Why Stain?

Alcian Blue, Alizarin RedChameleon Embryo

1

liz marchiondo

Inthefieldofembryonicresearch,scientistsneedtovisualizethedevelopmentofskeletalstructures.HowisitpossibletoseethedevelopmentofbonesortheformationofcartilagewithouttheuseofX-Rays?Howarescientistsabletotracktherecessionorgrowthofcalciumandcartilagedeposits?Isitpossibletoseeintoanorganismwithouttakingabladetoit?Theanswerliesinachemicalprocessthatallowsresearcherstolookattheorganisminanewlight. UtilizingtheAlcianblueandtheAlizarinredchemicals,thehardtissuesofmammals,rep-tiles,andevencephalopodscanbepigmented.Afterthestainshaveadheredtomarkersinthesolids,calciumandcartilage,trypsinisusedtoclearthesofttissue.Overaperiodoftime,dependingonthedensityofthespecimenstained,thestainsandnaturalpigmentationareslowlyleechedfromthespecimen.Whatisleftisasamplethathasatranslucent,fleshyenvelope.Encasedwithinthisclearcontainmentisthespecimen’spigmentedinternalstructure.

Photo Setup

2

Colorsrangefromdeepblue-violettobrightreds.Thesepigmentsdependonwhathasbeenstainedanditsdensity.Athinpieceofcartilagewillbeavividblue,whilethickerpieceswillappearasadarkerblue.Externalcharac-teristicscanalsobehighlightedwiththistechnique.Scalesandbarbsonaquaticorganismsaredistinguishedeasierwhensentthroughthestainingprocess. Creatingthestainedsamplesisonethingbuthowisaresearchersupposedtosharetheirfindings?Wordscanonlygosofar.Aresearchercanpublishallthewordstheypleasebutsubstance,impact,andthat“wow”factorarelackingwithouttheproperuseofimages.Sincethespecimenswereoriginallyembryos,amacroor“close-up”photographicconfigurationisbeneficialtocapturesmalldetails.Theinherentcharacteristicsofthesespecimensalsomakethemaninterestingpracticeinlighting.Withatranslucentexteriorand,sometimes,extremelydensestructuresaphotographermayhavetomakeadecisiontoeitherexposeforthesofttissueorforthestains. Becausethespecimensare,bydesign,moisttheyneedtobestoredinglyceroltopreventdryingout.Thismakesphotographingthechameleonachallenge–withitscontoursandmoisture,highlightsandspecularhighlightsarediffi-culttoavoid.

Equipment:NikonD300,Nikkor60mmmacrolens,NikonBellows,Zeiss16mmmacrolens,USBcable,lighttable,heavydutycopystand,Maccomputer,NikonCameraControlPro2,AdobeBridgeCS5.1,AdobePhotoshopCS5.1,ZereneStacker. ANikonD300wasattachedtoacopystandandtetheredtoaMaccomputerviaaUSBcord.NikonCameraControlPro2andAdobeBridgeCS5.1wereusedtocaptureandviewtheimagesinrealtime.Alighttablewasusedasthemainlighttobacklightthechameleon.Adheringtofundamentalphotographictechniqueswillpreventlonghoursofeditingpostprocessing.Beforephotographing,takethesesimplestepsthatwilltakeonlyafewmomentsandwillsaveyouhoursofscreen-staringlater.1.)Keepyouworkareacleanandfreeofdustandparticulateintheair. a.Thatmaymeaneitherreplacingtheglycerolthespecimenisin,buildingadusttentoverthephotographicsetupordoingacombinationofthetwo.2.)Makesureyourcamerasensorisfreeofdust.Ifneedbegetitcleanedbyprofessionalsbeforeyoushoot. a.Besuretokeepthecamerafacingdownwhenswitchingoutlensesandbellows.3.)Keepvigilantofanysortofdebristhatmayfallintothesetupwhileyou’reshooting.

How to Capture an Image with Extended Focus (Z-Stacking)

3

Withathickspecimenathighmagnifications,thereisonlygoingtobeathinksliceofthespecimeninfocus.Howisitpossibletogetaspecimenentirelyinfocus?Z-Stackinginvolvesphotographingmultipleframesofasinglecompositionandloadingthoseimagesintodedicatedstackingsoftware.Theimageseachhaveaparticularplaneinfocus,withtheprecedingandfollowingimagesinthesequencehavingincremen-taldifferencesinthelocationoffocus.Thesoftwareessentiallyfindsthegreatestcontrastbetweenpixelsandseesthatas“sharpness.”Theseslicesofsharpnessarecompositedtogethertocreateafocusedimage.

A B CA Single capture, point of focus is just below the tip of the tail.B Single capture, point of focus is midway through width of tail.C Single capture, point of focus is midway through the forehead.

The chameleon image on the front page is a stack of 32 images.

A B C

A Single capture, point of focus is just at the base of the image.B Single capture, point of focus is midway through head crest.C Single capture, point of focus is at the tip of the head.

This final image was composited from 13 individual images.

