color balance. If the light inten-sity is very low, a long exposure time is often neces-sary, resulting in a color shift. This color shift is dueto the reciprocity effect. To explain: under normalconditions total exposure equals illuminance multi-plied by exposure time (E = IT). As the illuminationlevel increases, exposure time decreases and viceversa, in a reciprocal relationship. Throughout anormal range of light levels and exposure times, thisrelationship holds true, but in very low or extremelyhigh light levels it may not. The photographic effectwill vary with changes in illuminance (I) and time(T). This is especially true for long exposures, whichare quite common in photomicrography.

Reciprocity effect is usually apparent as a decreasein emulsion speed at very low light levels. Since acolor film contains three emulsion layers, a changein color balance occurs unless all three layers changealike. All three layers, however, may exhibit different

44

reciprocity effects. Color balance can be quite errone-ous when a very long exposure time is necessary. Longexposure times are often necessary in fluorescencephotomicrography, as well as in photomicrographyat high magnification, with polarized light, or withan interference microscope.

Color shift due to reciprocity effect can often becorrected by placing a color-compensating filter inthe light beam before exposure. At very long expo-sures, two filters may even be necessary to compen-sate color variance.

Information on both exposure and filter compensa-tion for reciprocity effect is given in Table 8. Thedata given in the table for each film applies only tothe type ofillumination for which the film is balanced.

Miscellaneous FactorsSeveral reasons for poor colorbalance are attributableto "improper handling" of color film, not to anyspecific photographic exposure technique or condi-tion. In some cases, a permanent effect is produced,since no filter compensation can be applied. If aphotomicrograph shows an undesirable color effectthat is not due to any of the factors mentionedpreviously, one of the following conditions may bethe cause.Improper Storage: All photographic films are perish-able products that are damaged by high temperatureand high relative humidity. Color films are moreseriously affected than black-and-white films becauseheat and moisture usually affect the three emulsionlayers to different degrees. For color film, a changein color balance may be accompanied by a changein overall film speed and contrast. None of theseeffects is entirely predictable. Proper storage of colorfilm is necessary both before and after exposure forconsistent color balance. Of course, greater care isnecessary under hot and humid conditions. Recom-mended storage conditions are covered in KodakPamphlet No. E-30, Storage and Care of KODAKColor Films, and in the Data Book KODAK ColorFilms, Kodak Publication No. E-77; available fromPhoto dealers.

Chemical Fog: Color films not sealed in foil envelopesor screw-cap cans should be kept away from anyfumes, from formaldehyde, from paraformaldehyde,as well as from any other harmful gases or vapors.Such gases can influence color balance, speed, andcontrast; their effects on color film are erratic andunpredictable. Color balance can vary in any direc-tion. Film speed may increase, but contrast willusually decrease-especially with extended exposureto a particular gas, since maximum density is reduced.

TABLE 8Reciprocity Characteristics of KODAK Color Films

(Exposure* and Filter Compensation)

Exposure Time (Seconds)Film

1/1000 11100 1/10 1 10 100

KODAK Photomicrography + 1 stop + 2Y2 stops NotNone None None CC40Y+Color 2483 CC20Y CC10M Recommended

KODACHROME". None None + % stop + % stop + 1 stop NotProfessional (Type A) No Filter No Filter No Filter No Filter CC10Y Recommended

KODAK EKTACHROME6116 None None None NonetType B (Process E-3) No Filter No Filter No Filter No Filter

KODAK EKTACHROME6115 None None + % stop + 1 stop + 2 stops NotDaylight Type CC10B +(Process E-3) No Filter No Filter CC10B CC10B CC10M Recommended

KODAK High Speed None None None None + 1 stop NotEKTACHROME(Tungsten) No Filter No Filter No Filter No Filter No Filter Recommended

KODAK High Speed None None None + 1 stop + 1% stops NotEKTACHROME(Daylight) No Filter No Filter No Filter No Filter No Filter Recommended

KODAK EKTACOLOR None None NoneProfessional 6101. No Filter No Filter No Filter Not HecomrnendedType S

KODAK EKTACOLOR See Film Instructions for SpeedsProfessional 6102, Not RecommendedType L No Filter from 1/10 to 60 sec

KODACOLOR" None None None + % stop + 1 stop + 1Y2 stopsNo Filter No Filter No Filter No Filter No Filter No Filter

KODAK EKTACHROME-X None None None + 1 stop + 2 stops NotNo Filter No Filter No Filter No Filter CC15Y Recommended

KODACHROME25 tt None None tt tt ttNo Filter No Filter

KODACHROME64 tt None None t tt ttNo Filter No Filter

*The exposure increase, given in lens stops, includes the adjustment required by any filter or filters suggested.

tSee Supplementary Data Sheet packaged with the film.

ttAt the time of publication, specific recommendations for reciprocity effects with KODACHROME25 and KODACHROME64 Films are not available. However, individual tests using10 CC filter intervals are suggested. As a starting point, try red filtration for long exposures and cyan filtration for short exposures.

Note: The data for each film in the above table are based on average emulsions. They apply only to the type of illumination for which that film is balanced, and assume normalrecommended processing. The data should be used as guides only. The adjustments are subject to change due to normal manufacturing variations or subsequent film storageconditions after the film leaves the factory.

A loose camera back may allow some light to leakinto the camera. The extent of "flare" in this casedepends upon how long the condition exists.Outdated Film: When possible, color film should beused before the expiration date stamped on the boxbecause a change in color balance is possible afterthis date. The magnitude of change depends uponstorage conditions during the usable "life" of that

Light Fog: Color films should be processed either intotal darkness or in lighttight tanks. If a color filmhas receivedeven a very short exposure to a darkroomlight-leak or to a safelight, color balance will beaffected. For example, a dark green safelight, whichmay be used briefly with panchromatic black-and-white films, will impart a green fog on color reversalfilms.

45

TABLE 9Color-Balance Corrections

Appearance of Possible Cause RemedyPhotomicrograph

Slightly Yellow 1. Emulsion variance Use CC1OB filter.2. Colored mounting medium Use blue CC Filter

(CC10B or more).3. Low color temperature Use light-balancing filter

of light source of 82 Series.

Slightly Magenta Emulsion variance Use CC1OGfilter.(reddish blue)

Slightly Cyan 1. Emulsion variance Use CC1ORfilter.(bluish green) 2. Heat-absorbing filter in Use CC1OR,or possibly

light beam CC20R, filter.

Slightly Blue 1. Emulsion variance Use CC1OY filter.

2. Abbe condenser not Adjust condenser for Kohlerfocused correctly illumination.

Definitely Blue 1. Ultraviolet radiation present Use No. 2B filter to removeduring exposure with arc ultraviolet radiation.lamp Use appropriate light-

2. High color temperature of balancing filter of 81light source Series.

Slightly Green 1. Emulsion variance Use CC10M filter.

2. Heat-absorbing filter Use CC1OM, or possiblyin light beam CC20M, filter.

Slightly Red 1. Emulsion variance Use CC1OCfilter.

2. Abbe condenser not Adjust focus of condenserfocused correctly for Kohler illumination.

Slightly Yellow-Red Low color temperature of Use light-balancing filterlight source of 82 series.

Definitely Reddish Yellow Daylight film with tungsten Use No. 80A filter, plussource-no correction light-balancing filter of

82 series.

46

film. If a film has been properly stored under recom-mended conditions, the rate of change is decreasedbut not eliminated. When a quantity of film is storedin sealed containers in a freezer at low temperature(0 to -10 F, for example), the rate of change is greatlydecreased. In this case, film can be used beyond theexpiration date without excessive change in charac-teristics. Film stored in this way should not be openedfor use for at least 2 to 3 hours after removal fromthe freezer; otherwise, condensation might occurwhen the cold film is subjected to room temperature.

Radiation Exposure: Whenever a color film has re-ceived exposure from a source such as x-rays, radium,cobalt 60, or other radioactive materials, a changein color balance can occur. Such exposure can happenin hospitals, industrial plants, or research laboratorieswhere either radiography or radiotherapy is practiced.If color films are stored or used in areas adjacentto a room where such radiation is present, suitableprotection should be provided in the form of lead orconcrete shielding of adequate thickness.

Processing Errors: Some changes in color balancein KODAKEKTACHROMEFilm and KODAKPhotomi-

crography Color Film 2483can be traced directly toa departure from recommended processing conditions.The color change can be toward magenta, green,green-yellow,or blue. The causes of such changes canbe uneven or insufficient agitation, inadequate rever-sal exposure, one or more exhausted solutions, con-tamination of solutions, improper mixing of chemi-cals, or incorrect time or temperature. The resultingchanges in color balance cannot be compensatedadequately. The only remedy is correct processing,as recommended in instructions furnished with pro-cessingchemicals. Information on color variances dueto processing is included in the following Kodakpamphlets: Identifying E-3 Processing Errors (E-60)and Identifying E-4 Processing Errors (E-62). Bothare available on request.Viewing Conditions: The conditions under which atransparency is viewed also may have a noticeableeffect on its apparent color quality. For critical use,transparencies intended for projection should bejudged by projection. If a transparency is to be viewedon an illuminator, such as in an exhibit or otherdisplay, the transparency should be judged on anilluminator similar to the one that will be used.

BLACK-AND-WHITE PHOTOMICROGRAPHYBlack-and-white photographic materials are oftenused in photomicrography for a variety of reasons.Illustrations in scientific books, journals, and reportsare usually in black-and-white photography becauseof the additional expense involved in printing color.Although color might be more desirable, a goodphotomicrograph in black-and-white is always accept-able. (Figure 18.) Black-and-white materials havethe advantages over color of better contrast control,more consistent development, and quicker results.

PROPERTIES OF PHOTOGRAPHICMATERIALSA black-and-white photomicrograph is almost alwaysa negative, which must be printed onto a suitablepositive material. Reversal materials are seldom used.The selection of a negative material, usually a film,requires knowledge of photographic characteristicssuch as color sensitivity, contrast, granularity, resolv-ing power, speed, and exposure and developmentlatitude.

Color sensitivity is an inherent characteristic relat-

FIGURE 18-Same as cover, except photographed on KODAKEKTAPAN Film. A KODAK WRATTEN Filter No. 11 was used for correcttonal rendition of specimen colors.

ed to the response of a film to colors of the spectrum.A black-and-white film may be blue-sensitive, ortho-

. chromatic, orpanchromatic. Blue-sensitive materialsrespond only to blue light, and to some extent toultraviolet; orthochromatic materials have extendedsensitivity in the green; and panchromatic materialsare sensitive to all visible colors. Panchromatic films(or plates) are most often used in black-and-whitephotomicrography, since stained, colored specimensare common. Color sensitivity is a fixed property ofphotographic material; it is not subject to change byalteration in processing, as are other characteristics.

Generally, a film with very fine grain, high resolvingpower, and moderate contrast is selected for black-and-white photomicrography. Film speed is often aconsideration, but is of lesser importance unless thesubject is in motion and a film of high photographicspeed is needed. In this case, a sacrifice in granularity,contrast, or resolving power may be necessary in orderto record a satisfactory image.

The best procedure to follow in selecting a suitablematerial is to consider the specimen to be pho-tographed. If the image exhibits relatively low con-trast, a high-contrast film may be needed. Conversely,if the image has high contrast, a low- tomedium-contrast film may give best rendition ofdetail. If the negative must be enlarged, a film havingextremely fine grain can be an advantage.

Most films are classified as having low, medium,or high contrast. While this property can be alteredto some extent by changing development time in agiven developer or by changing the developer itself,the best practice is to follow the manufacturer'srecom,mendation as to developer and developmenttime. Devia ion from an established procedure mayhave a pronounced effect on graininess, resolvingpower, or speed. An experienced photomicrographer,with a good photographic background, may be com-petent enough to vary development in order to pro-duce a desired effect with a particular film, but thenovice would do well to follow recommendations.

Resolving Power and Graininess"Resolving power" refers to the ability of a pho-tographic material to record fine details distin-guishably. It is expressed as the number of lines permillimeter that are recognizable as separate lines ina photograph. Resolving power is determined for aparticular film by photographing-at greatly reducedsize-a parallel-line test chart with a high-qualitylens. The image is then examined through a micro-scope to determine the number of lines that can beresolved. Determination of resolution depends on the

48

test-object contrast; comparison of different films ismade only with test-objects of equal contrast.

Graininess is related not only to the size of silvergrains produced in a film after development but alsoto the irregular clumping of silver grains in the image.The degree of graininess will vary, depending on thetype of developer used and, somewhat, on develop-ment time. A "fine-grain" developer may produce lessgraininess, but speed and contrast will be reduced.Generally, fast films have coarser grain than slowfilms.

The graininess designation for a photographic ma-terial is usually an indication of inherent resolvingpower. A film with very fine grain is thus capableof high resolution. Remember, however, that resolu-tion in photomicrography is not dependent on theresolving power or on the graininess of a photographicmaterial. High resolution can be achieved only bya quality microscope used efficiently. Resolution ina microscope image cannot be improved pho-tographically. If the details in a specimen are notvisible in the microscope, there is no film or techniquethat will make them visible.

It is a common misconception that a film with veryfine grain is necessary to achieve high resolution inphotomicrography. The real advantage ofusing a filmwith very fine grain is the probability of good imagequality. Even though the recorded image cannot bebetter than the microscope image, the photographicmaterial should not degrade image quality. A filmwith high resolving power will show an image moreeffectively than one of coarse grain or one with lowresolving power. For example, a microscopic specimencontains fine details, whose separation is equivalentto about 2000 lines per millimeter. The microscopeoptics in use may resolve details at a magnificationof 500.The separation of details in the image is nowequivalent to 2000/500, or only four lines per milli-meter. Any film is capable of such low resolving power,but a fine-grain film will show the details to betteradvantage than a coarse-grain one.

Selection of a fine-grain film will also allow somephotographic enlargement without lowering imagequality. Too much enlargement, however, may resultin a state of empty magnification; that is, althoughthe image is larger, no more details are resolved. Thispossibility must be considered when small-size films,such as 35 mm, are used in photomicrography, Whenan image is recorded on a small film area, furtherenlargement is necessary in order to distinguish de-tails in a print. However, if maximum usablemagnification has been achieved in the microscopeand on the film, no further enlargement is possiblewithout degrading image quality. When enlargement

KODAK Sheet Films for Black-and-White Photomicrography

Color ASA Number ResolvingKODAK Film Sensitivity (3200K) Contrast Power Granularity

*EKTAPAN 4162 Pan 100 Medium Medium Very Fine

*PLUS-X Pan Professional 4147 Pan 125 Medium High Very Fine

"SUPER-XX Pan 4142 Pan 200 Medium High Fine

"TRI-X Pan Professional 4164 Pan 320 Medium Medium Very Fine

'"ROYAL Pan 4141 Pan 400 Medium Medium Very Fine

"Contrast Process Pan Film 4155 Pan 80 Very High High Fine

"ROYAL-X Pan 4166 Pan 1250 Medium Medium Fine

*Contrast Process Ortho 4154 Ortho 50 Very High Very High Fine

*TRI-X Ortho 4163 Ortho 200 Medium High Very Fine

Commercial 6127 Blue Sensitive 8 Moderately High High Very Fine

'Commercial 4127 Blue Sensitive 8 Moderately High High Very Fine

'All of these films have ESTAR Thick Base.

is contemplated, therefore, the magnification on thefilm should be lower than the maximum usable.

