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However, as lens and coatingstechnologies have improved overthe years, visible light microscopyhas slowly found applications insuch diverse disciplines aschemistry, geology, physics,materials sciences, and even thesemiconductor and computerindustries. Most biological microscopesare equipped only with brightfieldand darkfield illumination forexamining pre-stained or livespecimens. To enhance contrastof unstained samples, many of themore modern microscopes are also

APHOTOMICROGRAPHY

PRIMERIt’s worth a closer look

Light microscopy has classically been viewed

as an experimental tool for the biological and

medical sciences. In this respect, the

microscope has proven useful in countless

investigations into the mysteries of life.

equipped with phase contrastoptics. These optical techniquesare generally of little use forimaging specimens in the physicalsciences where alternativemethods such as light plane cross-polarization, differential interfer-ence contrast, and Rheinbergillumination are commonlyemployed. Unfortunately, thesealternative optical illuminationtechniques add considerably tothe initial expense when a micro-scope is purchased. In this article,I discuss how low-cost methodscan be implemented to convert the

biological microscopes found inmost high schools into polarizedlight microscopes useful forinstructional purposes in thephysical sciences. Also, the basicprinciples of photomicrographyare discussed.

MICROSCOPE SETUP Several science supply dis-tributors are an excellent sourceof high quality optical compo-nents at reasonable prices (see thelist in Figure 1). Polarizerssuitable for performing crossed-polarized light microscopy can bepurchased from these dealers forless than $25.00 and adapterswhich couple most cameras to themicroscope are available for$15.00 to $100.00. Expensivehigh-powered microscope objec-tives (lenses) have a very narrowdepth-of-field and are not reallyuseful for a majority of work inthe physical sciences, therefore

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OLYMPUS CORPORATION4 Nevada DriveLake Success, N.Y. 11042Telephone: 516-488-3880

CAROLINA BIOLOGICALSUPPLY COMPANY2700 York RoadBurlington, N.C.Telephone:800-334-5551

E. LEITZ, INC.24 Link DriveRockleigh, N. J. 07647Telephone: 201-767-1100

CARL ZEISS, INC.One Ziess DriveThornwood, N.Y. 10594Telephone: 914-747-1800

NIKON INC. INSTRUMENTGROUP623 Stewart Ave.Garden City, N. Y. 11530Telephone 516-222-0200

AO SCIENTIFICINSTRUMENTSP.O. Box 123Buffalo, N. Y. 14240

FISHER SCIENTIFIC50 Fadem RoadSpringfield, N. J. 07081Telephone: 201-379-1400

EXCEL TECHNOLOGIES90 Phoenix AvenueEnfield, Ct. 06082

EDMUND SCIENTIFIC CO.101 E. Gloucester PikeBarrington, N. J. 08007Telephone: 609-573-6250

McCRONE ACCESSORIESAND COMPONENTS850 Pasquinelli Dr.Westmont, IL 60559Telephone 312-887-7100

VVVR SCIENTIFICP.O. Box 330348Houston, TX 77233Telephone 800-392-3338Telephone800-527-1576

Figure 1. Microscope and accessory manufacturesand distributors

lower cost 5x and 10x objectives,which usually come as standardequipment on most microscopes,are sufficient. Two polarizers will be neededto convert the microscope. Thefirst polarizer is inserted into thelightpath at the base of thesubstage condenser (see Figure 2).This polarizer can be held in placewith tape, or if the microscope hasa built-in light source, the polar-izer can be placed directly overthe field lens. The second polar-izer, commonly termed theanalyzer, is place inside the bodyof the microscope between themain body tube and the eyepiecetube. There is usually a lens

mount at the top of the body tubeand the analyzer can be placeddirectly on this mount. Because ofthe restricted space within themain body tube, the analyzer mustbe limited in size to 1-2 centime-ters in diameter. An analyzer ofthe proper size can be acquired bycutting a piece of polarized sheetplastic or buying a small polarizerfrom a dealer (Figure 1). Afterinstallation of the polarizer andanalyzer, the microscope illumina-tion is turned on and the polarizerat the microscope base is rotateduntil the viewfield becomes verydark (total extinction). At this point, the polarizationdirection is perpendicular between

