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PHOTOMICROGRAPHY:COMMON GROUND FOR

SCIENCE AND ART

Michael W. Davidson and Randolph L. RillDepartment of Chemistry

and Institute of Molecular BiophysicsThe Florida State University

Ta l l a h a s s e e , F l o r i d a 3 2 3 0 6 U S A

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Figure 1. Title: none. Description: A single exposure of molecular weight calf thymus DNA in a slightlyhydrated condition. Awards: Honorable mention in the 1987 Nikon Small World Competition. Exposure 1: A900 milligram/millitre mixture of DNA and saline buffer was sandwiched between a microscope slide andcoverglass and sealed with a polymethylmethacrylate mounting medium. The sample was observed with a firstorder retardation plate (530 nm) inserted between the sample and the analyzer. Exposure was colour correctedwith a 20 CC magenta filter. The 10x objective was utilized with polarized light for the 0.17 second exposure.

Figure 2. Title: DNA Liquid Crystals. Description: A single exposure of Smectic-like liquid crystalline DNA.Awards: Photographs of the type have been published in numerous periodicals including Nature, the BritishJournal of Photography, Second Opinion, The Bethesda Research Laboratories catalog, Microscopy andAnalysis, and Functional Photography. Exposure 1: A 350 milligram/millilitre solution of nucleosome corelength (approximately 500 Angstroms) DNA in a buffered ammonium acetate (0.3M Ammonium acetate, 0.01MSodium Cacodylate, and 0.01M EDTA adjusted to pH 6.5 by addition of solid Cacodylic Acid) spontaneouslyforms smectic-like liquid crystals. The sample was sandwiched between a coverslip and a microscope slide andsealed with a polymethylmethacrylate mounting medium before examination. The exposure was taken with a10x objective for 0.12 seconds with a 10 CC magenta colour correction.

Figure 3. Title: Nebraska. Description: A multiple (4) exposure of ascorbic acid (the wheat in the foreground),stretched polyethylene (the morning sky), polybenzyl-1-glutamate spherulites (the stars), and the field dia-phragm defocused (the sun).

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Figure 4. Title: Neptune Beach, Florida. Description: A multiple (5) exposure of the antibiotic,chloramphenicol (the grassy foreground), stretched polyethylene with a blue filter (the morning sky andocean), the field diaphragm (the morning sun and its reflection), and polybenzyl-1-glutamate (the stars).

Figure 5. Title: Ascorbic Acid, Idaho. Description: A multiple (5) exposure of ascorbic acid (the wheatin the foreground), Xanthan gum base (the snow-covered mountains), stretched polythylene (themorning sky), polybenzl-1-glutamate spherulites (the stars), and the field diaphragm image defocused(the sun).

Figure 6. Title: Nuclear Sunrise. Description: A multiple exposure (4) of ascorbic acid (the desertforeground), stretched polyethylene (the morning sky), polybenzyl-1-glutamate (the stars), and the fielddiaphragm defocused (the rising sun).

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various textures formed by DNA as itentered the liquid crystalline state(illustrated in Figure 11-4). To thisend, we have used both light andelectron microscopy; however, thisarticle will be dedicated to our colourphotomicrography with a polarisinglight microscope.

In December 1986, we firstobserved a highly birefringent,beautifully coloured, fan-texturedsmectic (2-dimensionally ordered)liquid crystalline state of DNA(Figure 24) which we attempted tophotograph using standard E6transparency films processed bycommercial laboratories. The resultsthat we obtained were disappointingbecause the contrast and coloursaturation of our transparencies wereof an inferior quality to the speci-mens that we observed first-hand inthe microscope. To correct this, weinitiated a series of tests usingvarious daylight and tungsten-balanced films which we began

The escalating technology inoptics and optical coatings, coupledto the development of high qualitymicroscopes and deeply colour-saturated photographic films has, inpart, led to an explosion in theutilisation of photomicrography fornumerous fields. Many years ago, themicroscope was an exclusive tool ofbiologists who spent countless hours

observing and drawing variousspecimens of biological interest.Today, however, the microscope hasfound a home in disciplines asdiverse as Chemistry, Physics,Geology, Psychology and MaterialsScience, to name a few.