As an example, suppose that an image is recordedon film at x500 but that the microscope optics inuse are capable of X 1000. The recorded image couldstill be enlarged 2X without causing emptymagnification. If the same image were recorded atonly X 250, then a 4 X photographic enlargementwould be possible. If the image were recorded atX 1000, however, no more enlargement could be ac-complished efficiently. These facts are of particularinterest and importance when photomicrographs aremade for publication.

Since the virtual image in the microscope is at 10inches, the above comparisons refer to a glossy printviewed at 10 inches. Were a negative enlarged twice,the same detail would be observable at 20 inches, butno new detail would be opened up for study at 10inches. Another factor governing the initialmagnification is the N.A. of the system-a suitableobjective for resolving the structures should be chosenand magnification at the film plane controlled witha goodeyepiece.

Exposure LatitudeExposure latitude is the range of exposure allowable

TABLE 10

from underexposure to overexposure, in producing aprintable negative. More latitude is usually possibletoward overexposure. When in doubt as to correctexposure, then, it is safer to overexpose a negativefilm (within reasonable limits). The. amount of "ac-ceptable" overexposure, however, is limited by in-creased graininess and difficulty in printing. Ideally,of course, the optimum exposure will produce the bestnegative.

Generally, high-contrast films provide the leastexposure latitude, and low-contrast films the most.Also, latitude will vary, within limits, according toselection of developer and development time. Asdevelopment time increases, exposure latitude as seenin the processed negative decreases.

Development LatitudeA negative film (or plate) capable of a wide rangeof contrasts (by manipulation of developer or devel-opment time) is highly desirable in photomicrog-raphy. Such a material can be used in a great numberof applications. Very fewmaterials, however,have thiscapability. A film designated as low in contrast canseldom be used to produce high contrast; one desig-nated as high-contrast should be used cautiously forlow-contrast results. In both cases,undesirable effects

49

on graininess, speed, and tonal rendition may result.A film of medium or moderate contrast will usuallybe capable of the widest development latitude.

Film SpeedFilm speeds are arithmetic; the number assigned toa film as its ASA number is directly related to itssensitivity. A high-speed film of ASA 400 is twice asfast as one rated at 200, for example. Speed ratingsare usually given on instruction sheets supplied withthe different films or in Data Books, and apply fordaylight or tungsten illumination as indicated.

The rated speed for a film, given in the instructionsheet, only applies when the film is developed accord-ing to recommendations. Deviation from the recom-mended development procedure may have a pro-nounced effect on speed and contrast.

SHEET FILMS AND PLATESAlthough a variety of sheet films are available fromEastman Kodak Company, there are a few whosecharacteristics make them especially suitable for pho-tomicrography. One, in particular, is KODAKEKTAPANFilm 4162 (ESTAR Thick Base). Thispanchromatic film has very fine grain, medium re-solving power, and a speed of ASA 100. Contrast ismoderate, but can be varied over a wide range fromlow to high by selection of developer and developmenttime. EKTAPANFilm is capable of wide exposurelatitude when developed for low to medium contrast.Its emulsion is coated on ESTARThick Base, whichhas excellent dimensional stability and lies fiat. Be-cause its speed is similar to that of KODAKEKTACOLORProfessiona Film 6101, Type S, it is valuable whenboth color and black-and-white negatives are neededfor the same subject. EKTAPANFilm is available fromphotographic dealers, in all standard sheet sizes.

Table 10lists Kodak sheet films for black-and-whitephotomicrography and their pertinent charac-teristics. Development data for these films are includ-ed on instruction sheets contained in film boxes.These data can also be found in the Kodak DataBook KODAK Professional Black-and- White Films(Publication No. F-5), available from photo dealers.

There are two Kodak plate materials which areintended for use in photomicrography: KODAKMPlate and KODAKMetallographic Plate. KODAKMPlate is panchromatic, and is therefore useful forgeneral photomicrography with colored specimens.KODAKMetallographic Plate, as the name implies,is designed especially for metallography (photomi-crography of metals); it is orthochromatic in sensi-tivity. Both materials have medium grain and high

50

resolving power. By developer selection, they arecapable of a range of contrasts, from low to very high.Complete information about these plate materialsis available from Eastman Kodak Company,Department 412-L, Rochester, New York l4650.Ask for KODAK Metallographic and M Plates,Kodak Publication No. P-33.

ROLL FILMSThe selection of a suitable roll film for general photo-micrography is more limited than that of a sheet film.As in general photography, one of the most importantcharacteristics to consider is graininess, since therecorded image must often be enlarged in order todistinguish fine details. Most roll films either havevery fine or extremely fine grain. As previously stated,the condition of empty magnification must be consid-ered when a roll film is used in photomicrographyand a negative subsequently enlarged.

KODAKPANATOMIC-XFilm is considered an excel-lent material for general photomicrography. It isavailable in standard 120 roll sizes and in 35 mmmagazines of 20and 36exposures. PANATOMIC-XFilmhas extremely fine grain, panchromatic sensitivity,and moderate speed (ASA 32). An advantage peculiar .to the 35 mm size is that it can be "reversed" toobtain direct positives by special development. Infor-mation about "reversal" of this film can be foundin Kodak Publication No. F-19, Black-and- WhiteTransparencies with KODAK PANATOMIC-X Film(FX135). For a copy, write Eastman Kodak Company,Department 412-L.

KODAKPLUS-XPan Film is a 35 mm film availablein magazines of 20 or 36 exposures. It has extremelyfine grain, panchromatic sensitivity, and a speed ofASA 125. KODAKPLUS-X Pan Professional Film isavailable in packs and in 120 and 220 rolls. It hasthe same characteristics as the 35 mm film.

KODAKTRI-X Pan Film is a high-speed (ASA 400),panchromatic film with fine grain. It is especiallysuited to photomicrography of living, moving micro-organisms, where short exposure times are necessary.It can be obtained in 120, 127, and 620 roll sizes aswell as in 35 mm magazines of 20 and 36exposures.

KODAK Photomicrography Monochrome Film(ESTAR-AHBase) SO-410 is a 35 mm high-contrast,extremely fine grain negative film. It features ex-tremely high resolving power and moderate speed(exposure index 80 to 160,depending upon processing).This film is panchromatic, that is, it is sensitive toall colors. Photomicrographs of low-contrast speci-mens such as blood smears, tissue cultures, andkaryotypes of human chromosomes will benefit fromthe contrast enhancement of this film. The contrast

can be altered from high to medium by the develop-ment chosen. This film is available in 35 mm maga-zines of 36 exposures. Data on this film is availableon request. See the inside back cover for details.

Complete film-development data and the charac-teristics of all standard Kodak black-and-white rollfilms are included in Kodak Data Book KODAKFilms for the Amateur, available from photo dealers.

FILTERS IN BLACK-AND-WHITEPHOTOMICROGRAPHYIn black-and-white photomicrography, filters are usedprimarily for control of image contrast. An increasein contrast is often desired in order to make a speci-men stand out against the background or to differen-tiate between colored elements, which may appearto have equal brightness on black-and-white film. Inthe latter case, a filter might be employed to absorbone color more than the other. Otherwise, the colorscould record as equal gray tones, particularly if thepanchromatic film used had equal sensitivity to bothcolors. (See Figure 19.)

When a specimen color is a very pale one againsta bright background, it may record as a pale grayagainst a white background. A filter which absorbsthis specimen color efficiently will render it as a darkergray; increased contrast will result. Maximum con-trast occurs when the specimen color is completelyabsorbed; intermediate contrast, when it is partiallyabsorbed; and minimum contrast, when the filtercolor is the same as, or similar to, the color in thespecimen.

Increasing ContrastFilters are often used to increase contrast in photomi-crography of blood smears. In the United States,blood smears are commonly stained with Wright'sStain, a combination of methylene-blue and eosinstains. The first stain usually appears deep blue andthe second light red. A green filter, such as a KODAKWRATTENFilter, No. 58, will show more absorptionfor the eosin color and will render it with goodcontrast against the background. Another commonstain combination, hematoxylin and eosin, usuallyrequires a green filter also. The particular green filterdepends on stain concentrations. The No. 58 filterapplies if the eosin color is pale; a lighter green, suchas a No. 11 or No. 13,when the color is more intense.

Occasionally, a specimen may be stained with morethan two colors. In order to distinguish separate colorsin a black-and-white photomicrograph, one must se-lect a filter on the basis of its ability to adjust colorbrightness and to assist in recording the colors as

' ..,:-.- ~ -- •••~. "0

..

CFIGURE 19-Effects of KODAK WRATTEN Filters in black-and-whitephotomicrography. A. No. 25 Filter (Red). B. No. 58 Filter (Green).C. No. 47B Filter (Blue).

suitable tones of gray. A KODAKWRATTENFilter,No. 11, is recommended for this purpose when thefilm used is panchromatic. (See Figures 20 and 21.)

When a specimen color is moderately dense, detailwithin the specimen can be recorded if the filter color,is the same as, or similar to, the specimen color.

If maximum contrast is desired between a colored

51

Of'

FIGURE 20-Same as Plate IV (page 40) on KODAK EKTAPAN Film.A KODAK WRATTEN Filter, No. 11 (light yellow-green), was usedto lighten background and show detail in cells.

specimen and the background, the filter should absorbthe specimen color completely. In this case, the filtercolor is complementary to the specimen color.

One technique which can be used for selecting afilter is to examine the specimen through the micro-scope while trying different filters in the light beam.When the desired contrast or detail rendition isachieved, a photomicrograph can be made throughthat filter on a panchromatic photographic material.

When contrast between specimen and backgroundis desired, the following tabulation of specimen colorsand contrast filter colors will be helpful as a generalguide.

When selecting a filter for contrast, you must notobtain too much contrast in a photomicrograph. Theresult would be a blocking of dark areas and a lossof fine detail. Also, the use of high-contrast film isnot necessary if sufficient visual contrast is obtainedthrough the filter.

Specimen Color Contrast Filter Color

Blue ...Blue-Green .Green ..Red .YellowBrown ..PurpleMagenta (Blue-Red)Violet ..

· Red· Red· Red· Green· Blue· Blue· Green· Green· Yellow

52

KODAK WRATTEN FiltersWell over 100 different KODAKWRATTENFilters arecurrently available as 50, 75, and 125 mm gelatinfilm squares. These gelatin film squares can be cutby the customer to fit specific equipment; they areavailable through photographic dealers.

Table 11 contains a list of the filters most oftenused in photomicrography. These filters can be com-bined for narrow-band transmittance or used individ-ually to transmit the indicated broader bands of color.

TABLE 11

KODAK WRATTEN Filters for General Photomicraphy

Filter VisualNumber Color Spectral Transmission

25 Red From 590 nm into the infrared58 Green From 470 nm to 610 nm and 700 nm

into the infrared47 Blue From 360 nm to 520 nm and 710 nm

into the infrared35 Purple From 320 nm to 470 nm and from

650 nm into the infrared

22 Deep Orange From 540 nm into the infrared29 Deep Red From 610 nm into the infrared15 Deep Yellow From 510 nm into the infrared45 Blue Green From 430 nm to 540 nm11 Yellowish Green From 300 nm to 350 nm and from

420 nm into the infrared

It is not always possible to recommend a specificfilter, even when stain colors or other specimen colorsare known. Visual inspection of a specimen throughdifferent filters is usually the best guide when select-ing a filter for photomicrography. An experiencedphotomicrographer can examine a specimen and oftenselect the best filter with little trouble, since he isusually familiar with stains and filters and with theireffects in a photomicrograph.

The transmission and absorption characteristics ofall Kodak filters are included in Kodak PublicationNo. B-3, KODAK Filters for Scientific and TechnicalUses. This Data Book, published by Eastman KodakCompany, can be purchased through photo dealers.

PROCESSING THE NEGATIVEAfter photomicrographic exposures have been made,the next step is to process the film. This includesdeveloping,rinsing, fixing,washing, and drying.

Correct development is most important, since thispart of the procedure causes the image details toappear on the film. For high negative quality, selec-tion of the correct developer and developing timeshould be made according to the manufacturer's

•A

BFIGURE 21-Megaloblastic blood cells, pernicious anemia, x 550.(a) No filter; (b) with KODAK WRATTEN Filter, No. 58 (Green).

recommendations. While it is true that many devel-opers can be used with a given film, the influenceof each developer on contrast, graininess, speed, andtonal rendition must be considered.

With continuous agitation, single sheets of film orsingle plates can be developed efficiently in a tray.When several exposed films or plates must be pro-cessed simultaneously, however, they should be de-veloped in a tank with suitable hangers. Since inter-mittent agitation is used in tank development,developing time is usually about 25 percent longerthan in tray development.

Development of a 35 mm roll film is commonlycarried out in a reel-type tank, with agitation at30-secondintervals. The reel should be loaded in totaldarkness or with suitable safelight conditions, insert-ed into the tank containing developer, and the top

set in place. Processing of the film can then becontinued in room light.

After films or plates have been washed, it is recom-mended that they be immersed in KODAKPHOTo-FLOSolution for about 1 minute before drying. This helpsto minimize the formation of water spots and unevendrying.

The following Kodak Data Books contain furtherinformation about handling and processing black-and-white films. They are available through pho-tographic dealers: Publication No. F-5, Kodak Pro-fessional Black-and- White Films, Publication No.J-l, Processing Chemicals and Formulas for Black-and- White Photography.

PRINTINGBlack-and-white photomicrographic negatives can beprinted to a positive either on a suitable paper (pro-ducing a print) or on a positive film (producing atransparency). A negative can be printed by contactto produce a print the same size as the negative orby projection to produce a smaller print, one the samesize as the negative, or a print on which the imageis larger than the negative. For either contact orprojection printing, a selection ofKodak photographicpapers is available. Details concerning the charac-teristics of these papers are contained in Kodak DataBook G-l, KODAK Photographic Papers, availablethrough photographic dealers. Two other Data Books,Professional Printing in Black-and- White (KodakPublication No. G-5) and Enlarging in Black-and-White and Color (Kodak Publication No. AG-16),willalso be of value.

For paper prints, it is common practice in photomi-crography to use glossy-surface papers. Glossy paperwill produce prints with the highest brilliance andbest rendition of details. Glossy prints are also com-monly required for reproduction in journals, books,and other publications.