the polarizer and the analyzer andyou then have what is termedcrossed polarizers. When pur-chasing polarizers, it is importantto select polarizing materials thatare very close to a neutral gray incolor such as the threadedpolarizers made for the front of acamera lens. Avoid polarizingmaterials that are green or amberin color as these will not producetotal extinction. Some microscopemanufacturers offer a low-budgetpolarization kit ($150.00 to$300.00) which is easily installed.It is advisable to contact yourmicroscope’s distributor on theavailability of these items if yourbudget allows. The adjustments describedabove apply only to transmittedlight microscopy where polarizedvisible light passes through thesample. An alternative method ofmicroscopy utilizes reflected lightwhere a beam of light is reflectedoff the surface of the sample to beexamined. To avoid investing inexpensive reflected light attach-ments, oblique illumination froman external light source can besubstituted to achieve a reflectedlight effect. A high-intensity lightsource such as a fiber optics lampprovides an excellent substitute. Attaching a camera to themicroscope is the last step.Microscope viewing heads comein three varieties: monocular (oneeyepiece), binocular (two eye-pieces), and trinocular (twoeyepieces and a photographytube). A camera can be adapted toeach of these viewing heads.Commercial aftermarket cameraadapters usually are attached toone of the viewing tubes with athumbscrew and adjusted to beparfocal with the eyepieces bysliding the adapter up or down onthe viewing tube. A simplecamera back will be sufficient forphotomicrography because thecamera is only required to store,expose, and advance the film. Themicroscope itself acts as thecamera lens.

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the slide to cool slowly beforeexamination, or place the meltedchemical on the microscope stageand examine the crystallizationprocess as it occurs. Certain chemicals recrystallizevery rapidly (within a few min-utes) while others may recrystal-lize slowly over a period of days,weeks, or even months. Urea andbenzoic acid are excellent ex-amples of common laboratorychemicals that will recrystallizerapidly enough to be examineddirectly after melting. Theinstructor and students shouldexperiment with safe, familiarchemicals that are available toidentify those that are optimal formicroscopic analysis. Another very effective methodof preparing crystals is to dissolvethe chemical in a suitable solventsuch as water, ethanol, or mineralspirits. A drop of the solution issandwiched between the micro-scope slide and coverslip and thesolvent slowly allowed to evapo-rate, resulting in formation ofcrystalline patterns. This methodis especially useful for chemicalsin the salt family that usuallydecompose upon heating andleave a tar-like mess. Chemicalscan display a wide spectrum ofpolymorphic crystalline patterns

depending on whether they aremelt-recrystallized or recrystal-lized by solution evaporation. Samples for reflected lightmicroscopy usually require verylittle preparation. Reflected lightmicroscopy can be likened totopographical surface examina-

For photomicrography, it is veryimportant to ensure that yourmicroscope is aligned to produceeven illumination across theviewfield. Information on micro-scope alignment is available in theowners manuals or in textbooksdealing with microscopy.

SAMPLE PREPARATION The Laboratory chemicals foundin high school chemistry stock-rooms provide an excellent sourcefor samples. The chemicals listedin Figure 3 are easily obtained andare known to produce goodcrystals for viewing with a polar-ized light microscope. Mostcrystals are anisotropic andbirefringent, which means that willrefract plane polarized lightemitted from the polarizer and will“bend” it until it is visible throughthe analyzer under crosspolarizedillumination.

