How to improve photography through the microscope
Transcription
How to improve photography through the microscope
Howto lmprovePhotography Through theMicroscope ,-:.;:. a G ",.1,;..' O ru K'..,. ^ a o Howto get the mostout of this booklet Thisbooklells divdedintofourman sections. ltemsmarkedwitha a I st the minmuminformationthat newcomers to the art of photomicrography needto know,whie llemsleft unmarked are addressed to userswhohavealreadytakenphotoswiththe mlcroscope butare notsatisfied withthe results.By relerringto sections1 to 3 for picturetakingand section4 for checking, youwill findthis bfochurea reliablehelpthe nexttimeyouwantto takea photomicrograph. FrompEparationto observation lvlicroscopes suitabletor photomicrography . . . . . . . . . . . .8 Equipment suitabletor photomicrography. . .. . . . . . . . . . .9 Varioustypesot photomicrographic equipment a n dp e r f o r m a n c e ...........10 aEquipmentneededfor photomicrography .. . . . . . . . . . . . 11 Suitablelocalionsfor settingup thiemicroscope .......12 aObjectivesand photoeyepiecessuitablefor photomicroOraphy ..........13 aDifferencesin the peripheralimagesdependingon o b i e c t i vdee s i o n .. . ... . .14 aDiffeiencesin re-solution dependingon lne lype of objective . . . . . . . . . . . . . . . . 15 asome hintsaboutthe objective. . . . .. . . . . . . . . . . . . 16- 18 Selection of a condenser. .. . . . . . . . .. .. . . . . . . . . . . . . . 19 a o b s e r v a t iporno c e d u r. e. .s. . . . . . . . . . . . . . . . . . . . . m - 2 1 a H a n d l ionf g s p e c i m e. .n. s. . . . . . . . . . . . . .. . . . . . . . . .. 2 2 Useof the fieldirisdiaphragm . . . . . . . . . . . . . . . . . . . . . .23 lJseof the apertureiris diaphragmand its effects . .24 - 25 aBasic focusingmethods . . . . . . . . . . . . . . . .. . . . . . . .6 - 27 How to spot dirt and specksof dust in the optical systemsof the microscopeand the Ohotomicrographa i cl t a c h m e n t .........28 aHow to cleanthe microscopeframe . . . . . . . . . . . . . . . . . 29 a H o wt o c l e a n t h eo p l i c aslv s t e m . . . . . . . . . . . . . . . . . 3 0 3 -l a H o w t o c l e a na n o i i . i m m e r s ioobnj e c t i v e..... . . . . . . . . . 3 2 aHow to cleanspecimens. . . . . . . . . . . . . . . . . . . . . . . . . . .33 Sectionl FrompEpaaationto obseryation Sectlon2 Whenlakingpictur€s in photomicrography SectionS Howto obtain good pdnls In the caseof poorphotos How to obtain good prints How to obtaingood color prints . . . . . . . . . . . . . . . . .60- 61 p nts . . . . . . . . . . . . . . . . . . . . .62 aPreparingblack-and.white N l a r kosn t h ep h o t o ...........63 H o wl o a v o i dm a r k sd u r i n g d e v e l o p m e. n . .t. . . . . . . 6 4 - 6 5 $ection4 Trcuble€hooting When taking picturesin photomicrography tr:oJclion ol eouiDmenisuilable lor GE-Jal on andphoiomicrcgraphy, basic lti.€dge, observaiion procedures, cE rc methods,etc. are explainedin .esylsundeGlandmanner. t"*'** l*, l-r"= lt<r, and exprafanon or p'oceduresneededfof pholG drfferences in filmsandpurchase can ruin lhe best +ct processing ' This seciiondealswilh film de nl, howlo ordercolorPrlnls, elc. Basicinlomalion in photomicrography Operatinginstructionsfor modelsP[,4-1oAD and P[,4IoADS(with35mmcameraback) . . . . . . . . . . . 36- 37 M a g n i f i c a toi fopnh o t o g r a pehqi cu i p m e.n. .i . . . . . . . . . . 3 8 Differencesin resolutionaccordingto the combinaiions o f o b i e c t i va en sd e y e p i e .c.e. .s. . . . . . . . . . . . . . . . . . 3 8 Settingot photographicmagnification(effective m a g n i f i c a .t .i .o.n. .) . ..............39 F r a m i on fgt h es p e c i m .e. n. . . . . . . . . .- . . . . . . . . . . . . 3 9 C h e c k i nogf t h ef i n i s h epdh o t o m i c r o g r a p h s . . . . . . . . 4 0 - 4 1 Pholomicrography techniques E x p o s uar d e j u s t m el en ct h n i q u e. .s. . . . . . . . . . . . . . 4 2 - 4 3 ..........44-45 U s eo f t h eA El o c k C o m p e n s a t ifnogr a f i l m ' sr e c i p r o c if tayi l u r e . . . . . . . . . . . 4 6 color photomicrography ...........41 O C o l of ri l m O T v D eosf f i l t e r s .............48 6 Differences in colorrendition depending on the of tilm and lightsource. . . . . . . . . . . . . . . 49 combination E Differences in colorreproduclion depending on differencesin color temperature. . . . . . . . . . . . . . . . 50 on lhe lype a Diflerences in colorrendition depending ........5'1 o ff i l m. . . . . . . . . . . . . ........52 a P u r c h aos fec o l ofri l m. . . . . . . . . . (cc) filterswhentaking Useof color-compensating .....-.........53 c o l opr h o l o s .............54 a T e sp l hotography phologEphy Black-and.white 55 a Black'and-while tilrn . 