If a photomicrographer already has negatives, butneeds positive transparencies, he can use one of twoKodak films to obtain transparencies: EASTMANFineGrain Release Positive Film 5302 or KODAKFineGrain Positive Film 7302. The first is a 35 mm filmavailable in lOO-footrolls only; the second is availablein sheets and rolls. Both films are blue sensitive andof low speed. They can be handled and processedunder the illumination from a KODAKSafelight Filter,No. lA (light red), in a suitable lamp.

For a complete guide to all Kodak black-and-whitematerials, processing, printing, enlarging, and copy-ing, all in one publication, we recommend theKODAKDarkroomDATAGUIDE, available throughyour photographic dealer.

53

EXPOSURE METHODSAs in other forms of photography, exposure in photo-micrography is the result of light acting upon asensitized emulsion-the film. Exposure is influencedby light intensity (image brightness) and by exposuretime. In order to record an image of brightness, aspecific exposure time is necessary, depending uponthe film speed.

Exposure time can be determined either by usinga sensitive exposure meter or by making an exposuretest series. Because image brightness in photomi-crography can be quite low, particularly at highmagnification, a preferable exposure meter should besensitive to very low light levels. To determine expo-sures with a hand-held meter, hold the meter abovethe eyepiece at a distance such that the diameterof the emerging light-beam will be slightly greaterthan the diameter of the photocell. A meter readingis made with the meter in this position in a dimly-lighted room. Some professional exposure meters havespecial microscope attachments. The manufacturer'smanual gives instruction for these attachments.

When an exposure test series is to be made, anynecessary filters should be placed in the light beam,whether a color film or a black-and-white film is tobe exposed.

EXPOSURE TESTSRoll FilmWhen a 35 mm film or other size of roll film is tobe exposed, an exposure test can include severalframes, each exposed for a different length of time.Image brightness, of course, should remain constant.The exposed film is then processed and the individualframes are examined over an illuminator to determine

54

which one received the correct exposure.The most convenient series would include all of

the exposure times available with the camera shutter.These can range from 1 second to 1/125 second. Ifthe shutter has a Time (T) setting, a few long expo-sures could also be made (2, 4, and 8 seconds, forexample). One out ofthis wide range of exposure timeswill be correct for that film and the existing imagebrightness.

Another convenient method of determining properexposure is to make an exposure series on a roll ofblack-and-white film, process it, and evaluate thenegatives. The best negative is the one that exhibitsthe most detail throughout, in both shadows and

FIGURE 21-Pholomicrographic setup with automatic exposuremeter attachment

highlight. The film speed of the black-and-white filmcan then be correlated with any other film that mightbe used. As an example, suppose the taking film wasKODAKPANATOMIC-XFilm with a speed of ASA 32and the new film to be used is KODAKHigh SpeedEKTAcHRoMEFilm (Tungsten) with a speed of ASA125.The proper exposure on the PANATOMIC-XFilmturned out to be 1/30 second. Since High SpeedEKTAcHRoMEFilm has about four times the speedof PANATOMIC-XFilm, it will require only one-fourththe exposure, or 11125second. (Exposure time variesinversely as film speed rises.)

Sheet FilmWhen a camera accepts sheet films, a series of testexposures can be made on one film sheet by thefollowingprocedure:1.Pull out the dark slide of the film holder until the

entire sheet is uncovered in the camera. Make anexposure for 1 unit of time; for example, 1 second,1/2 second, 1/125 second, or whatever initial timemay be indicated by the image brightness level.

2. Push in the slide about an inch and repeat theexposure for the same unit of time.

3. Push in the slide another inch and give an exposurefor 2 units of time.

4. Continue to push in the slide to cover approxi-mately Linch steps, exposing each step for twiceas long as the previous one. The successive stepswill then have received exposures of 1,2,4,8 timesthe initial time unit.

Develop, fix, and wash the film as recommended inthe instruction sheet. Examine the resulting negativeto select the step which shows the best reproductionof the subject.

EXPOSURE CALCULATIONOnce the correct exposure time has been found fora particular set of optical conditions, the correct timesfor new conditions with the same microscope andillumination can be calculated. The factors whichaffect image brightness are changes in magnificationor in numerical aperture. Magnification, of course,will change if a different eyepiece, a different objec-tive, or a new eyepiece-to-film distance is involved.Also, if the objective is changed to one of higher orlower magnifying power, the numerical aperture willbe different.

If the optical conditions for which the exposure timewas originally determined by test are called "stan-dard,'.' they can be compared mathematically withthe "new" conditions to calculate the "new" exposuretime, according to the following formula:

New Exposure TimeStandard Time (

StandardN.A.)' X ( New Magnification )'New NA. Standard Magnification

The application of this formula is illustrated bythe following example: The correct exposure forKODAKHigh Speed EKTACHROMEFilm was deter-mined to be 11100second (0.01second). Suppose thata 10x eyepiece and a 20x objective (N.A. = 0.50)were used. Magnification is x 200. These are the"standard" conditions: exposure is 0.01 second,magnification is x 200, and N.A. = 0.50. The samefilm will be exposed, of course, but the new exposurewill be made at X 430, with the same 10X eyepieceand a 43X objective of N. A. = 0.65. What is thenew exposure time under these conditions?

New Exposure = (0.50)' X (430)'.01 0.65 200

New Exposure = .0276second = .!. second30

In this way exposure time can be calculated whenany of the optical exposure factors are changed. Itis even possible to set up a table to include all exposuretimes for a color film used under different conditions.This would assume, of course, that the microscopeand the illumination were correctly adjusted for eachcondition.

In black-and-white photomicrography, specificlight filters are often used for contrast control. Whendifferent filters are used, as they often are, their "filterfactors" must be considered in working out exposuredetermina tions.

Judgment of ExposureA good photomicrographic exposure should alwaysshow the subject to best advantage, whether theimage is on a reversal color film or on a negativematerial. It is easier, however, to judge correct expo-sure on a reversal material, since this type of filmhas very narrow exposure "latitude." If an exposuretime is not right, this is immediately apparent.Overexposure will wash out the light areas, with aloss of "highlight" detail. Underexposure will causea darkened appearance overall, with loss of detail inthe darkest, or "shadow," areas. A good exposure ona reversal film will show detail in all areas wheredetail was evident in the specimen.

Negative materials, both color and black-and-white, exhibit much more exposure latitude than do.reversal color film. Therefore, judgment of the bestexposure time is more difficult. In general, exposure

55

should be controlled to provide best rendition of detail. in the darker areas of the subject. These areas willbe represented on a negative by light areas. If thefilm is underexposed, very little detail will be evidentin these areas of the negative. When such a negativeis printed, the background and highlight areas mayprint very well, but the darker areas will be too dark,with few, if any, details. Considerable overexposureof a negative may cause loss of detail in both thehighlight areas and the darkest areas. With negativematerials, more overexposure than underexposure canbe tolerated.

Filter FactorsWhen a colored filter is placed in the illuminationbeam in black-and-white photomicrography, it willabsorb a certain amount oflight. The amount oflightabsorbed depends upon the particular filter. One mayabsorb more, or less, than another. If the exposuretime for a black-and-white film is determined with"white" light-that is, without any filter-it must beadjusted (increased) when a filter is used, since theimage brightness will be decreased. The amount ofincrease in exposure time is known as the "filterfactor." The exposure time without the filter is multi-plied by the filter factor to determine the exposurewith the filter in place. Filter factors for all Kodakblack-and-white films are published in the instructionsheets packaged with the films.

If exposure time is determined for a black-and-white film by making exposure tests, the filter canbe in position. Correct exposure will then be achievedwithout resort to the filter factor. If a new exposureof a different subject is to be made, and a differentfilter is necessary, the new exposure must considerthe change in filter factors. For example, suppose thatan exposure was made on a black-and-white film,witha filter having a factor of 4.The new exposure requiresa different filter, whose factor is only 2. The newexposure time will be one-half the previous one. Or,if the new factor were 8, the new exposure time wouldbe twice the original exposure.

If exposure is determined with an exposure meter,only white light (unfiltered) should be used for black-and-white films. The photocell in an exposure meterdoesnot have a uniform response to all spectral colors,so measurement of exposure with the filter in placecould be erroneous. Thus, the best practice is tomeasure the exposure time for white light and thenapply the filter factor for finding the correct exposure.

If color films are to be exposed, filter factors donot apply. Light-balancing filters and color-compen-sating filters are used here. They are already in placefor any exposure determinations.

56

Determining Filter Factors: Because of variance inthe spectral response of different photographic mate-rials and the difference in color quality of differenttypes of illumination, published filter factors can onlybe considered as approximate. If an exact filter factoris necessary for specific conditions, it must be deter-mined experimentally. This can be accomplished bymaking a "step-exposure" series on the film to be used,with the illumination to be used, and of course with"white light." It is not necessary to have a specimenin place on the microscope stage for the exposureseries-the background brightness will suffice. Theexact series of exposures will be governed by theactual brightness level. If the level is low, a "powerof 2" series-such as 112, 1, 2, 4, 8, and 16 seconds-could be used. Shorter exposure times would applyfor a high brightness level. Microscope adjustmentsmust not be changed during the test.

If a corresponding step-exposure series were thenmade with a filter in place, both series could becompared to determine which steps matched fordensity. If no two steps matched, either another serieswith closer steps could be made or the filter factorcould be determined by interpolation of density be-tween the two steps most closely matched. Thisrequires a knowledge of densitometry, but is the mostexact technique.

Suppose, for example, that 2 seconds of exposureproduced a medium density in a series made withwhite light. When the filter was placed in the lightbeam and another exposure series made, it took 8seconds to produce a density equal to the 2-secondexposure in the first series. The filter factor wouldthen be 8 divided by 2-which, of course, is 4.

Experimental determination of filter factor is espe-cially useful when filters are used in combination,since factors are seldom published for combinations.Once the combination filter factor has been deter-mined, it can then be applied in all future exposures,provided that the same film and illumination are used.

Exposure MetersSome types of exposure meters are made specificallyfor photomicrography and are sensitive enough torespond to light through a wide range, from very lowto very high brightness. A meter scale may be precali-brated by the manufacturer to give a direct readingof exposure time, with various settings for film speed.Obviously this is the most convenient type of meter.Other meters may be very sensitive to light but havereadings in terms of units of illuminance, such asfootcandles. In this case, the manufacturer oftenprovides a simple calculator, table, or graph so thatbrightness readings can be converted to exposuretimes.

If a meter reads brightness but no deviceis providedfor correlating brightness and exposure time, themeter must be calibrated. This can be done by makinga brightness reading of the image at lowmagnification(about X 100).The brightness reading is recorded, asindicated on the meter scale, for later reference. Thena series of exposures is made at various shutter speedson a reversal color film. After the film is processed,the correct exposure time is selected. A table (or agraph) can then be made that includes the brightnessreading, the correct exposure time for that reading,and the speed of the color film exposed. If anotherreading were made at a different magnification, onecoulduse the existing data to find the correct exposuretime. For example, if the new reading at lower powerindicates twice the brightness, then the new exposuretime will be one-half the previous one; if a new readingat higher power is only one-half the first one, thenew exposure time will be twice as much, etc. Expo-sure time for a given film will vary inversely as thebrightness of the image increases. Table 12 is shownas an example. The figures are arbitrary and do notrefer to any particular conditions. Film speeds areincluded to make the table complete, but they donot refer to particular films.

TABLE 12

Brightness Shutter Speeds

Readings ASA 32 ASA 64 ASA 125 ASA 250

500 1/125 1/250 1/500 1/1000250 1/60 1/125 1/250 1/500125* 1/30 1/60 1/125* 1/25064 1/15 1/30 1/60 1/12532 1/8 1/15 1/30 1/6016 114 1/8 1/15 1/30

-Exposure time determined by test for a brightness reading of 125 (exampleonly).

This system could be used for any light meter notcalibrated in terms of exposure time or film speed,including meters normally intended for conventionalphotography outdoors and indoors.

"Probe" Meter: One type of meter is equipped witha "probe" containing a photocell. The probe is placedin the microscope eyepiece tube, with the eyepieceremoved, in order to read image brightness. Whena probe type of meter is used, the same eyepieceshould always be used in the microscope for pho-tography. Otherwise, the meter readings will notalways be valid.

When an eyepiece-camera with a beam splitter isused over the microscope, light readings can be madefrom the light emitted from the eyepiece of the beamsplitter. However, image brightness here will often

be less than that "seen" by the film because thedivision of light varies with different beam splitters.In some systems, 90 percent of the light goes to thecamera and only 10 percent is seen visually throughthe observation eyepiece.The actual division,howev-er, may be 80-20,70-30,or even 50-50,depending onthe specific eyepiece camera in use. Hence, theamount of light divisionwill influence the calibrationof a light meter.Making Exposure ReadingsExposure readings can be made in various positions:at the film plane of the camera, anywhere betweenthe eyepoint of the ocular and the film plane, in theeyepiece tube of the microscope with the eyepieceremoved, or from the light emitted from the observa-tion eyepiece of a beam splitter. The best position,of course, is at the film plane, since the image bright-ness there is the same as would be recorded on thefilm. This position, however, is not always accessiblebecause the camera may be closed during pho-tography. The ground-glass screen of a sheet-filmcamera allows this type of reading very nicely.

If the exposure reading is made at some positionabove the microscope eyepiece, the image brightnesswill always be greater than that at the film plane.This fact must be considered in calibrating an expo-sure meter. Also, the light reading should always bemade at the same distance above the eyepoint.

Methods of Measurement: Wherever the reading ismade, there are two methods ofmeasuring brightness,or exposure time. One method is to read just thebackground brightness with the specimen slide movedaside on the stage. This system provides a large areaof uniform brightness on which to make a reading.The method is especially useful with reversal colorfilm,where exposure time is dependent on the bright-est part ofthe specimen-which is essentially the sameas the background brightness.

The second method of measuring brightness is toread the actual brightness of the specimen image.Thissystem also works efficiently for color films, as longas the specimen imagehas average brightness. If denseareas predominate the field, overexposure may occa-sionally be encountered, and the bright, highlightareas may appear washed out. This method shouldalways be used, however, when negative films, eithercolor or black-and-white, are to be exposed. Thisproduces good rendition of detail in the darker areasof the subject as previously described in "JudgmentofExposure."

Whichever method is used to make the reading, .the meter must be calibrated to conform with it, ifaccurate readings are to be made consistently.

57

Integrating Meters: Many current photomicroscopesdesignedfor brightfield photomicrography, and othersinvolving phase and interference systems, areequipped with photocells or photoresistors that inte-grate and measure the brightness of the image. Thisis integrated with other control data, like film speed,to automatically time the exposure. A wide range ofbrightness is accommodated. Allowances for filterfactors are made by the integrating element. But ASAratings, reciprocity departures, and subject densitycan be preset by the photographer. Those who prac-tice photomicrography extensively would do well tostudy the various types of these meters offered bymanufacturers.