To prepare crystals for examina-tion in the microscope, deposit afew milligrams of the appropriatechemical on a glass microscopeslide and carefullyplace a glass cover-slip over the powder.Next, heat the bottomside of the micro-scope slide carefullywith a bunsen burneror hot plate until thepowder has com-pletely melted.(NOTE: Some chemi-cals decompose uponheating and willprovide poor subjects for micro-scopic examination. Othersproduce harmful vapors andshould be avoided.) When molten,the chemical will flow underneaththe coverslip and fill the entirevolume between the coverslip andthe microscope slide. Either allow

CLASSICAL PHOTOGRAPHY

ASSIGNMENTS CAN BE COUPLED

WITH SCIENCE MICROSCOPY

STUDIES TO PROVIDE A

MULTIDISCIPLINARY PROGRAM

IN PHOTOMICROGRAPHY

Figure 2. Schematic illustration of a visible light microscope equippedfor photomicrography.

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tion with a high-power magnify-ing glass, and almost anything canbe examined in microscopic detailwith this technique. For example,the fine details of surface struc-ture can be revealed on leaves,coins, printed paper, insects, and avariety of other specimens. Perhaps the most interestingsubjects for reflected lightexamination are integratedcircuits. These electronic “chips”generally are packaged either bybeing molded into plastic cases orcemented into ceramic cases. It isvirtually impossible to recover anintegrated circuit from a plasticcase because the epoxy resinflows into the microstructure onthe circuit surface and cannot beeasily removed. However, thecement that secures the two halvesof an integrated circuit ceramiccase can be scored with a hacksawand split with a fine chisel toreveal the internal chip. The chipis cemented into a depression onthe bottom section of the ceramiccase and can be viewed directlywithout removal from the case.Leaving this portion of the casingintact also serves to protect thedelicate silicon surface of theintegrated circuit. Most program-mable read-only memory (E-PROM), random access memory(RAM), microprocessors, andmany other digital and analogintegrated circuits are protectedwith ceramic cases. Defectiveintegrated circuits are quite

satisfactory for examinationbecause the defect is usually notapparent on the surface of thecircuit. An excellent source for non-working integrated circuits iscomputer or electronics repairshops. These shops normallystockpile a large quantity ofdefective integrated circuits andwill usually give them away at nocost. Alternatively, many newintegrated circuits are availablefrom dealers for less than $2. Besure to specify that you requireceramic cases when ordering newintegrated circuits. Examination of integratedcircuits with reflected light canserve two purposes. Details of aparticular circuit structure arereadily apparent and, by observ-ing differences in the architectureof various integrated circuits,students can begin to get a handleon the complex electronicsinvolved in modern devices suchas radio, television, and comput-ers. Reflected light microscopyhas become an indispensable toolfor the semiconductor industrydue to its usefulness in character-izing manufacturing defects andmonitoring the successive stagesof integrated fabrication.

PHOTOMICROGAPHY A necessary responsibility ofmicroscopy is to capture theimages seen in the microscopeonto photographic film to obtain

“hard copy” for research records.In a high school environment,classical photography assign-ments can be coupled with sciencemicroscopy studies to provide amultidisciplinary program inphotomicrography. Photomicrography encompassesthe techniques of both black-and-white and color photography.Black-and-white film processingis substantially lower in cost thancolor film, if processing is donein-house. Many commercial filmprocessors no longer offer black-and-white processing services orcharge exorbitant amounts for thisservice. If budget restrictionsforce the exclusive use of black-and-white photomicrography, it isadvisable to invent in darkroomequipment so students candevelop and print their ownphotomicrographs. A green filter should be insertedinto the microscope lightpathbetween the light source and thefirst polarizer (see Figure 1) forblack-and-white photomicrogra-phy. I recommend the use ofKodak™ Technical pan film withHC-110 developer for crispimages with excellent resolution.Printing can be done on KodakPolycontrast™ paper withDektol™ developer. After pro-