Our interest in photomicrographyoriginated from a need to view, atrelatively high magnification, the

Figure 9 Figure 10

Figure 7 Figure 8

Figure 7. Title: Sulphur Canyon. Description: A multiple (4) exposure ofrecrystallized sulphur (the canyon), polybenzyl-1-glutamate (the stars), and thefield diaphragm defocused (the moon) with a blue filter (the sky).

Figure 8. Title: Sunday. Description: A multiple (3) exposure of the antibioticchloramphenicol (the grassy foreground), hardened epoxy resin (the purplish sky),and the field diaphragm defocused with a red filter (the rising sun).

Figure 9. Title: Cety Alpha 5. Description: A multiple (4) exposure of smecticliquid crystalline DNA (the foreground), polybenzyl-1-glutamate spherulites(stars), and the field diaphragm defocused with a blue filter.

Figure 10. Title: Tornadoland. Description: A multiple (6) exposure of ascorbicacid (Vitamin C-the desert foreground), cholesteric liquid crystalline DNA (thetornado), and Cibachrome bleach crystals (the clouds and dust) with a blue sky.

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Figure 11. Title: Westworld. Description: A multiple (2) exposure of epoxy resin (the clouds and the mountains)and the field diaphragm defocused (the red sun).

Figure 12. Title: The Stand. Description: A multiple (4) exposure of ascorbic acid (the wheat in the foreground),polybenzyl-1-glutamate (the stars), the field diaphragm defocused with a mask (the new moon), with a blue sky.

Figure 12

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processing in-house with commerciallyavailable Kodak E6 kits. After manymonths of tests and comparisons, wefinally settled on Fujichrome 64 T andhave been using this film almost exclu-sively, with the exception of PolachromeHC 35 mm instant colour transparencyfilm, which we employ for the annualPolaroid International Instant Photomi-crograph Competition.

Our entry into the photomicrographcompetition circuit started with the 1987Nikon Small World contest in which wereceived 6th and 9th prizes and twohonorable mentions for photomicro-graphs of liquid crystalline DNAsamples. The Nikon contest heralded anew era for our photomicrographybecause one of the winning micrographswas a multiple exposure which weintended to resemble an alien landscape.Photomicrographs of this type have nowwon many competitions5 and we haveextended our emphasis to make thesephotographs more life-like. We nowterm these types of photomicrographs,Microscapes. Several examples of ourmost recently fabricated Microscapesare illustrated in Figures 3-12.

FABRICATION OF MICROSCAPESNikon’s UFX-II digitally controlledexposure monitor allows for a double ormultiple exposure mode which isemployed in the construction of multipleexposure Microscapes. Our microscopeis a Nikon Optiphot-pol in which lightintensity and distribution are regulatedby the field diaphragm (a leaf-typeshutter) and emitted through a lens in thebase of the microscope. By carefullymasking a portion of the field diaphragmlens, selective areas of film can beexposed to light captured through themicroscope.

The sun or moon is added by closingthe field diaphragm almost completelyand defocusing the image until theindividual leaves merge to form acomplete circle. Next, an orange or redfilter is inserted into the light pathwayand the substage condenser isdecentered, by realignment of theadjustable centering pins, to move thediaphragm image to the appropriatelocation and an exposure is recorded.Long exposures yield a bright, white

Figure 13. Title: Vitamin C-horse. Description: A single exposure ofascorbic acid recrystallized from the melt. Exposure 1 (see Figure 16).

Figure 14. Title: The Many Faces of Vitamin C. Description: A singleexposure of ascorbic acid recrystallized from the melt. Awards: Honorablemention in the 1988 Nikon Small World Competition. Exposure 1(See Figure 16).

Figure 15. Title: Man in the Moon-Greencheese. Description: A singleexposure of ascorbic acid recrystallized from the melt. Exposure 1(See Figure 16).

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Figure 16. Title: The Ghost. Description: A singleexposure of ascorbic acid (Vitamin C) recrystal-lized from the melt. Exposure 1: A 20 milligramsample of ascorbic acid was sandwiched between amicroscope slide and a cover slip and heated untilmelted (with some decomposition) in a bunsenburner. Upon crystallization (1 to 9 weeks), theimage was photographed with a 10x objective witha 20 CC magenta colour correction filter for aperiod of 0.14 seconds.