EXPOSURE RECORDWhen photomicrographs are made often, it is advis-able to make a record of the conditions involved. Adetailed record will permit duplication of a setup forfuture use either in rephotographing a particularspecimen or for photography of similar specimens.This information could be recorded on the envelopecontaining a photomicrograph, or in a notebook. Thefollowingtable includes pertinent data for one partic-ular situation, as an example.

Photomicrograph No. 69-104

Subject _Pollen grains-hibiscusStain • _ _ _None. normal colorObjective .•. _ .• 10x apochromatEyepiece _ _ _ _ _ _ _ _15 X compensating

Distance (Eyepiece to Film) _10 inches (fixed)Magnification X 150Light Source __ __ Tungsten halogen. 12 voltsFilm _ _ KOOAKEKTACHROMEFilm 6116. Type BFilter _CCl ORExposure Time _ _1/25 secondDevelopment Process E-3Remarks Above filter used to neutralize color

of heat filter.

The data may differ for some photomicrographs.Exposures for black-and-white films, for example,should include specificdeveloper, time, and tempera-ture. If the eyepiece-to-film distance is always fixed,as in most eyepiece-cameras, this datum can beomitted. The purpose of the photomicrograph couldalso be included (for whom it was made, why it wasmade, etc).

COMMON FAULTS IN PHOTOMICROGRAPHYEven the most careful photomicrographer sometimesmakes mis akes and produces a photomicrograph ofless than the best quality. In order to correct suchmistakes in future work, he must be aware of causesand effects. There are many factors which can affectresultant image quality. Some are related to low-quality optics, while others are related to improperadjustment and alignment of the illumination andoptics. The specimen itself can influence image quali-ty; it may be too thick or be improperly stained. Dirt,dust, or grease on any of the optical components canaffect the resultant image. Incorrect use of filters inblack-and-white photomicrography can reduce con-trast or detail rendition. In color work, the wrongfilters will affect color balance.

MORE-COMMON FAULTSUnsharp ImageThis is probably one of the most common undesirableeffectsin photomicrography. There are several causes:

Possibly the image was focused sharply but there

58

was some "slippage" in the fine-focus adjustment onthe microscope. If this happens often, the microscopemay be too old or worn or the adjustment may beloose. You can find out whether the image focuschanges by watching it for a short while after criticalfocus. If it stays in focus, something else is causingthe trouble.

The camera shutter may have been actuated toohard or too fast-causing vibration which made theimage go out of focus. Always use a cable release andpress it slowly.

If the image is focused on a ground-glass screen,it may be that the surface is too coarse,making criticalfocus difficult. Use a ground glass with a clear centerso that focus is achieved on the "aerial" image. Thissituation was discussed under "The Ground Glass"on page 20.

Most eyepiece-cameras include a reticle in theobservation eyepiece. This reticle must be sharplyfocused.A young person's eyes will accommodate thefocus of this reticle even when it is not in sharp focus.

Check before making the exposure to be sure thatboth the reticle and the specimen are in focus. If thereticle is out of focus, then the specimen image mayalso be unsharp when it is recorded on film. A focusadjustment for the reticle is provided on the observa-tion eyepiece.

If a high, dry objective is used in the microscope,and if the cover glass on the specimen is too thin,undercorrection for spherical aberration is intro-duced. The image can never be focused sharply. Thereare only two remedies. (1) If the cover glass can beremoved and replaced with one of correct thickness(No. 1 112), a better image will be obtained. Thisremedy, however, is not always possible, since thecover glass may be cemented firmly in place. (2) Anoil-immersion objective of comparable magnifyingpower may be used to obtain a better image. Thenthe cover-glass thickness is less important. Objectivesof this type are available from several firms. If oneis to be purchased, make sure it can be used efficientlywith the microscope in use.

Possibly the objective in use was designed for adifferent microscope with a different tube-length.This may also introduce spherical aberration, affect-ing image sharpness.

People who weal' glasses or contact lenses some-times have difficulty achieving sharp focus in a micro-scope or through an observation eyepiece on a beamsplitter. The only thing that can be done here is tocheck with the manufacturer. Possibly he is awareof this problem and has a means for solving it.Sometimes a high eyepoint eyepiece can be usedeffectively to correct this problem.

Vibration will cause a recorded image to be unsharp,particularly at high magnification or with long expo-sure times. If any steady bench vibration occurs, itwill be noticeable in the microscope at highmagnification; the image will appear to be in motion.A fast shutter speed (11100 second or faster) orelectronic flash will minimize the effect, but will notremove the cause. If the reason for the bench vibrationcan be found, possibly it can be eliminated. Thealternative is to mount the microscope or the standon suitable shock absorbers. Shutter vibration, too,must be suspected. A slowshutter speed will minimizethis effect because if the cause arises in the shutter,this vibration is only prevalent during the first partof the exposure.

Hazy ImageAn image appears hazy when a grease spot is presenton the front lens of the objective, on the top lensof the eyepiece, or on the specimen slide. Thesesurfaces may have been touched accidently. All glass

surfaces should be inspected frequently and cleanedwhen necessary. A piece of lens tissue can be moist-ened with xylene (EASTMAN Organic Chemical No.P460) or with the more volatile ligroine (EASTMA

Organic Chemical No. P950) to clean a greasy surface.Too much solvent, however, can affect the cementin the objective lens or the mounting medium on theslide.

A small drop of immersion oil on a dry objectivewill also produce a hazy, unsharp image. This isusually an accident and can occur when a dry objec-tive is placed in position with oil on the slide. Theoil can be removed with tissue moistened with xylene.Oil should always be removed from a slide after use.

Uneven IlluminationWhen the objective and substage condenser are notaligned satisfactorily, or when the illuminator andthe light source within it are not correctly alignedwith respect to the microscope, the illumination willbe uneven. The effect will be quite noticeable in arecorded image. The background, or the specimenimage, will be darker on one side than the other.

The objective in a microscope is usually fixed inposition, so no adjustment of its position is possible.The substage condenser, however, can and shouldhave centering screws so that it can be centered withrespect to the objective. To find out if it is off-center,look down the tube of the microscope with theeyepieceremoved. A circle of light will be seen whichis the back lens of the objective. Then, if the substagediaphragm opening is decreased, the circular imageof the diaphragm imposed on the image of the backlens will be seen. If the two images are concentric,then the condenser ·.isaligned with the objective. Ifthey are not concentric, adjust the screws on thecondenser until they are.

When the objective and condenser are properlycentered with respect to each other, but the illumina-tion is still uneven, either the illuminator is off-centeror the lamp in the illuminator is off-center. Very oftenthe lamp itself is off-center, particularly with built-inillumination. The position of the filament may varyin different tungsten lamps, even in those of the sametype. Most microscopes with built-in illuminationhave centering screwson the lamphouse for centeringthe lamp. This centering can be checked by placinga piece of white paper in front of the field lens andadjusting the centering screws. When the lamp fila-ment is off-center, the illumination on the paper willbe uneven. Adjust the screws until the light is even.This effect may also be seen by looking in the micro- .scope without a specimen in place-just the clear fieldwill be visible. If the light is' too intense, place a

59

neutral density filter in the light beam to reduce thelight level. The effect of uneven illumination, what-ever the cause, is most noticeable at low magnifica-tion, since a large field of viewis recorded.

Low ContrastThere are three principal reasons for low contrastin a photomicrograph. First, the substage diaphragmmay be opened too far, creating flare and reducingimage contrast considerably. The setting of this dia-phragm should be made according to the principlesof Kohler illumination. (See page 28.) The effect isnoticeable in both color and black-and-white photo-micrography.

Second, a filter may sometimes be used in black-and-white work for detail rendition with stainedspecimens resulting in low contrast. If the color ofthe filter is similar to that of the specimen-it trans-mits the color of the specimen. The effect is to reducevisual contrast between the specimen and the back-ground. Contrast in this case can be enhanced by(1) using a filter which has partial absorption for thespecimen color, (2) using a film of higher than normalcontrast, or (3) selecting a developer that produceshigher contrast.

Third, the subject itself may exhibit very littlecontrast. This contrast can be improved to somedegree by the previous mentioned methods. Differentlighting methods may improve this contrast also.

LESS-COMMON FAULTSToo Much ContrastToo much contrast is not likely to happen with colorfilm,since processing conditions are fixed and contrastfilters do ot apply. It occurs in black-and-whitephotomicrography (1) when a high-contrast film isused and no effort is made to select a low-activitydeveloper, (2) when a high-contrast developer is usedwith a regular film, (3) when development time isunduly prolonged, or (4) when a contrast filter is usederroneously.

Poor ResolutionProbably the principal cause of poor resolution isimproper use of the substage diaphragm-when it isreduced to too small an opening. Resolution is de-creased considerably, artifacts and diffraction areintroduced, and a generally poor image results. Followthe technique of Kohler illumination in adjusting thediaphragm to the correct opening.

If the substage condenser is not correctly adjustedand its position is too low, the effects are similar.The objective is not used at full numerical aperture.

Poor resolution also occurs when a photomicro-

60

graph is enlarged too much. Empty magnificationresults, and the image appears unsharp. When select-ing the magnification, follow the rule of 1000 timesthe N.A. of the objective. At times, overmagnificationmay be permissible when the purpose is to make finedetail larger and therefore, easier to see-a psy-chological benefit.

Bright Spot in FieldA bright spot in the field can occur when one usesa conventional camera with an integral lens or aneyepiece-camera with a compensating lens above themicroscope eyepiece. Ideally, the eyepoint of theocular should be at, or very near, the front surfaceof the lens. If the front surface of the lens is tooclose to the ocular, an out-of-focus image of the backlens of the objective may be recorded as a bright spot,when a reversal color film is used. It would, of course,be a dark spot on a negative, becoming a bright spotin the print. If the camera can be moved up a littlefarther above the microscope, the spot will disappear.If it is moved too far, however, image quality maysuffer, or the image may be vignetted.

Shutter-Blade ImageIf the camera shutter is too far above the eyepoint,a silhouette image of the shutter blades may berecorded as they open, particularly with fast shutterspeeds. The shutter (leaf type) opens from the centeroutward, then closes toward the center. If theeyepoint of the ocular is positioned near the center,this effect cannot occur. Of course, if a lens is in thecamera one has little control of the eyepoint position,other than to place it at or near the front surfaceof the lens. In this case, fast shutter speeds (1/60second or shorter) should not be used. If necessary,a neutral filter can be used to reduce the light, thuspermitting slower shutter speeds. When the cameradoes not contain a lens, the eyepoint can be positionedin the center ofthe shutter.

Out-of-Focus SpotsDust and dirt particles on the front surface of thelamp condenser lens, on filters in the light beam, oron the top surface of the eyepiece will record asout-of-focus spots. They may be colored on a colorfilm or gray (or black) on a black-and-white film.Scratches on a glass surface will record as unsharpspots. Bubbles in a lamp condenser or in a heat-ab-sorbing filter in the illuminator will show as spotsalso, and may be bluish in a color film record. Cracksin those elements will produce streaks. Check all glasssurfaces frequently, especially if unidentified spotsshow up.

SPECIALIZED TECHNIQUESAND APPLICATIONS

SPECIALIZED TECHNIQUESA majority of the subjects examined or photographedthrough a conventional microscope appear either darkor colored against a light background. When theyappear dark, it is due to light absorption within theirelements. When they appear colored, it is usuallybecause they have been treated with biological stainsto produce color contrast. This color contrast is aresult of differential absorption of various stains bythe elements of the specimen. Whether the subjectappears dark or colored, the effect is called"brightfield illumination."

In an unstained condition, many specimens exhibitlittle or no contrast when viewed against a brightbackground in a compound microscope. They arecolorless and comparatively transparent. Conse-quently, they are practically invisible. When staininga specimen is either impossible or undesirable, con-ventional brightfield microscopy cannot be used. An-other microscopical technique must be used to makethe specimen visible. The particular method selecteddepends upon the specimen itself and the particularresults desired.The following discussions are intendedto present brief descriptions of those special tech-niques that will either produce better optical contrastbetween the elements of a specimen and its back-ground or allow images to be viewed or photographedwith finer resolution.

Contrast SystemsSeveral simple and sophisticated optical arrange-ments have been worked out for the purpose ofenhancing the contrast between specimens and back-ground and for delineating structures in transparentand translucent subjects. The photomicrographerdoes not need a doctorate in optics to use suchmicroscopes. Manufacturers' instructions are explicit,operationally. However, their technical discussions dorequire some knowledge of the physics of light forunderstanding. The designs involve numerous slits,plates, prisms, and lens and condenser combinations.The photomicrographer can best juggle these, andmost confidently evaluate his results, when he hasa grasp of the underlying principles. When he doesnot know them, he is in the position of someone

FIGURE 23-Alpha particle tracks, x 500, with darkfield illumina-tion. Photographed on a KODAK Metallographic Plate.

driving a car without knowing of the existence andfunction of the transmission.

There is another benefit that comes from beingknowledgeable. Many of the devices are quite intri-cate. Tolerances of the order of a wavelength of lightare involved. Therefore, educated prudence demandsthat the photomicrographer confine himself to mak-ing only the operating adjustments and alignments.Any other manipulations or repairs should be leftto the manufacturer or his representatives. The pho-tographer should know what to leave alone.

Darkfield MethodMany transparent and semitransparent specimens-such as microorganisms, cell structures, and crystalinclusions-are not readily visible in a brightfieldmicroscope. Their visibility can be improved greatlyby a method called "darkfield illumination," in whichthe specimen is seen as a bright object against a dark,or even black, background.

For the darkfield method, the cone of light normally .illuminating the specimen must not enter the micro-scope objective. In the darkfield microscope, only light

61

that is scattered or reflected by the specimen is "seen"by the objective. This is achieved in the conventionalmicroscope by use of darkfield diaphragm stops orspecial darkfield substage condensers called "parabo-loid" or "cardioid" condensers. The darkfield stop isinserted underneath the condenser and the aperturediaphragm is opened to its maximum. It may be usedat low and medium power with dry objectives, whilethe special condensers are used only at high power.The N.A. of this condenser is greater than the N.A.of the objective. The cardioid and paraboloid con-densers use the oil-immersion technique, which re-quires that oil be placed between the bottom of theglass slide and the top of the condenser. Great caremust be taken that bubbles are not introduced eitherin the oil under the oil-immersion objective or in theoil between the slide and condenser. A single bubblewill introduce considerable flare and reduce bothimage contrast and optical quality.

Since special darkfield condensers are to be usedwith oil, particular care must be taken in regard toslide thickness. The correct range of slide thicknessis usually specified on the condenser mount. If theslide is too thin, the oil layer collapses when thecondenser is focused critically. If the slide is too thick,it is often impossible to obtain correct focus of thecondenser.