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cessing a roll of film, carefully cutthe negatives into sections of 5frames each and store in speciallymade polyethylene storage sheets.You can make contact prints byplacing a sheet of negativesdirectly onto an 8" x 10" piece ofpolycontrast paper and exposingfor a few seconds with the en-larger lens aperture wide open.Contact sheets are an ideal way ofcataloging data and they provide acompact method for storing orsorting through many images.When an enlargement is needed,simply remove the appropriatenegative strip. Color photomicrography isconsiderably more complicatedthan black-and-white photomicro-graphy because color film emul-sions are color balanced for aparticular spectrum of light. Theterm “color temperature” refers tothe wavelength spectrum emittedby a particular light source. Forinstance, films intended to be usedoutside in ordinary daylight orunder fluorescent lighting arebalanced during manufacture for acolor temperature of 5500º Kwhile films made for indoortungsten light bulb use arebalanced for a color temperatureof 3200ºK. The majority of microscopes usea tungsten-halide lightbulb as a

light source that emits a wave-length spectrum centered in the3200º K color temperature region.Therefore, films color balancedthe best results. All major filmmanufacturers haveone or several 3200ºK films available in35-mm transparencyformat. Transparencyfilm is preferable tocolor negative filmfor several reasons:Color negative filmsare balanced for5500º K and must bemanipulated duringprinting to avoid a decidedlyyellowish cast. Mostphotoprocessors cannot or willnot produce satisfactory resultswith photomicrographs on colornegative film. The contrast andcolor saturation in transparencyfilm cannot be equaled by colornegative film. Color transparen-cies are easier to label, store, andcatalog, and they can be projectedat seminars. With a 20-to-50 watt tungsten-halide bulb in your microscope,exposure times are usually veryshort and allow the use of slowfilms such as Ektachrome 50 orFujichrome 64T. Using slow filmsreduces the grain in photomicro-graphs. If tungsten-balanced

films are not available, a Kodak80A or equivalent filter can beinserted into the lightpath betweenthe light source and the firstpolarizer to allow the use of

daylight balanced films withminimal color shift. But, if thisfilter is used, exposure times mustbe increased one to three f-steps toallow for a reduction in lightintensity. When photographing newsamples or after making changes tothe microscope (such as installa-tion of polarizers), the new expo-sure characteristics should bedetermined on a test roll of film.Bracket several exposures of thesame viewfield at least one andpreferably two f-steps over andunder previous exposure times.This will assure at least one orseveral good exposure times. Thiswill assure at least one or severalgood exposures and will yieldexposure time information usefulfor photomicrography of futuresamples. The best film, in my opinion, isFujichrome 64T, a highly color-saturated E-6 transparency filmwith excellent contrast. Recently, anew emulsion of this film wasintroduced that is designed toallow push processing with verylittle reduction in image quality.Push processing is a methoddeveloped to increase contrast(inherently low in photomicro-graphs) and color saturation. Thisis accomplished by underexposingthe film one to two f-steps andincreasing the process time in thefirst developer during the E-6process.

COLOR PHOTOMICROGRAPHY

IS CONSIDERABLY MORE

COMPLICATED THAN

BLACK-AND-WHITE

PHOTOMICROGRAPHY

6

INDEPENDENT STUDENTRESEARCH PROJECTS A variety of specific applica-tions are available to teachers andstudents in the form of individual-ized student research projects.The following paragraphs shouldserve as an introductory guide tothe myriad of projects which arepossible.

1. By examining a number ofdifferent integrated circuit types,the student can become familiarwith details of various electronicdesign motifs. For instance,microprocessor integrated circuitsgenerally contain a ROM andRAM section for internal calcula-tions and storage of information.These memory sections differ

from one circuit to another andalso from the same type of circuitsprovided by different manufactur-ers. Also, transistor size hassteadily decreased as moretransistors are packed onto asingle integrated circuit. Manyolder, non-working integratedcircuits are available from elec-tronics repair facilities, and thesecircuits can be examined fortransistor size versus manufac-turer date. In addition, memorycircuits can be studied to relatemicroscopic features to the actualstorage capacity of the integratedcircuits. These types of investiga-tions should be undertaken bystudents who have an interest inengineering and electronicstechnology.