Figure 17. Title: Swirlaway. Description: A singleexposure of Niacin (a member of the B complex)recrystallized from the melt. Exposure 1: A 20milligram sample of niacin was sandwichedbetween a microscope slide and a coverglass andwas heated in a bunsen burner until melted. Uponrecrystallization, the sample was photographedunder polarized light with the 10x objective and a20 CC magenta colour correction filter for a periodof 0.10 seconds.

Figure 18. Title: Welcome to the Jungle. Descrip-tion: A single exposure of ascorbic acid (VitaminC) recrystallized from the melt. Exposure 1 (seeFigure 17).

Figure 19. Title: Sunflowers. Description: A singleexposure of ascorbic acid (Vitamin C) recrystal-lized from the melt. Exposure 1: A 40 milligramsample of ascorbic acid was sandwiched between acoverslip and a microscope slid and heated untilmelted with a bunsen burner. After recrystalliza-tion the sample was photographed with the 4xobjective after a 530 nm retardation plate had beeninserted between the sample and the analyser.Colour correction was 20 CC magenta and theexposure time was 0.07 seconds.

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centered sun or moon, while shortexposures give more colour-saturatedimages with a yellow-to-red transi-tion from centre to edges. The newmoon illustrated in Figure 12 iscreated by placing the tip of a ball-point pen in the light path afterclosing down the field diaphragm anddefocusing.

The mountains are maskedexposures of unrefined Xanthan-gumdissolved in aqueous solution andallowed to concentrate while sand-wiched between a microscope slideand a cover slip. This polysaccharidetends to undergo a transition tocholesteric liquid crystalline states, inconcentrated solutions, whichresemble mountainous formations.

The morning sky is created bycutting a rectangular portion of apolythylene film (plastic storage bag,thickness~100 micrometers) andstretching it in a uniform mannerbefore pressing onto a microscopeslide. Upon stretching, the polyethyl-ene molecules align to a formbirefringent diffraction gradientwhich casts a yellow-to-red-to-bluevisible light spectrum to simulate amorning sky. After masking previousexposures, the film is exposed to amasked section of the diffractionspectrum in order to obtain the effectdesired.

Stars are created by imaging smallcholesteric liquid crystalline spheru-lites of polybenzyl-1-glutamatedissolved in dimethyl formamide.Previously exposed areas of the filmare masked for these exposures inorder to eliminate burning of thespherulite image.

The clouds present in Figure 9 aregenerated by defocusing colourlessbirefringent crystals of Cibachromebleach.

Various foregrounds are obtainedby photographing crystalline forma-tions of a wide spectrum of drugs,vitamins, and other assortedbiochemicals. For instance, theforegrounds in Figures 3 and 4 areexposures of ascorbic acid (VitaminC) and chloramphenicol (an antibi-otic) respectively, recrystallized aftersandwiching the pure biochemical

between a microscope coverslip andslide and heating until melted. Insome instances, the chemicalsrecrystallize rapidly within a fewminutes. However, many take weeksor even months to completelyrecrystallize. The composition of theforeground in each photomicrographis discussed in the respective figurelegends.

Generally, after the selectedforeground is exposed, the exposedarea is carefully masked by placing ablack card over a large enough areaof the field lens to completely stopany additional light from reachingthe exposed portion of the film.Next, a second exposure portion ofthe film, Next, a second exposure ismade usually either with Xanthan

Figure 20. Title: Hot Water. Description: A single exposure ofThiamine (Vitamin B1) partially recrystallized from the melt. A20 milligram sample of Thiamine was sandwiched between amicroscope slide and a coverglass and was heated in a bunsenburner until melted. Upon recrystallization, the sample wasphotographed under polarised light with the 4x objective and a 20CC magenta colour correction filter for a period of 0.16 seconds.

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gum mountain exposure, stretchedpolyethylene, or through a blue filterin the brightfield mode (See Figures 3-12). In some instances, where a blacksky is desired, no filter is used (seeFig. 6 which illustrates a photomicro-graph entitled Liquid Crystal Land).The field diaphragm image is the nextexposure with either a yellow or a redfilter inserted into the light pathway.After this exposure is taken, thedecentered condenser is left in placeand the field diaphragm is openedfully. Next, a microscope slide with asolution of polybenzyl-1-glutamatespherulites is brought into focus, anarea devoid of spherulites is placedover the previously exposed fielddiaphragm image, and an exposure istaken in the polarising mode. Thisinsures that stars do not becomeimaged in the center of the sun ormoon. Careful masking of previouslyexposed areas (mainly the mountainsand foreground) is essential, manytimes with selectively cut masks, as theexceedingly long exposure timesnecessary to image the spherulites willcreate a bleached area (burned) in themountains or the foreground.