The essential principle of the darkfield optics is theformation of a hollow cone of light whose apex occursin the plane of the specimen. When the light iscarefully focused at the plane of the object but noobject is present, the hollow cone of light passedthrough the condenser is invisible, because the objec-tive is inside the dark base of the hollow cone.When a sp cimen is present, the light is deviated, orscattered, into the objective by structures on thespecimen. A bright image of these details is thenvisible against a dark background. Because of thehigh contrast of the image, the system is capable ofdetecting extremely fine particles.

Color is seldom produced in a darkfield microscope,except in fluorescence work; thus, black-and-whitefilms are used predominately. Since considerable lightis lost in this system, a high-speed film,such as KODAK

TRI-X Pan Film, is appropriate-particularly whenthe subject is in motion.

A darkfield microscope is an excellent tool for usein biology and medicine. It can be used effectivelyat high magnification to detect and photograph livingbacteria. Similarly, at low magnification, wholemounts and tissue sections can be viewed and pho-tographed. In marine biology, a darkfield microscopeat very low power is used extensively for recordingsea life such as algae and plankton.

62

Stop-Contrast MethodDarkfield illumination provides the means for thegreatest enhancement of image contrast. It is espe-cially valuable for particles, but is extremely wastefulof light. Wilska worked out a "stop-contrast," or"anoptric," method for improving the image contrastof unstained specimens. While his method was devel-oped after the advent of phase-contrast systems (tobe described in the next section), it is easier tounderstand, and therefore is presented first. Also, thebasic scheme ofmanipulating "direct" and "deviated"beams is common to both systems.

The anoptral arrangement is quite similar to abrightfield system, but the image effect is one ofdarkfield (or semi-darkfield, as pointed out furtheron). A ring-shaped, "annular" slot is located near,or projected to, the condenser. This is illuminatedby a solid-source lamp (ribbon filament or arc). Thering of light becomes the source for the microscope.It is focused, by the condenser and objective, nearthe periphery of the rear aperture plane of the objec-tive (not in the plane of the primary image). SeeFigure 24.

Thus, the potential path of this ring of light (thedirect beam) is:• Through the condenser to the slide, where it is

spread (defocused) evenly over the field, for illumi-nating the specimen and background.

• Directly through the background to the objective,where it is gathered back into a ring in the rearaperture plane of the objective. (Any rays goingdirectly through a thin specimen pass through thisring to the primary focus.)

• Defocused in the plane of the primary image, wherethe specimen is in focus.Finally, the ring of light is integrated by the

eyepiece without structure (i.e., no bright ring ap-pears), but the direct light from the specimen, beingin focus at the primary image, is refocused.

This was specified as the potential path of theannular rays. However, in the anoptric system, thehollow beam is partially blocked in the aperture planeby a semiopaque (usually 90 percent) ring coated onthe rear element of the objective. The shadow of thering mask matches the focused aperture-plane imageof the ring source, thereby allowing only 10 percentof the illumination from the source to be integratedby the eyepiece.

It should be remembered that the annular sourceis integrated in the slide, so that a specimen cantransmit some of the direct rays to the primary plane,where they are not completely masked off. Since atransparent specimen has such lowstructural opacity,the direct beam through it contributes only a little

-.-----------+--,.--.---~PRIMARYIMAGEOF OBJECT

REAR APERTUREPLANE

OBJECTIVE

OBJECT PLANE,SPECIMEN

CONDENSER

:""t":::::I::::::: tI

I

ANNULAR SLOT

BEAM FROM SLOTDEVIATED RAYSDIRECT RAYS

FIGURE 24-Schematic path of rays through an annular slotplaced in the rear focal plane of a condenser for phase-contrastmicroscopy. The slot comes to a focus (S) in the rear apertureplane of the objective. From there the beam of rays is spread(defocused) over the plane of the primary image of the specimen.The object, a small detail in the specimen, transmits part of thebeam (direct rays) and scatters the rest (deviated rays). Both setsof rays from the object come to a focus in the primary-imageplane. If an opaque ring were placed over S, the illumination beamwould be blocked from the primary image and a darkfield situationwould prevail. However, the deviated rays from the object wouldget around the opaque ring. In the anoptric system, a partiallyopaque masking ring is coated on the objective. In the phase-con-trast system, a transparent compensating plate, carrying a 1/4-wave, phase-changi ng ring, is inserted somewhere in the objectivetrain. This changes the phase of the direct rays so that they canproduce an interference pattern with the deviated rays.

to the final image detail. As in a darkfield microscope,scattered light from the specimen also forms an image.In essence, a darkfield situation is presented to theeyepiece.The background brightness depends on thedegree of opacity of the annular mask.

The optical arrangement is not the same as thatof conventional darkfield illumination, however. In-stead, as in Kohler illumination, the source passesdefocused throughout the specimen plane. When atransparent specimen is in place in a phase-contrastmicroscope, edges and very fine detail diffract and

scatter the light. This deviates many of the rays fromthe specimen structures, and that is why they arenot refocused into the annular ring image,where theywould be masked off. Instead, they appear in theprimary image (and eyepiece), just as particles dounder darkfield illumination. The scattered light fromsuch structures furnishes the "deviated" beam.

Contrast is improved partly because of thisdarkfield tone arrangement, but chiefly because cen-tral, direct illumination that would come up througha fully lighted condenser is not present with anannular source. Such central rays flood a field fromall angles, whereas the obliquity of peripheral raysprovides modeling for the specimen. This modelingappears in the deviated beam, but would be too weakto be seen with transparent specimens were a centralbeam present.

However, when a specimen is stained, the dyepattern is strong enough under Kohler illuminationto modulate a central beam, minimizing the need foroptical contrast enhancement.

In some anoptric arrangements the annulus is notblocked off. Instead, it is left clear, but the rest ofthe rear surface of the objective is coated-usuallywith a 50 percent opacity. Thus the contrast rolesof the direct and deviated beams are reversed. Thefirst mode is useful for recording specimens of lowvisibility such as unstained chromosomes; the second,for highly refracting, bright subjects like yeast cells.

By varying the thickness (opacity) of the coatingsthat do the masking, the intensity of the directbackground beam and the amount of light deviatedby the specimen can be adjusted. Wilska found thatan even balance (equal intensities) provided the bestdelineation for most subjects.

Phase-Contrast MethodThe phase-contrast microscope is probably the mostoutstanding contribution to microscopy in recentyears. It can be used to produce excellent contrasteffects with a wide variety of otherwise-transparentspecimens. Since it permits visualization of interiordetails in cell structures, it has a definite advantageover the darkfield microscope. Probably its widestapplication is in the field of tissue culture, where itpermits one to examine and photograph living, grow-ing cells. Here, time-lapse cinephotomicrography isof definite advantage, since a specimen can be pho-tographed at intervals timed to be synchronized withchange. Subsequently, the photographs can be pro-jected at increased rates of speed. The study andphotomicrography of livingblood specimens by phase'contrast also becomes possible, providing a means ofvisualization previously impossible.

63

Phase Optics: The phase-contrast method was intro-duced by Zernike. Fundamentally, the procedureinvolves a direct and a deviated beam, as in theanoptric system. (See Figure 24.) The difference isthat the masking and balancing of the beams areaccomplished by phase-interference modulation,rather than by opacity modulation.

A word on this interference phenomenon may behelpful here. When a ray of light from a single pointsource is split in two and passed through (refracted)a transparent medium, the two rays can be recom-bined without interference. But if one passes througha medium of different refractive index, it is speededup or slowed down, depending on the index. Thenthe two, when recombined, may be out of phase. Ifso, interference occurs and the recombined beam isnot as intense as the original. To appreciate this,visualize the two split rays as waves traveling sideby side. If neither one is altered, they can combine"in phase." Peaks will meet peaks and valleys willmeet valleys; there is no destruction of intensity. Butif one component is altered in velocity, the wavesmay no longer match in configuration. When theconfigurations differ by less than a wavelength, thewaves are out of phase and their recombinationresults in a loss of intensity. When they are out ofphase by 1/2 wavelength, peaks will meet valleys andthe rays will cancel each other out, extinguishing thebeam.

In addition to refraction and retardation by media,diffraction at edges and scatter from very fine detailscan also change the configuration of the waves. Dif-fraction is largely responsible for the edge effects seenin phase-contrast microscopy.

In the phase-contrast system, an annular source,again, is utilized (although linear and crossed slitscan be employed). But instead of an annular densitymask behind the objective, a "1/4-wave" ring is used.This retards a wave by 1/4 wavelength. To form thismask, a transparent medium is coated in a ring ona "compensating plate," which is also transparent.The plate is positioned behind the objective in itsaperture plane, where the annular source comes toa focus. The 1/4-wave ring is aligned with the focusedring source.

As in the anoptric system, the beam from theannular source is defocused in the slide and in theprimary image. But its light, not being masked withan opacity, can be integrated by the eyepiece.Howev-er, neither the eye nor the film can detect the 1/4wave change in"phase.

As before, the specimen, in the defocused directbeam, deviates much of the light, depending on index,thickness, and fine structure. Again the deviated light

64

goes to the eyepiece via the primary image. However,the deviated rays have been changed in phase by thespecimen. The change plays a vital role in forminga high-contrast image, even though it may be as smallas 1/20 wavelength.

A direct beam is passed by the background. Somedirect light is not deviated by the specimen. Thus,when the illumination reaches the specimen, it is splitinto direct and deviated rays by the specimen itself.The direct rays go into the 1/4-wave ring. But whilethe direct part through the specimen has no moreopacity modulation than it does in the anoptricsystem, it now does have an added phase modulationproduced by the ring. The deviated rays also havea phase change dependent, in degree, upon the dif-fraction of the specimen.

When the beams of direct and deviated rays fromthe specimen are focused in the primary image, inter-ference occurs between the two recombined, speci-men-image components, and between edgesand back-ground. The amount of intensity reduction in anygiven structure depends upon the phase difference ofthe two rays imaging that structure. When this is1/2 wavelength, maximum reduction (darkness)occurs.

The brightness of the background (in a given setup)is always the same tone, corresponding to a mediumgray in photography. But the brightness of the speci-men image relative to this tone can be altered byseveral means. Phase plates and mounting mediacontrol this aspect.

Some phase control is done with an annular com-pensating plate. As in the anoptric system, the con-trast roles of the two beams can be reversed. In thephase method, the compensating plate can have aring ground thinner than the rest of the plate, ratherthan a 1/4-wave coating. Then the direct beam isadvanced 114 wave instead of being retarded, becauseof the shorter path through the plate. With a retard-ing phase plate, if the retardation of the medium andthe phase ring add up to an amount that is notsufficiently revealing, a compensating plate with anadvancing ring can be substituted. This will makea II2-wave difference in the configurations of thewaves going through the phase-ring area.

Other plates are sometimes used to alter the phaseof the deviated beam by specificamounts. This is donebecause it has been found that the best rendition ofdetail usually occurs when most of the details in thetwo beams are about 1/4 wave out of phase. Also,for goodresults, the brightness ofthe two componentsof the specimen image should be equal. So platescoated with thin opaque metallic films can be usedto balance the intensity of the beams. Plates are

selected from knowledge of the indices of mediumand specimen, or quite often by trial.

A somewhat confusing point in the nomenclaturearises from the difference between the designationsof European and American equipment manufac-turers. The first group defines a "positive" phase plateas one which advances the direct beam, and a "nega-tive" phase plate as one which retards this beam. Theopposite is elected by the second group. It is betternot to mix these terms with "contrast" designations.The reason is that a given phase plate can producebright or dark contrast images, depending uponwhether the index of refraction of the specimen ishigher or lower than that of the medium. In general,designations are based on the specimen having thehigher index. But an objective marked "dark con-trast" can yield the opposite effect when the mediumis changed. It is obvious that the photomicrographershould fix his mind on what his particular equipmentdoes, rather than go entirely by what it is called.

The image appearance, regardless of the opticalelements used to modify it, is quite indicative of thetype of contrast that has been obtained. When thespecimen is generally lighter than the background,"bright-phase" contrast exists; when darker, "dark-phase" contrast exists. The outermost fringe, or halo,between the specimen and background is black in thefirst instance and white in the second. (Figure 25.)

Effects of Mounting Media: Contrast can be manipu-lated in degree and "sign" to a practical extent byselection of the medium in which the specimen is

FIGURE 25-Epithelial cell, bright phase contrast. In this illustra-tion, note the dark halo present around the cell and the artifacts.In dark phase-contrast records, the halo is almost white. KODAKEKTAPANFilm.

mounted. If the refractive index of the medium istoo close to that of the specimen, very little contrastwill result. It is suggested that the medium differsufficiently in refractive index to provide adequatecontrast for both visual examination and photomi-crography. When tissue specimens are examined, forexample, a medium other than balsam is needed.Balsam is a common mounting medium, but itsrefractive index is very much like that of unstainedtissue. A medium such as glycerine, for temporarymounts, or Diaphane and white Karo syrup for per-manent mounts, will provide much better contrast.The refractive index of balsam is about 1.53; whileDiaphane and glycerine have a lower index, about1.47. (See mounting media, Table 3, page 24.)

Filtration: In phase-contrast photomicrography, agreen filter is commonly used in conjunction witha black-and-white film. The green filter is appropriatebecause phase objectives' are most of en correctedoptically for green light. An excellent filter for thispurpose is KODAKWRATTE Filter, No. 58. When asomewhat deeper green is desired, use a KODAKWRATTENFilter No. 61.

Color films are not normally used, since the phase-contrast method is most frequently used with color-less, or almost colorless, images of low original con-trast. Moreover, optimum phase-contrast effect isobtained with a single color, normally green. Possibleexceptions to this may be photomicrography of verylightly stained objects or photomicrography withpolarized-light. A green-sensitive, black-and-whitefilm of moderate contrast is quite suitable for phase-contrast photomicrography. When sheet film is used,KODAKEKTAPANFilm 4162 (ESTARThick Base) ishighly recommended. This film has very fine grainand wide development latitude, with moderate speed.When a higher speed film is necessary in order tostop the motion of living specimens, KODAKROYALPan Film 4141 (ESTARThick Base) or KODAKROYAL-X Pan Film 4166 (ESTARThick Base) is indicated.

For 35 mm still photomicrography, KODAKPLUS-XPan Film, KODAKTRI-X Pan Film, and KODAKPANATOMIC-XFilm are recommended. If higher con-trast is desired, KODAKPhotomicrography Mono-chrome Film (EsTAR-AHBase) SO-410 and KODAKHigh Contrast Copy Film 5069 are applicable. Thesefilms have extremely fine grain and have provenexcellent for this application.

In cinephotornicrography, 16mm EASTMANPLUS-XNegative Film 7231 is recommended when extremelyfine grain is desired; EASTMAN4-X Negative Film7224, when extremely high emulsion speed (El 500)is essential.