The light table could not be easily maneu-vered in small increments so the only alternative I couldthink of was to move the camera via the copy stand. AsIshot,Ihadthefilesgotothecomputer’s“picture”folder.Eachcompositionwaslabeled,(ex:forthefullbodystack, “Chameleon_stack_01_001, …_002, 003, etc.).ThatwayIdidnothavetodistinguishone“IMG_####”fromanother“IMG_####”whenloadingthefilesintothestackingprogram.Foreachcompisition,IphotographedtheseriesandwentbacktocheckthatIhadthenecessaryamountofimagesandoverlappingplanesoffocus.Itisannoying and time consuming tophotograph for hoursonlytofindthatinthestackingsoftware,thereisagapinfocus.Thisgapusuallyhappenswhentherewereframesthatshouldhavebeenphotographedbutthecamerawasmovedtoomuchwithoutphotographinginbetween. IkeptthecamerasettingsatISO200andhadshutterspeedanywherefrom1/2secondto1/160thseconddependingonthemagnificationandwhatparticularfeatureofthechameleonIwasfocus-ingon.ThegreatthingabouttheNikonD300,andnowmanycameras thatareavailable, isitsLiveViewmode.Icansimplyswitchthecameratothatmode,clickthe“LV”buttonintheNikonCapturesoftwareandIhaveanim-agethatwentfrombeingtwoinchesontheLCDtooverfourinchesonthecomputer

screen.ThebestthingIcansuggesttowork slowly and thoroughly withyourimaging.Keepaneyedropperofglycerolonhandtorehydrateyour specimen ever so often.Thismaybescientificim-aging but don’t forget tohave fun with the speci-men. Don’t just thinkand wonder what thespecimen may looklike at “this” or “that”angle, photograph it!

Photographing

File Format

Image Optimization

Itispartofmyworkflowtophotographinthecamera’sRAWfile.Ifyourcameraiscapable,IhighlysuggestconvertingittoworkwithRAWfilesforoptimizationinpostprocessing.TheRAWfileswereloadedintoAdobePhotoshopCS5.1whereglobalbatcheditsweremade.Isavedthoseimagesinanewfolderas.tifffiles.Please,ifyouhaveeverneverusedastackingprogrambefore,donotuseRAWfiles,eveniftheprogramacceptsthem!Itwillslowdownandbogdownthesoftwareandyourcomputer.Afterusingmultiplestack-ingprogramsinthepastforvariousprojects,IchoseZereneStackerforthechameleon.InZerene,IutilizedtheDMapstackingoption;ithasathresholdthatallowstheusertochoosewhatareasoftheimageto“ignore”whenstacking. Whenmakingeditsorchangestomyimages,flatteninglayers,makingmajorchangestowhitebalance,etc.Ialways“SaveAs.”Keepthemaster(RAW)filessavedandworkoffofthembutdon’tmakeanydamagingchangestothem.Workinlayersandusenon-destructivemodesofimageediting.Insertmetadatatotheimagessothatotherscanfindyouthroughyourwork(File-FileInfoinPhotoshop).Backupyourimagesindifferentandmultiplelocations;investinaharddrive,aportableharddrive,anonline“safe”orsomecombinationofthesemethods.

BeforeIstacktheimages,IusePhotoshoptofine-tunethewhitebalance.Iftheimagesneedtobestacked,IgothroughthestepsinZereneStackerandexportthefinalimageasa.tifffile.Fromthere,IworkmoreinPho-toshop.Icroptheimage,sometimesZerenecreatesimageartifactsincludingelongatingportionsoftheimagetofittheframe.NomatterhowvigilantIamwhenIphotograph,thereisalwaysdustortinyhairs/fibersonthespecimen.Ijustcan’tavoidit!ThisiswheretheSpotHealingtoolbecomesanasset.Smallparticlesthathavefallenonoraroundthespecimencanberemovedwithoutdestroyingtheintegrityoftheimage.Finalstepsforthechameleonincludedesaturatingtheoverallimagejustabit.Thisallowsforbetterprintsand,sometimes,thereare

detailsthatcan’tbeseeneasilywhencolorsaresupersaturated.The lastedit Imade to the imagewascopying thefinal,

flattened layerandapplyaHighPassfilter (Filter–Other-HighPass,between4.0-5.5)andputthat

top layer into “Overlay”mode.The im-agewasthenflattenedandFileInfo

wasaddedbeforeSavingAs.

6

I’mLizMarchiondo,fourthyearforBiomedicalPhotographicCommunicationsattheRochesterInstituteofTechnology.AnticipatedgraduationdateisMay2012withaBachelorofScienceDegreeandaMinorinArchaeology.Inthesummerof2010,Icompletedaco-opattheHarborBranchOceanographicInstituteandin2011Icompletedaco-opwithCarlZeissMicroImaging,Inc.

Pleasefeelfreetocontactmewithanycomments,questionsorconcernsat: eam9974@rit.edu

Toviewmoreofmywork,feelfreetovisit: lizmarchiondo.smugmug.com

Thisprojectwouldnothavebeenpossiblewithout: TheRochesterInstituteofTechnology SchoolofPhotographicArtsandSciences TheBiomedicalPhotographyLaboratory Dr.AndrewGillis TeresaDeForge