In Figure 4, the ocean was createdby placing a blue filter with a selec-tive mask in the light path (at the fieldlens) and carefully defocusing themicroscope until an ocean skylineeffect was obtained. The fielddiaphragm image was then moved tocoincide with the skyline and re-corded with a partial mask to resemblea rising sun. The field diaphragmimage was subsequently moved to aslightly lower portion of the film and,with careful attention to alignmentwith the first field diaphragm expo-sure, exposed after placing a diffrac-tion grating in the light path betweenthe substage condenser and theobjective. The diffraction grating wasconstructed using Polachrome HC 35mm instant colour transparency filmexposed to an extremely bright lightto generate a very fine series of lines.This yields a shift in the wavelengthsrecorded on film to longer (more red)wavelengths and spreads out theimage to resemble a reflection of thesun on the water.

THE MANY FACES OFUBIQUITOUS VITAMIN COur various photomicrograph portfo-lios are each constructed around acentral theme as described for theMicroscapes collection in detailabove. Additional portfolios include avitamin collection, an antibioticcollection, as well as selected collec-tions of other chemicals andbiochemicals.

However, probably the mostunusual and visually exciting collec-tion is our vitamin collection (Figures13-20 are selections from this group).A sub-portfolio from the vitamincollection has been named The ManyFaces of Vitamin C due to the fact thatmany of the wide spectrum ofcrystalline morphologies displayed byrecrystallized ascorbic acid resemblefaces in one respect or another.Figure 16 is an example of an unusualmorphology which possesses ahaunting appearance. The patternillustrated in Figure 13 was titledVitamin C-Horse by StevenRosenbaum of Modern Photographydue to its striking resemblance to asea horse. Likewise, surrealistic facescan be found in Figures 14 and 15.Figure 15, a photomicrograph of anisolated crystallite of ascorbic acidhas been titled The Man in the Moon-Greencheese due to its uncannyresemblance to the fictional Man inthe Moon on one side of the innercircle and to green Swiss cheese onthe other side. Currently, we haveabout 40 members in the Faces ofVitamin C portfolio and constantlysearch out additional morphologicalformations from recrystallizedascorbic acid samples.

The needle-like morphologydemonstrated in many recrystallizedsamples of ascorbic acid can befurther enhanced in appearance by theaddition of a 530 nanometer retarda-tion plate between the sample and theanalysing polariser as is illustrated bythe photomicrographs in Figure 17and 18. By scanning microscopeslides and photographing formationsin this manner, we have succeeded ingenerating a large collection of thisunusual type of artwork.

Additional members of TheVitamin Collection are shown inFigures 19 and 20. Niacin, a watersoluble member of the B family,when recrystallized from the melt,yields a highly unusual, but beautiful,crystalline morphology as is depictedin Figure 19. Figure 20 is a photomi-crograph of Thiamine (Vitamin B1,)partially recrystallized from the melt,imaged with a 530 nanometerretardation plate inserted. It stronglyresembles a boiling solution withchips at the bottom. These are only asmall sampling from The VitaminCollection intended to illustrate theinherent beauty to be found inpolarized light photomicrographs ofrecrystallized vitamins.

CONCLUSIONBy coupling a well-developedphotographic methodology to acreative imagination, there is virtu-ally no limit to the types of photomi-crographic artwork possible with thelight microscope. Here we havedemonstrated a few examples of thewide spectrum of possibilitiesavailable to the microscopist who iswilling to take the time and effortnecessary to compose micro-art.

EXPERIMENTAL METHODSAll photomicrographs were com-posed using a Nikon Optiphot-polpolarizing light microscope in eitherthe brightfield or polarizing mode. AUFX-II digitally controlled exposuremonitor measures light intensitythrough a 30 per cent central portionof the viewfield by employing aphotomultiplier and calculatesexposure time based on this reading.Illumination is provided by a tung-sten-halide 12 volt bulb operating at8.5-9 volts. Images were recorded onFujichrome 64 T, a 3200 Kelvincolour-balanced (tungsten) transpar-ency film operating at approximatelyISO 64. Exposures were usuallymade at 1 to 3 f-stops below therecommended exposure of the UFX-IIsystem and Kodak E-6 processing(done in-house) was extended 25-50per cent in the first developer. Slightmodifications were made to the

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chemical composition of the firstcolour developers to enhance contrastand colour saturation.