65

Interference-Contrast MethodsThere are several other types of microscopes thatutilize the interference effect. The most useful onesfor use in general fields are those based on theinterference-contrast principle. The method does notyield the contrast enhancement of a phase-contrastmicroscope, but it does eliminate most peripheral andstructural edge halos, thereby delineating fine detail.It also introduces color effects and gives a three-dimensional appearance ofrelief. (See Plate II-E.)

Apart from the fact that the condenser is fullyilluminated, the basic difference in the optics of thetwo systems is this: In the interference-contrast mi-croscope, it is the optical system that produces thetwo interfering beams; it is not the specimen, as inthe phase-contrast method. The interference-contrastbeams are called the "object beam" and the "referencebeam." The first carries the image, and the secondis either a homogeneous or an asymmetrical beam.Interference takes place between two types of beams,not between two image components, as in the phase-contrast system.

Again, several analogies may be helpful. In thephase-contrast system, the configurations of the twobeams are like the wakes oftwo boats merging courseson a smooth lake. The interference pattern theycreate is sharpest when the boats have the same sizeand speed (balanced beams). In an interfer-ence-contrast system, the wake of a single boat inter-feres with waves on a rough lake. Balance is bestwhen the lake is no rougher than the wake of theboat. To apply the analogy to the Nomarski system(to be discussed), the wakes of two boats travelingside by side interfere to produce the pattern; the wakeof the boat <studiedhas the wake of the other asym-metrical to its course.

Once the object and reference beams aTesplit, theyare separate in the plane of the specimen. They arerecombined for interference before they reach theeyepiece.There are two commonly used methods (dueto Smith) for affording this separation. They are the"double-focus" system and the "shear" system.

In the first, both beams are focused by the condens-er along the optical axis of the microscope.The objectbeam is focused in the plane of the specimen. Thereference beam is focused above or below the slideand forms no structured image. In effect, the specimenis seen through the even blur of the reference beam.The two beams are displaced vertically. The wanteddetail is imaged and interferes with the same detailout of focus.

In the shear system, both beams are focused in theplane of the specimen, but they are optically displacedlaterally. In effect, only the subject beam illuminates

66

the specimen-the reference beam is moved aside forsubsequent recombination below the eyepiece. Thisbeam should pass through a relatively clear area ofthe slide in order to minimize the imaging of unwant-ed detail, which would intrude on the subject detail.Thus this system is not as suitable as the double-focusmethod of studying tightly packed slides.Nomarski Optics: A commonly used interferencesystem for all powers of objectives was worked outby Nomarski. It employs a modified shear system.The illumination from the lamp passes through thefollowing components (seeFigure 26):• A polarizer below the condenser produces a beam

of plane-polarized light.• A Wollaston prism splits the beam in two before

it enters the condenser. It also rotates the planesof polarization of the two beams so that they aremutually perpendicular. They cannot interferewhere they cross at various foci in the optical train.

• After passing through the condenser, the slide, andthe objective, the beams are recombined by anotherWollaston prism with oppositely cut faces. However,they still cannot interfere.

• A diagonally oriented polarizing analyzer plate cir-cularly polarizes (seepage 67) the two beams so thatthey can interfere before entering the eyepiece toform the interference contrast image.

Operational Details: Photography of interfer-ence-contrast images is similar to that used forbrightfield illumination. However, it may be necessaryto reduce the aperture diaphragm to obtain more evenillumination. If this is done, the resolving power ofthe objective will decrease. Consequently, the bestaperture setting is largely dependent on the specimenitself, and some experimentation will be necessary toobtain the best compromise.

The Nomarski system is very efficient in that itusually permits a full aperture. It is able to resolvefiner structure than the phase-contrast method.However, the Nomarski image may be somewhatdeceptive with respect to topography, depending uponwhether the object beam or the asymmetrical refer-ence beam dominates the image. For this reason, thephotomicrographer should have some idea of thesurface contours of the specimen. (For example, noticethe change in appearance that occurs when Plate II-Eis viewed upside down.) Then, by means of a rotatingstage (which he ought to employ), he can orient theslide until the image conforms. In this way the fullbenefit of the system for delineating transparentsubjects can be obtained.Francon SystemCertain three-dimensional specimens ID the metal,

plastics, and glass industries and in crystallographyand metallography can be examined by reflectedinterference with a system developed by Francon. Apolarized-light microscope is adapted for axial illumi-nation. Instead of Wollaston prisms, Savart plates,which are double, flat plates of quartz crystal suitablycut, serve to produce divided beams. The degree ofseparation of the beams depends upon the thickness

T EYEPIECE

10ANALYZER

WOLLASTON PRISM

IIII

II

OBJECTIVE

-----',.. •••..--- OBJECT PLANE,SPECIMEN

CONDENSER

WOLLASTON PRISM

~~ •• ~ •••••• ANALYZERt - OBJECT BEAM

LIGHT --- - REFERENCE BEAM

FIGURE 26-Nomarski System. Schematic path of a single rayfrom a lamp fully illuminating a condenser. The ray is first plane-polarized and then split into two rays by the Wollaston prism.The two travel separately through condenser, slide, and objective.A beam of rays going through the structure to be studied in thespecimen is called the object beam; the other (which goes asym-metrically through another part of the specimen) is the referencebeam. A second Wollaston prism recombines the beams. Sincethey have been plane-polarized perpendicularly to each other,they cannot interfere. So a polarizing element (analyzer) with itsplane of polarization at 45 degrees to each of the incoming beamsis introduced. The analyzer circularly polarizes each beam sothat they can combine to form interference patterns.

of the Savart plates chosen and can be made quitespacious. (See Plate II-E.)

The films and processing conditions recommendedfor the brightfield illumination technique on page 32apply in interference-contrast microscopy. Exposuresettings should be determined experimentally.

Polarizing MethodPolarized light has many valuable properties thatmerit understanding by the photomicrographer. Mostillumination sources for microscopy emit beams ofheterogeneous light-the waves are vibrating in alldirections perpendicular to the axis of propagation,like paper streamers tied to the guard on an electricfan. If the fan were brought close to an open venetianblind, only those streamers vibrating in the horizontalplane would pass through the slits. If the blind werehung sideways,only the vertically vibrating streamerswould pass through. Two blinds, crossed in thisfashion, would block all the streamers. Polarizingmedia present optical slits that perform in a waysomewhat like these blinds. They pass half of thelight "plane-polarized" according to the orientationof the slits. The other half is absorbed.

Transparent "isotropic" objects pass plane-polarized light unchanged. "Anisotropic" materials,such as crystal quartz, can split a plane-polarized rayinto two rays. A Wollaston prism, made up of twoopposed quartz crystals, serves to rotate the planesof the two ray components perpendicular to eachother. And of course, an oppositely oriented prismcan recombine the two rays. (See Figure 26.)

In the Nomarski microscope, both beams are circu-larly polarized as they recombine. They are able tocreate interference effects, which two rays cannot dowhen plane-polarized perpendicularly to each other.This is accomplished in the following way: In thepath of a plane-polarized ray, when another polarizeris placed with its slits (plane of polarization) at 45degrees to the vibration of the waves, the ray becomes"circularly" polarized. (A circularly polarized beamshould not be confused with a heterogeneous beam.)If an increment of plane-polarized light can be visu-alized as a sheet of paper moving (parallel to theoptical slits) along the beam, then an increment ofcircularly polarized light would be a sheet of paperedgewise spinning around its axis of propagation asit moves along the beam. A heterogeneous increment,however, could be represented as a moving bookopened up so wide that the covers are touching.)

In the ordinary polarizing method, the microscopesystem is quite simple-a "polarizer" is placed belowthe slide and an "analyzer" at the eyepiece. Theseare mounted with their slits perpendicular to each

67

other; they are set for "extinction." Now, the objectitself, when it is anisotropic, plays a role somewhatlike that of a Wollaston prism. It splits and rotatesthe plane-polarized rays from the condenser by asmall amount, so that when they reach the perpen-dicularly oriented analyzer, they are not extinguishedby it. But rays going through an isotropic object areso changed; thus they are extinguished. One obviousbenefit is differentiation of anisotropic and isotropicareas within a transparent specimen, say, in a thinslice ofrock for petrographic study.

But there is another, better-known effect producedby the method-interference colors. These are createdby a growing anisotropic crystal, for example. Thissubject consists of structures of varying thickness.A crystal is a medium which retards the phase ofrays passing through it, and an anisotropic crystalalso modifies the polarization. Retardation and thedegree of anisotropic splitting depend upon thicknessand wavelength; hence, the spectral colors in whitelight are not all affected alike. When two adjacentrays pass through a given spot in the crystal andare split by it, they are close enough for interference,and this may cause the blue components to augmenteach other. In another spot, of different thickness,a red color may appear. Because of the concomitantrotation of their planes of polarization, these colorscan pass through the analyzer and form the imagepattern. Rays through the background of the slideare not rotated and hence are extinguished, prevent-ing the background from flooding the image.

A "tint" plate is a thin lamina of a birefringentsubstance like mica. The thickness is accurately con-trolled so that the effective retardation is a specifiedfraction OD a wavelength. It has a transmission axisof maximum refringence. This axis can be orientedat various angles to the plane of the polarized beamfor controlling the region of the spectrum at whichmaximum wavelength augmentation takes place.Also, its retardation can be added to that of a weaklybirefringent specimen for an enhanced image.

Some isotropic materials can become anistropicunder strain, the degree depending upon the stress.Such objects also exhibit interference patterns incolor when white illumination is employed. Mono-chromatic light yields dark and bright strain lines.

Very striking effects can often be obtained witha brightfield microscope equipped with polarizingfilters. Some transparent substances, principally cer-tain chemical crystals, have an optical property called"birefringence," or "double refraction." When suchsubstances are viewed in a microscope betweencrossed polarizers, they may appear bright or evencolored against a dark background. Place one po-

68

larizer in the illumination beam and the other some-where behind the objective, usually on the eyepiece.

The formation and growth ofbirefringent chemicalcrystals is a fascinating subject that can be pho-tographed on color film, with either a still or moviecamera. A low-powermicroscope is sufficient. Crystalsare formed by dissolvinga chemical in distilled water,placing a drop of the solution on a microscope slide,and spreading the solution thin. As the water evapo-rates, the crystal growth begins. Either of thepolarizers can be rotated to produce the crossedposition, which results in a dark background on whichthe colored crystal pattern forms. Sodium thiosulfatecrystals (commonly known as "hypo") work extreme-ly well for this study. Abright light source is generallyneeded. (See cover illustration.)

Very slight birefringence can be detected and pho-tographed by placing a sensitive tint plate betweenthe polarizer in the light beam and the analyzer. Inthis case, the background will be colored; the exactcolor depends upon the orientation of the plate.

Other subjects which show polarization effects arerock sections ground thin for petrography, fibers,hairs, starches, and many components of plant andanimal tissues.

Stereo MethodWhen one looks at any object, the image recordedby the optical system, in conjunction with the coor-dinating facility of the brain, has a three-dimensionaleffect. This effect is the result of two different viewsof the object, separated by an angle dependent uponthe interpupillary distance of the individual, and theninterpreted as one image. This is the principle of theGreenough binocular dissecting microscope, designedin 1897for low-powerwork. In this type ofmicroscope,each eye is provided with its own complete micro-scope. The paired objectives are mounted togetherat the approximate angle of binocular vision: 15°.Therays from the objective pass through a Porro prism,which produces an image that is not inverted. Theprisms in the paired objectives are similar to thoseused in field binoculars. This microscope has beenmodified and improved in recent years. The objectiveshave been incorporated into a drum, or slidingnosepiece. The use of wide-field oculars and prismsproduces a large, flat field of view, with the rangeof magnification n-om X 3 to X 100. This type ofmicroscope has the advantages of great depth of focus,long working distance, large, flat fields, and simplicityof operation.

The "stereo" microscope, as it is commonly called,has become invaluable in industry, medicine, teach-ing, or wherever a microscope of low power is needed.

Most newer stereo microscopes now have zoom capa-bilities and a maximum magnification of X 200. Theuse of stereo microscopes varies from general scientificinvestigation in all fields to production examinationin quality control. The most important use in relationto photomicrography is in examining the specimento determine the best area to photograph. This isespecially necessary in photomicrography of biologi-cal specimens-where dissecting, staining, and selec-tive mounting are performed.

This microscope has several limiting factors whenspecimens are to be photographed through one of thebody tubes. The principle limiting factor to producingquality photomicrographs with stereo microscopes isthe low N.A. of most objectives. Additionally, sinceonly one objective, at a slight angle to the specimen,is used, the depth and resolution typical of stereoviews is not recorded on film. Many microscopemanufacturers have accessories to correct this situa-tion. Paired stereomicrographs can be made in severalways that are described in The Practical Use of theMicroscope by G. H. Needham. The best film to useisKoDAcHRoME64Film (Daylight) orKODACHROMEIIProfessional, Type A, depending upon the color tem-perature of the light source. These filmshave fine grainand good resolving power.

FIGURE 27-Paramecia photographed at low power in a binocularmicroscope.

SPECIALIZED APPLICATIONSFluorescence PhotomicrographySome materials will emit light of a longer wavelengthwhen excited by short wavelengths of radiation. Thisphenomenon is called "fluorescence." Either ultravio-

let or blue light is often used as an exciting radiationto produce visible light of longer wavelength. If asubstance does fluoresce, the effect is called either"primary fluorescence" or "autofluorescence." Somematerials do not fluoresce by themselves but can beimpregnated with chemicals, such as certain dyes (seeTable 5, page 33), that will fluoresce. Dyes of thistype are called "fluorochromes"; the effect in theoriginal material is called "secondary fluorescence."For example, when excited with ultraviolet, chloro-phyll will fluoresce with a deep red color. Whenstained with dilute acridine orange, human epithelialcells will glow an orange-red under ultraviolet radia-tion. (See Plate II-D, page 38.)

Fluorescence microscopy and photomicrographyhave acquired great importance in recent years in thefield of exfoliative cytology. A very appropriate appli-cation is in early detection of cancer in smears,exudates, and tissue sections. Fluorescence tech-niques are used to advantage in other fields of medicaland biological research also.

light Source: A very efficient light source for mostfluorescence work is the high-pressure mercury-vaporlamp, which emits very bright radiation in bothultra-violet and short-blue wavelengths. When a lightsource having a continuous visible spectrum as wellas ultraviolet is needed, the xenon arc serves thepurpose very well. Most fluorochromes are excitedby ultraviolet and produce fluorescence somewherein the visible spectrum. The fluorescein dyes com-monly used in antibody techniques have a maximumabsorption for long blue wavelengths, however, andrequire a continuous source, such as a xenon arc.

When a substage mirror is used, one with analuminized surface should be selected. The reason forthis is that silver, often coated on these mirrors, isa poor reflector of ultraviolet radiation.