2. The crystallization patterns of asingle set of biochemicals-vitamins, for instance-can beobserved by assembling a collec-tion of recrystallizedbiochemicals. In a large bio-chemical family, such as thevitamins, many different types oforganic chemical groupings arepresented. This leads to a largespectrum of different crystallinemorphologies. By combiningspecific information about theindividual vitamins with photomi-crographs of the vitamins, thestudent should succeed in produc-ing a interesting and informativephotographic display. Studentsinterested in biology, chemistry,and biochemistry would benefitfrom this experiment.

3. For students who are primarilyinterested in photography, thebeautiful colors provided bypolarized light microscopy canserve to sharpen color photogra-phy skills. Because the micro-scope has a relatively fixed settingwhen compared to standardphotography, students can com-pare the results produced bydifferent films under indenticalconditions. Also, students caneasily see the effect, on photomi-

Figure 3. Common chemicals suitable for recrystallization.

CHEMICAL COMMENTS

Alka-Seltzer Best crystals from aqueous solution.

Ascorbic acid (vitamin C) Can either be melt-recrystallized orrecrystallized from alcohol.

Aspartame (Nutra-sweet) Good crystals from melt-recrystallization or aqueous solution.

Aspirin (acetylsalicylic acid) Equally good crystals from melt-recrystallization or aqueous or ethanolsolutions.

Acetaminophen (Tylenol) Equally good crystals from melt-recrystallization or aqueous solution.

Benzoic acid Best crystals from melt-crystallization.

Biotin (vitamin H) Best crystals from melt-cystallization.

Citric acid Good crystals by evaporation fromaqueous ethanol solutions.

Epsom salts Recrystallize from aqueous solutiononly.

Glucose and sucrose Crystals quickly obtained by evapora(common sugars) tion of solution in water. Verybeauti

ful crystals after melt-crystallization.

Ibuprofen (Advil) Best crystals after evaporation of

rubbing alcohol from ethanol.

Kodak Dektol paper developer Best crystals after evaporation ofworking stock solution.

Kodak D-76 film developer Best crystals after evaporation ofworking stock solution.

Kodak rapid fixer Best crystals after evaporation ofworking stock solution.

Niacin (a B vitamin) Beautiful crystals after melt-crystallization.

Nicotinic acid Good crystals from melt-orevaporation from alcohol solutions.

Tartaric acid Very good crystals from ethanolsolutions.

Urea Crystals form rapidly after melt-recrystallization.

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Michael W. Davidson is a research associate

at the Nat ional High Magnet ic Field

Laboratory and the Supercomputer

Computat ions Research Inst i tute in the

Department of Physics at the Flor ida State

Universi ty, Tal lahassee, Florida 32306.

crographs, of small differences incolor processing variables.

4. The topic of art in science isbecoming increasingly morepopular. Students interested inthis area can benefit by producinga selection of color photomicro-graphs and mounting them fordisplay in local art galleries andlibraries. This project will providestudents with experience inphotomicrography as well as thedetails pertaining to preparingphotographs for display.

By introducing polarized lightmicroscopy and photomicrogra-phy to high school students, yougive them experience with atechnique that is becoming amainstay of modern science andindustry. Students will find thattheir creativity in photomicrogra-phy is limited only by the bound-aries of their own imagination.

NOTE The author would like to thankthe Nikon Instrument Group forproviding photomicrographyequipment and the FSU Center forMaterials Research and Technol-ogy for continued support.

REFERENCESDelly, J.G. 1988. Photography

Through the Microscope. NewYork: Eastman Kodak Companypublication.

Davidson, M.W., and R.L. Rill.1989. Photomicrography: Com-mon ground for science and art.MICROSCOPY and Analysis 4:7-12.

Davidson, M.W. 1991. Fasci-nating photography with a simplelight microscope. PHOTOgraphicMagazine (April): 92.