ACKNOWLEDGEMENTSThe authors wish to thank KayeMerchant for her enthusiasm andinspiration during the early days ofthis study. In addition, we would liketo acknowledge the assistance of TomFellers (FSU Department of Physics)for advice concerning photographicmethods and David Van Winkle (FSUDepartment of Physics) for advice onliquid crystal behavior. This workwas supported, in part, by the NIH.

REFERENCES1. Rill, R.L., Hilliard, P.R., and Levy,G. C. (1983): Spontaneous Orderingof DNA. Effects of intermolecularinteractions on DNA motionalDynamics monitored by 13 C and 31 PNuclear Magnetic Resonancespectroscopy. J. Biol. Chem. 258,250-256.

2. Rill, R.L. (1986): Liquid crystal-line phases in concentrated aqueoussolutions of NA+ DNA. Proc. Natl.Acad. Sci. USA, 83, 342-346.

3. Strzelecka, T.E. and Rill, R. L.(1987): Solid-State 31 P NMR Studiesof DNA Liquid Crystalline Phases.The Isotropic to Cholesteric Transi-tion. J. Am. Chem. Soc. 109, 4513-4518.

4. Strzelecka, T. E., Davidson, M.W.,and Rill, R.L. (1988): Multiple liquidcrystalline phases of DNA at highconcentrations. Nature 331, 457-460.

5. The authors collectively have wonprizes in the following competitions:Nikon Small World Contest, 1987,held by Nikon Inc. Instrument Group,623 Stewart Avenue, Garden City,New York 11530. Prizes: 6th, 9th, 2honorable mentions.Photographic Magazine MonthlyContest, 1987, Held by Peterson’sPhotographic Magazine, 8490 SunsetBoulevard, Los Angeles, California90069. Prizes: May, 1987: Honorablemention, July, 1987: Honorablemention. Polaroid International InstantPhotomicrography Competition,1987, held by the Polaroid Corpora-tion, 575 Technology Square, 9PCambridge, Massachusetts 02139.Prize: 3rd place. Olympus Visionage InternationalPhotography Contest, 1988, held bythe Olympus Optical Co., Ltd., San-EiBldg.,22-2 Nishi-Shinjuku, 1-Chome,Shinjuku-Ku, Tokyo 163-19, Japan.Prize: Merit Award. American Society of ClinicalPathology 1988 Medical Photogra-phy Competition Sponsored byNikon, Inc., held by the AmericanSociety of Clinical Pathologists, 2100West Harrison Street, Chicago, Illinois60612. Prize: 1st in the MicroDivision. Nikon Small World Contest, 1988,held by Nikon Inc. Instrument Group,623 Stewart Avenue, Garden City,New York 11530. Prizes: 7th andHonorable Mention.Polaroid International InstantPhotomicrography Competition,1988, held by the Polaroid Corpora-tion, 575 Technology Square, 9PCambridge, Massachusetts 02139.Prizes: 1st and Honorable Mention incolour transparency category, andoverall best of competition grandprize.

6. Microscopy and Analysis, Novem-ber 1988, Issue 8. Note: Additionalinformation relating to the micros-copy of liquid crystals can beobtained from: The Microscopy ofLiquid Crystals by Norman H.Hartshorne. Published by TheMcCrone Research Institute Inc.,2820 S. Michigan Ave., Chicago,Illinois 60616 USA (Telephone 302-842-7105).

Michael W. Davidson is aresearch associate in theInstitute of Molecular Biophys-ics at The Florida StateUniversity. His main researchinterests are: photomicroscopy,liquid crystalline DNA, thepackaging of DNA into viruscapsids, synthetic heterocyclicchemistry, and the interactionof small molecules with DNA.

Randolph L. Rill is a Profes-sor of Chemistry and holds ajoint appointment with theInstitute of Molecular Bio-physics at The Florida StateUniversity. His main researchinterests (in addition ofphotomicroscopy) are: liquidcrystalline DNA, and thepackaging of DNA in nucleo-somes and chromosomes.