When ultraviolet radiation is used to producefluorescence, such as with a mercury arc, an exciterfilter is needed in the light beam to transmit thisradiation freely and to absorb visible light not neededin producing fluorescence. KODAKWRATTE FilterNo. I8A (glass only) has a high transmittance forthe 365 nm (ultraviolet) line of the mercury-vaporspectrum. It absorbs all visible light. A barrier filteris also necessary. This type of filter should absorbultraviolet, such as that transmitted by the specimen,and transmit visible radiation freely, including fluore-scence colors.The selection ofan efficient barrier filtercan be critical. A KODAKWRATTENFilter, No. 2B,2A, or 2E, can be used for this purpose. Althougheach filter will completely absorb ultraviolet, thedifference is in their absorption of short-blue wave-

69

lengths. If in doubt, the No. 2A Filter is suggested.

Fluorescein Dyes: In a fluorescent-antibody tech-nique, where fluorescein dyes are used asfluorochromes, secondary fluorescence usually occursin the green at about 540 nm. Maximum absorptionof these dyes is at 480 to 490 nm in the blue regionof the spectrum. A filter which transmits blue freelyis normally employed as an exciter filter. KODAKWRATTENFilter No. 47B is suggested. A barrier filterfor this application must transmit wavelengths longerthan blue and must absorb blue completely. A KODAKWRATTENFilter, No. 12 or No. 15, will suit thispurpose. Both filters are yellow and will transmit thegreen fluorescence color freely.

When fluorochromes are used to stain specimens,a certain amount of short-blue "autofluorescence"occasionally occurs also, particularly with tissue sec-tions. This autofluorescence often must be absorbedin order not to degrade the color of the secondaryfluorescence produced by the fluorochrome. Both theNo. 2A and o. 2E Filters have absorption for short-blue radiation, the No. 2E having the greater absorp-tion in this respect.

A barrier filter must be used behind the objective.If it is not, the residual ultraviolet will record as blueon color film and will degrade all fluorescence colors.A barrier filter of appropriate size can often be useddirectly behind the objective in the microscope bodytube, or a smaller-size filter can be placed in or onthe eyepiece of the microscope.

The selection of a mounting medium is also impor-tant. A medium must be chosen which has little orno autofluorescence. Autofluorescence of a mountingmedium may be either pale blue or pale green in colorand will degrade fluorescence colors. Temporarymounts can be made with either pure glycerine orCargille's Immersion Oil, Type A, or Crown Oil (ob-tainable through scientific supply houses). These havevery low fluorescence. Care should be taken whenhandling these oils, since both contain high percen-tages of toxic polychlorinated biphenyls. If glycerinecontaining such impurities as "acrolein" is used, alight greenish autofluorescence may occur. Most per-manent mounting media also produce autofluore-scence; therefore, they should not be used in fluore-scence work.

The pale-blue autofluorescence ofa mounting medi-um can be absorbed to some extent by either a barrierfilter which absorbs some blue or a pale-yellow KODAKColor Compensating Filter, such as a CC20Y. Pale-green autofluorescence can be neutralized by a pale-magenta color-compensating filter, such as a KODAKColor Compensating Filter CC20M. Heavier filters

70

may also absorb some of the desirable fluorescencecolor. Quite often the KODAKWRATTENFilter, No.2E will absorb ultraviolet as well as the pale-blueautofluorescence of a mounting medium.

Darkfield illumination is highly recommended forboth fluorescence microscopy and photomicrography.By this technique, the darkest background is achievedso that fluorescence colors stand out brightly. Aspreviously stated, oil-immersion darkfield condensersare most efficient for medium and high power. Takegreat care that no bubbles are introduced into theoil and that the microscope slide is of the correctthickness for the condenser in use. A darkened roomis highly essential to efficient darkfield work in fluore-scence microscopy in order to exclude extraneouslight, even from the slide surface.

Even though a fluorescent image may appear brightto the eye, long exposure times are often necessaryin photomicrography. Color films, of course, are sug-gested in order to record the image in color, as itappears to the eye. Therefore, a high-speed film-suchas 35 mm KODAKHigh Speed EKTACHROMEFilm,(Daylight)-will minimize exposure time and willrecord most fluorescence colors with reasonable accu-racy (see Plate II-D on page 38). Daylight-type filmis suggested because of its balanced sensitivity to red,green, and blue. This film can also be specially pro-cessed for higher speed (see page 34). Other daylight-type, color-reversal roll films such as KODACHROME64 or KODAKEKTACHROME-XFilm, although oflowerspeed, can also be used to record particular fluore-scence colors,

Ultraviolet PhotomicrographySince the limit of resolution attainable in photomi-crography depends upon the wavelength of light, thehighest resolution is obtainable with ultraviolet. Itis possible to make photomicrographs in the nearultraviolet (365 nm) by using a conventional micro-scope with a glass lens. Apochromatic objectivesperform best. Optical glass, however, will not transmitultraviolet radiation below a wavelength of about 310nm. In order to transmit the shorter wavelengths,a microscope equipped with fluorite optics or reflect-ing optics must be used. One of the principal advan-tages of this type of microscope is its ability to focusan image in visible light and to keep the image infocus throughout the ultraviolet region. In conjunc-tion with this microscope, a light source such as ahigh-pressure, mercury-vapor lamp is needed to pro-vide radiation in the far ultraviolet. For photomicro-graphy, a "monochromator" is also required in orderto isolate a particular wavelength or narrow bandof wavelengths in the ultraviolet.

Quartz optics and other types of microscopes andillumination, as well as a means for focusing theinvisible ultraviolet radiation, are discussed fully byLoveland (1970).

Photomicrographs in the near ultraviolet can alsobe made with this ultraviolet microscope. It providesa higher degree of resolution than is obtainable witha conventional microscope. In addition, the ultravio-let microscope allows one to study the selectiveabsorption of ultraviolet by living cells, tissues andfine particles.

What types of films are used in ultraviolet photo-micrography? All photographic emulsions have aninherent sensitivity to blue and ultraviolet. Sensitiv-ity in the ultraviolet actually extends far into thisregion, but the response of a film or plate is somewhatlimited, due to absorption of ultraviolet by gelatin,the medium in which the silver-halide crystals of theemulsion are suspended. For long wave ultravioletphotomicrography, almost any film or plate can beused to record the image. However, only black-and-white films need be considered, since color film hasno advantage. In 35 mm ultraviolet photomicro-graphy, KODAKPANATOMIC-XFilm is especially rec-ommended because of its extremely fine grain whichpermits excellent enlargements. When a higher con-trast is needed, KODAKHigh Contrast Copy Film 5069may be used. If sheet film is to be used, a very finegrain film of medium contrast should be the firstchoice. Films such as KODAKEKTAPANFilm 4162(ESTARThick Base) and KODAKPLUS-XPan Profes-sional Film (ESTARThick Base) are quite suitable.

Images formed by ultraviolet photomicrographyare low-contrast. Therefore, if a high-contrast filmis not used, medium- to high-contrast developmentcan achieve satisfactory results. To obtain medium-to high-contrast development, either extend the rec-ommended development time m use developers thatproduce higher contrast. It should be noted that thecolor-blind and orthochromatic films (that is, theblue- and blue-green sensitive films) generally providea higher contrast than panchromatic materials.

When higher sensitivity to ultraviolet is needed,or when sensitivity is desirable in medium-wave andshort-wave regions of the ultraviolet, it may be neces-sary to resort to special emulsions. Eastman KodakCompany can supply information about special ma-terials that are available. Also, information aboutthese products can be found in KODAK Plates andFilms for Scientific Photography (Kodak PublicationNo. P-315) and in Ultraviolet and Fluorescence Pho-tography (Kodak Publication No. M-27). These DataBooks can be obtained through photographic dealersor from Eastman Kodak Company by direct order.

Infrared PhotomicrographyMost of the subjects examined through a microscopetransmit light to some extent, even if treated withstains. Some subjects, however, are relatively opaquewhen viewed through a microscope; few,if any, detailsare visible. Increased visibility can often be obtainedby the use of infrared, the long wavelengths beyondthe visible spectral range. Infrared photomicrographyis especially useful in the field of entomology. Manyinsects have chitinous and dark-pigmented structuresthat are opaque to visible light but are penetratedfreely by infrared radiation. Heavily stained speci-mens, many textiles, forged and altered documents,dark-colored crystals, and many other subjects canbe photographed with infrared radiation. With a fast,color infrared film, photography in the infrared canbe used for many applications. Some of the mostcommon applications are in plant pathology, pollu-tion control, criminology, and histology. Infraredcolor photography has been used fOTdifferentiatingbiologic pigments, tissue structures, and inclusions,as well as for finding new criminal detection methods.

Because of the long wavelength of infrared radia-tion and because of lens aberrations, in that region,the infrared image cannot be as sharp as in ordinaryphotomicrographs. The benefits comes from the pene-tration of many visually opaque specimens. (SeeFigure 28.)

FIGURE 28-Dense animal hair photographed with infrared-sensi-tive black-and-white film and a KODAK WRATTEN Filter No. 87.

A color film used for infrared photomicrographyis KODAKEKTACHROMEInfrared Film; it is availablein 20-exposure rolls for 35 mm cameras and lOO-footrolls for 16 mm cameras. This film is a false-colorreversal film that was originally designed forcamouflage detection by aerial photography. It isbalanced for daylight-type exposures, (or for expo-sures by electronic flash) with a KODAKWRA'ITENFilter, No. 12 (yellow), over the camera lens.

For use in infrared photomicrography, Kodakblack-and-white infrared films with broad infraredsensitivity, are available in sheets or in 35 mm rolls.One ofthese highly sensitive films,KODAKHigh SpeedInfrared Film 2481 (ESTARBase), is available in 16

71

mm and 35 mm, lOO-footrolls as well as in sheetform [KODAKHigh Speed Infrared Film 4143 (ESTARThick Base)] for applications requiring high speedfilm. Twenty-exposure magazines of KODAKHighSpeed Infrared Film are also available. Also, specialblack-and-white emulsions with sensitivity to about1150nm can be obtained from Eastman Kodak Com-pany. More information about these films may befound in Applied Infrared Photography (Kodak Pub-lication o. M-28) and KODAK Infrared Films (N-17)available through photo dealers.

Since normal exposure-meters have low sensitivityin the infrared, exposure times are usually determinedby trial. It is common practice to make a series ofexposures, either on sheet film (by withdrawing thedark slide by definite amounts and varying exposuretime) or on 35 mm roll film (by making severalexposures of one frame each).

However, before making infrared photomicro-graphs, test film holders and plateholders, includingthe draw slides, to make certain that they are opaqueto infrared radiation. Failure to do this may resultin fogged emulsion and complete deterioration ofimage contrast. Processing recommendations for in-frared films are included with the materials.

Because infrared photography is quite specialized,it is not feasible to go into details here. Instead, theapplications have been given. Also, the basic require-ments have been outlined so that you can see thatthere is no particular complexity involved. everthe-less, certain careful but straightforward proceduresare needed in focusing the photomicroscope. Wheninfrared color film is employed, special attention mustbe paid to light sources and color balancing withfilters. Tho e who wish to carry on the technique arereferred to Kodak Publication M-28.

Photomicrography of ChromosomesThe study of human chromosomes is of widespreadinterest in cytogenetic laboratories throughout theworld. Since photomicrographs of chromosomepreparations must always be made for karyotyping,it is of great importance that the recorded image beof high quality in order to provide the most informa-tion. (See Figure 29.)

Basically, chromosome preparations are usuallymade from blood cultures in which the arrest of celldivision is accomplished with colchicine at the meta-phase stage of the mitosis. Smears are then made onmicroslides and are stained to produce contrast.Aceto-orcein and Giemsa staining are quite common.The preparation is viewed and photographed, usuallywith a green filter, both for additional contrast andfor high image quality. Because of the minute size

72

I

FIGURE 29-Human chromosomes. KODAK PLUS-X Pan Film(35 mm). KODAK WRATTEN Filter, No. 61.

of chromosome, a veTYefficient microscope with high-quality optics is essential. A bright light source isalso a necessity because the photographic materialsused often have high contrast but low speed.

SeveTa135mm high-contrast black-and-white filmsused for the photomicrography of chromosomes areKODAKHigh Contrast Copy Film 5069 and KODAKPhotomicrography Monochrome Film (EsTAR-AHBase) SO-410.Both of these films possess extremelyfine grain and high resolving power. They will produceexcellent results for this application. KODAKPhoto-micrography Monochrome Film also features variablecontrast, depending upon processing conditions.

When a sheet film is to be used, KODAKContrastProcess Ortho Film 4154 (ESTARThick Base) hasproven suitable for the purpose. Contrast may be toohigh when the recommended developer is used. Forsuitable contrast, the use of KODAKDeveloper DK-50(diluted 1:1 with water) is suggested. Developmenttime is 7 minutes at 20 C (68F) for tray development,or 8 minutes for tank development.

The negatives are printed on single-weight, high-contrast photographic paper so that the individualchromosomes can be cut out for assembly as a karyo-type. When unstained chromosomes are to be pho-tographed for study rather than for karyotyping, thestop-contrast method (see page 62) is recommended.

Photomicrography of AutoradiographsAutoradiography is a technique by which radioac-tivity is detected using photographic materials. Spe-cial photographic emulsions with high sensitivity tocharged particles are employed to trace the path ofradioactive-labeled substances in metabolic studies ofplants and animals. Also, it is useful in studying cells,

nuclei, and chromosomes in which the uptake andlocation of tracer substances in the various stagesof cell division can be recorded or quantitativelyevaluated. For further information on autoradio-graphic techniques, see, for example, Rogers, A. W.,"Techniques of Autoradiography," Second Edition,Elsevier Press, New York, 1969.

The contact image produced on a film or plate byan object that contains a radioactive substance iscalled an autoradiograph, In practice, the specimensection on a microscope slide is covered with thephotographic emulsion which, after exposure andprocessing, contains the silver grains of the autoradio-graphic image.

When making a photomicrograph, it is preferredthat both the specimen image and the autoradio-graphic image be in focus together so that the infor-mation in each image can be correlated. At lowmagnifications the available depth of field may besufficient to produce both images in focus at the sametime. Standard photomicrographic techniques as pre-viously described in this book can therefore be uti-lized. At higher magnifications-generally utilized inautoradiographic evaluations of biological speci-mens-both images must be separately focused forcomparative evaluation. A double exposure tech-nique, then, is required to obtain both pieces ofinformation in focus in the same photomicrograph.Simply determine the proper exposure for one of theimages and also determine the correct focus positionon the micrometer scale for each image. Use one-halfthe exposure time to photograph each image at itsdetermined focus position.

Photomicrographs of autoradiographic specimensmay be made in either black-and-white or color. Theblack-and-white films that can be used are KODAKHigh Contrast Copy Film 5069and KODAKPhotomi-crography Monochrome Film 80-410, which are con-trast-dependent upon development. The color filmthat can be used for normal exposures is KODAKEKTAcHRoME-XFilm; for low lighting conditions,KODAKHigh Speed EKTACHROMEFilm (Daylight)can be used. Color films can be processed by the useror can be sent to a color-processing station. (See"Processing Service for Increased Speed," page 34.)

Metallographic PhotomicrographyMetallography is the science of studying, interpreting,and recording details of the physical structure ofmetals and other opaque surfaces. The preparationof samples for metallography necessitates theirmounting, polishing, and etching. This science differsradically from other branches of microscopy. It -in-volves many different metals and alloys, both ferrous

and nonferrous. These samples may range from ex-tremely hard to very soft. The preparation of eachrequires its own technique; however, all specimensexamined or photographed that are opaque must beilluminated by incident light. The light, of course,comes from or near the optical axis of the microscope.A focused beam can be directed axially (see Figure31), with the beam-splitting mirror below the objec-tive in very low-power work. There is not enoughspace for this mirror when a higher-power objectiveis used. Thus, a special illuminator that incorporatesa mirror and holds the objective is used. The objectiveserves as a condenser. Provision is often made fordecentering the beam for oblique incidence, whenrecording surface irregularities rather than the specu-lar appearance of the object. Most metallographicmicroscopes have "built-in" vertical illuminators andan attachable light source.

The metallographic microscope differs from anordinary microscope, in that it includes a focusingstage and that the vertical illumination stays con-stant. The Le Chatelier type of inverted microscopeis most commonly used in metallography.

With this microscope, the specimen is held perpen-dicular to the optical axis of the objective by placingit face down on the stage. The stage can then bemoved up or down by both coarse and fine focusingadjustments. The objective is held in a vertical posi-tion beneath the stage (Figure 30). Alignment of the

FIGURE 30- The metallographic microscope allows photographs'to be taken of surface details of many opaque specimens. (Photocourtesy of Bausch and Lomb, Inc.)

73

camera, microscope, and illuminator is comparativelypermanent, so speed and accuracy are assured forproduction work. Various light sources are adaptablefor use with this type of microscope.

Some types of inverted microscopes include built-infacilities for polarized-light and darkfield work,as wellas the conventional brightfield arrangement. Acces-sories are available for phase contrast and for inter-ferometry. Inverted metallographic microscopes aremade commercially by several manufacturers, bothforeign and domestic.

More information about the factors involved inmetallography can be found in Kodak PublicationNo. P-39,Photomicrography of Metals, available fromphoto dealers.

Photography of Microelectronic CircuitsThe microelectronic circuit, with its many bondingwires, its highly reflective surfaces, and its intricatedetail, presents a problem to photograph. Unlessspecially illuminated with supplementary diffuselight, the bonding wires reflect the light source andappear black in the photograph. The highly reflectivesurfaces tend to obliterate any fine detail present.The high contrast of the sample is likely to causestrong diffraction patterns. However, for very lowpower and with a simple microscope, these problemscan be overcome by a method of illumination calledaxial lighting.

With ordinary lighting, the light source is directedon the specimen at approximately a 45-degree angle,giving a raw lighting. This usually causes too muchcontrast and, therefore, lack of detail. Axial lighting,however, is produced when the beam impinges on thespecimen along the subject-lens axis, in the directionfrom the lens to subject. This arrangement is shownin Figure 31. Note that the light beam comes in ata 90-degree angle to the axis. It is then partiallyreflected to the subject by the mirror-like action ofthe 45-degree microscope cover glass. The rest of thelight is transmitted through this glass and is lost.The photomicrograph is taken through the coverglass.

To photograph miniaturized electronic componentsat high magnification, where the objective and thespecimen are very close, it becomes impossible toilluminate the specimen by direct lighting. Therefore,a vertical illuminator is needed. Such an illuminator,called an epi-condenser, has a special condenser builtas a ring around the objective. The illuminating beamis projected to the specimen by this ring condenser.Several microscope manufacturers supply models invarious designs.Also,a Lieberhuhn mirror can be used

74

around a regular objective.To enhance the effect of the axial lighting and to

show all the detail possible in the bonding wires, anoblique, low-angle diffuse beam can be added. Thiswill show texture in the specimen. The ratio of thislighting to the axial lighting is usually one diffuseto twice the quantity of axial lighting. However, thiscan change depending on the depth of the specimen.

Other methods of lighting that can be used are thevertical illuminator, which directs the illuminatingbeam down the optical axis and through the objectiveto the specimen, and the convergent cone of lightfrom around the objective onto the specimen (e.g.,Ultropak Illuminator manufactured by E. Leitz, Inc.,468Park Avenue, New York, New York 10016).Thesemethods give a low contrast lighting but render gooddefinition without the problem of flare.

CAMERA

ANGLE MUST BEACCURATELY 45°

SPECIMEN

FIGURE 31-Elements for axial lighting. If a focused beam is used,the coil should be imaged in the plane of the camera lens.

Any lighting on the surface of the specimen tendsto produce flare light. This degrades the image andreduces the contrast. Protecting the objective fromany stray light, whether it is external or internal,will improve the contrast and image quality.

An excellent high-speed color film for this low levelof lighting is KODAKHigh Speed EKTACHROMEFilm.All other materials of photomicrography apply andwill not be repeated here.

Recording ReplicasIn some applications, a thin plastic replication of asurface is mounted on a slide. This is valuable whenthe specimen is too opaque for brightfield photomi-crography or when it is too bulky to place on themicroscope. The transparent replica is illuminatedwith the condenser slightly off-canter. Replicationmethods and materials and photomicrographic proce-dures are discussed in the literature on this highlyspecialized field.

LAMP

GLOSSARY OF TERMS USED IN PHOTOMICROGRAPHYAberration-Any inherent deficiency of a lens oroptical system which is responsible for imperfectionsin the shape or sharpness of the image as a resultof failure to image a point or a straight line as suchor an angle as an equal angle. The various forms ofaberration can be reduced but not completely eliminat-ed by assembling a lens from a number of differentelements of compensating characteristics.

Achromat-A lens which brings light from two partsof the spectrum (strictly speaking two specified wave-lengths) to the same focus. Modern achromatic lensesbring the blue and red regions of the spectrum tothe same focus, thus reducing chromatic aberration.

Anisotropic-Exhibiting different properties whenmeasured along different axes. A transparent, ani-sotropic material such as a crystal of calcite possessesdifferent refractive indices in different directionsthrough its mass (birefringence) and polarizes trans-mitted light.

Aperture-The lens opening through which lightenters an optical instrument. The area of this openingis sometimes adjustable by an iris diaphragm or stop.

Apochromat-Lens which brings three chosen colorsto the same focus. Apochromats used for microscopeobjectives are corrected for chromatic aberration forprimary red, green, and blue light.

Bertrand Lens-Removable positive lens which canbe fitted above the objective of a microscope to forman image of the objective's rear focal plane in thefront focal plane of the eyepiece. It enables the exitpupil to be observed without removing the eyepiecewhen setting up Kohler illumination.

Birefringence- The refraction of light in two slightlydifferent directions to form two rays. Polarizing filtersthat contain crystals oriented in one direction arebirefringent.

Chromatic Aberration-Faults in the performance oflenses due to light of different colors coming todifferent planes of focus, the blue image being nearestthe lens, or yielding images of different magnification.

Compensating Eyepiece-An eyepiece corrected pri-marily for use with apochromatic objectives, eliminat-ing the color fringes found when ordinary eyepiecesare used with such objectives.

Condenser-An optical assembly which concentrateslight from a source into a beam. The beam is an evenlyilluminated image of the source used in photomicrog-

raphy, projection of slides, and printing negatives.

Contrast-The ratio of the amount of light transmit-ted or reflected by the most transparent and mostopaque areas of an image.

Curvature ofField-A lens aberration in which imagescan be sharply focused only on a curved surface.Stopping down the lens reduces the visual effect byincreasing the depth of focus.

Depth of Field-The region in front of and behindthe focused distance within the subject in whichobject points still produce an image of acceptablesharpness.

Depth of Focus-Tolerance in the positioning of theimage plane of a lens within which the lens formsan acceptably sharp image of an object at a givendistance. In focusing, it represents, in effect, a focusinglatitude.

Distortion-Aberration of a lens which causes theimage to appear misshapen and deformed due to agradual increase or decrease in magnification fromthe center to the edge of an image.

Empty Magnification-Magnification achieved by in-creasing the size of the image but not detail. Thisis caused by limited resolving power of the opticalsystem.

f-Number-The ratio of the focal length of a lens toits effective aperture. A measure of the "speed" ofa lens or its ability to gather light.

Interference Microscopy- Technique by which theillumination is divided into two beams, one of whichpasses through the transparent subject matter. Thesecond beam is passed around, rather than through,the specimen. Recombination of the two beams re-sults in interference patterns corresponding to thelocal thickness variations in the specimen.

Kohler Illumination-A method of brightfield illumi-nation used in photomicrography and cinemicro-graphy. A collector lens focuses an image of the lightsource on the condenser, which in turn focuses thefield aperture in the plane of the specimen. Thistechnique provides a uniformly illuminated field fromnonuniform light sources such as coiled filamentlamps.

Nanometer-SI unit of measure for electromagnetic:radiation. Equals one meter times 10-9. Visible lightwavelengths range from 400 to 700nanometers.

75

Numerical Aperture (NA.)-A method of specifyingthe relative aperture of an objective lens and itsresolving power. The numerical aperture value refersto the angle of the cone of light emitted by thecondenser and accepted by the objective of the micro-scope. The formula is N.A. = ND times the sine ofD, where ND is the refractive index of the objectiveand U is one-half the angular cone of illuminationrequired to fill the aperture of the front lens of theobjective. About 1,000 times the numerical apertureindicates the approximate limit of useful magnifica-tion of an objective.

Parfocal- The property of several lenses of differentfocal lengths of coming to focus at the same position.

Phase Contrast Microscopy-A technique for reveal-ing the structural features of microscopic transparentobjects whose varying but invisible differences inthickness result in varying differences in the phaseof transmitted light. These phase differences areconverted to visible intensity differences when partof the transmitted light has its optical path changedby about % wavelength.

Polarizer-A transparent material which absorbsfrom light passing through it all vibrations exceptthose in a single plane.

Polarizing Filter-A filter which transmits light po-larized in one particular plane. Two such filters canbe used to distinguish crystals of different types in

photomicrography. If visually identical crystals differin their birefringence, they will show differences incolor and tone patterns if one polarizing filter is belowand the other (which is capable of being rotated) isabove the specimen.

Refraction-Change in direction of a ray of lightpassing from one transparent medium into anotherof a different optical density, that is, from air intoglass. As the wavelength of the light shortens, theamount of refraction increases.

Refractive Index-The ratio of the speed of light inair to its speed in some other medium. This ratiodetermines how much light rays are bent.

Resolving Power-The ability of a lens to distinguishfine detail in the structure of a specimen. This abilityis assessed by counting the number of closely-spacedobjects or lines that can be recognized as separatein the final image. The formula is resolving powerequals lambda divided by two times the numericalaperture, where lambda equals the wavelength ofgreen light.

Spherical Aberration-A lens defect in which lightrays passing through the outer regions of a lensconverge and cross the lens axis nearer to the lensthan rays passing through the central part of thelens. An object point at the lens axis is recorded asa disc of light.

REFERENCESAllen, R M. 1958.Photomicrography. New York: D.Van Nostrand.Bennett, A. H., Jupnik, H., and Osterberg, H. Phasemicroscopy. New York: John Wiley and Sons, Inc.Chamot and Mason. 1958. Handbook of chemicalmicroscopy. New York: John Wiley and Sons, Inc.Engel, C. E. 1968. Photography for the scientist.London and New York: Academic Press.Feinberg, P. 1968.Photomacrography, procedures andapparatus. Photo Meth. for Ind. 11, No. 2, 51-60.Gander, R 1969. Photomicrographic Techniques.New York and London: Hafner Publishing Co., Inc.Gude, W. D. 1968. Autoradiographic techniques.Englewood Cliffs, New Jersey: Prentice-Hall, Inc.Gumpertz, W. E. 1967. How to get good photomicro-graphs. Laboratory Management, 5, No. 1,28.Hassett, G. F., Hurtgen, T. P. 1972. Photomicrog-raphy in dentistry, Dental Radiography and Pho-tography 45,51-53,59.Klosevych, S. 1964. Photomicrography-resolutionand magnification. J. Bioi. PhotoAssoc., 32, 4.

76

Klosevych, S. 1964. Photomicrography-exposure de-termination, J. Biol. PhotoAssoc., 32, 1.Lawson, D. F. 1960. The technique of photomicrog-raphy. New York: MacMillian Co.Loveland, R P. 1970. Photomicrography, a compre-hensive treatise. New York: John Wiley and Sons,Inc. (2 volumes).Loveland, R P. 1971. "Characteristics and Choice ofPhotographic Materials for Photomicrography," TheMicroscope 19,177-203.Needham, G. H. 1958.The practical use of the micro-scope. Springfield, Illinois: Charles C. Thomas, Pub-lisher.Rogers, A. W. 1969. 2nd ed. Techniques of autoradio-graphy. Amsterdam, London, New York: ElsevierPublishing Co.Spinell, B. M. 1961. Simplified 365 mp, photomicrog-raphy with improved results. J. Bioi. Photo Assoc.29,4.Vetter, J. P. 1973. Practical considerations in colorphotomicrography, American Laboratory 5,4.Wall, L. C. 1966. Day light- type color films in photo-micrography, Medical Radiography and Pho-tography 42, 12-13.

Other Kodak Publications of Interest toReaders of this Data BookEastman Kodak Company publishes more than 800Data Books and technical pamphlets that provideauthoritative, up-to-date technical information aboutKodak products and their applications in many fields.

hese publications are listed in the Index to KodakInformation, Kodak Publication No. L-5, and can beobtained from your photo dealer, or ordered throughthe order form contained in the Index. You canrequest a complimentary copy of the Index by writingto Department 412-L.

Below are some of the publications listed in theIndex that are of particular interest to scientificphotographers:

KODAK Films for the Amateur (AF-l)KODAK Filters for Scientific andTechnical Use (B-3)r""ODAKColor Films (E-77)"OD_-Ur Black-and- White Films for

ionai Use (F -5)Processing Chemicals and Formulas (J-l)

~. __ .<-H- uet and Fluorescence Photography (M-27)nfrared Photography (M-28)

rJ.-ri•.- Infrared Photography (N-l)ph)' of Gross Specimens ( -5)

- Photography, Volume I (N-12A)E+=r=t.:;;~~rzraphy,Volume II (N-12B)

"rared Films (N-17)- - and Films for

.ography (P-315)icrophotography (P-52)- and Filmstrips (8-8)

::::::.:::=:::. -=?t=ph __\ oj J1 etaZs (P -39)