56 aComparisonof ditterentfilm brands . . . . . . . . . . . . . . . . .56 57 4 F i | t e .r. s. . . . . . . . . . . . . . . . ........58 P h o t o g r a pwh iyt hP o l a r o if di l m . . . Trouble-shooting ranging linishedphotomicrcgnphs youcan Cngto theircharacteristics :-€ causesfor iailureand take core action. In many instances,the shoollngseclion can also serve Problemsin tinishedphotosand thok co.reclion . . . . . . . . . . . . . . . . . . . . . . . .68-71 Poorcolorreproduction . . . . . . . . . . . . . . . . . . . . .72-73 Blurredimage i s i n l o c u sb u tn o ts h a r p . . . . . . . . . . . . . . 7 4 ' 7 9 T h ei m a g e imaqeappeared Oojeclsolherrha" the specimen ...............80-81 o n t h ei i l m . . . . . . . . . . . . 82- 83 lJnevenbrightness Sectionl From pr€paralionto obseryation lvlicroscopes suitable for photomicrography .. ... .. .. .. .8 Equipment suitable fo. photomicrography.. ... .. .. .. .. .9 Varioustypesof photomicrographic equipment a n do e r f o r m a n c e ...........10 aEquipmentneededfor photomicrography . . . . . . . . . . . . . 1'l Suitablelocations for settingup the microscope . . . . . . .12 aobjectivesandphotoeyep;eces suitablefor photomlcrography ..........13 in the peripheral lmagesdepending aDiff€rences on o b j e c t idvees i g n ............14 in resolution depending on the type aDifferences of obiective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 a s o m eh i n t sa b o utth eo b i e c t i v e. . . . . . . . . . . . . . . . 1 6 - 1 8 S e l e c t ioof n a c o n d e n .s.e. .r . . . . . . . . . . - . . . . . . . . . . . . 1 9 a o b s e r v a t iporno c e d u r.e. .s. . . . . . . . . . . . . . . . . . . . . n - 2 1 s p e c i m e. .n. s. . . . . . . . . . . . . .. . . . . . . . . . . 2 a H a n d l ionfg Useof the fiefdirisdiaphragm . . . . . . . . . . . . . . . . . . . . . .23 useof the aperture irisdiaphragm andits eftects..24-25 a Baslcfocusing methods . . . . . . . . . . . . . . . . .... .. ..26-27 Howto spotdirt andspecksof dustin the optical systemsof the rnicroscope andthe photomicrc g r a p h iact t a c h m e n t ,,,......m frame. . . . . . . . . . . . . . . . .29 aHow to cleanthe mlcroscope aHow to cleanthe opticalsyslem. . . . . . . . . . . . . . . . . 30- 31 objective . . . . . . . . . . . . . .32 aHow to cleanan oifimmersion . . . . . . . . . . . . . . . . . . . . . . . . . . .33 aHow to cleanspecimens o Microscopes suitablefor photomicrography 1. Thephotolub€acceptsthe pholomicrc graphiccameraattachmenl. 2. -Ihe image can be focused throughthe binocul lube. matching 3. Thedesiredlighlinlensily tre specimenconditionsc€n be set by the odical palh seleclor, D 4. Objectivesshouldhavehigh resolution andgoodllatness(iat imageall the way to lhe peripheryol lhe Msualfielo. 5. Theslageis rolalable. 6. Thecondenseris equippedwilh an aperlurei s diaphragmand cemeiing devicelor aligning10the oplicala.xis. z$""[if;ffffi;'lXll""*'ipd-r'h ------ 8. Sufiicientlighl intensityis assured. Lightinlensilyis adjuslable10matchthe observalionand pholographing condilions. lJnilormilluminationfrom low 10highmagnific€tion. 9. The microscopeslandis resislant 'The phoio shoE m.del BHS again$ enemal vibralions. 10.Provision lor insenion of filters o Equipment suitablefor photomicrography 1. Thec€rnea attachmenlaccepls35rnm or large{omatfilms.The35mmcamsra back is equippedwith automaticfllm 2. Theoplicalpalhcan be changedlo condilio.rs. matchphotographic 3. The measuringarca c€n be changed to matchthe specimen,(lntegraled metefing 3oor-sfDt meiering1%) 4. The cameraattachmentis fmly clampedon the microscope, colortemperalure. 5. Devicefor measuring 6. Widerangeforsettinglso/AsAsensilivity for reciprociylaw failure 7. Coanpensation is carriedout automaljcallyfor long 8. E\po€ureadjuslment,matching sp€cimenconditions,is possible. 9. Manualexposureis possible. suchas 16mm 10.Specialapplicalions. cine and35mmlime lap6epholography, can be performed. (A specialcorfrcl unil is reqrifedin 11. An exposurelock mechanism{AElock) is built'in. I''rne phoroshm mod€ PM.1oAos. o rii Various typesof photomicrographic equipment andperformance Typesol pholomicrographic oquipment ' Concernlng theioclsingscreenior olympusoM systemcameras: TTre screencanbechanged to sul dflerenl (OM.] uses 2. 4) Forpholo OM Olv-3. OM The',ocus ngscreenmLrsl berelse screenNo 1 12 placedior pholomicrography' ' mcrography Sh|]lterb !r mayoccufli magnificaton s increased Whenusng40Xand100X obleclives. adlustlhe lightinlenstyso llrat Shutlerb urmayresullwhen40X lheshltterspeedis 1-2 sec.In thiscase, and1mXobjeclvesareused' sometypesof coor fim mayrequnecoor Pi|nOADS/ OM s€desSLRcame6 E se ol locuslng Photographic quallty Easeol op€ralion Useol large forfial lilrn .Manualwinding oi 35mmlim .Use of cablefe ease .Manual windng of filrn .Use of cable release .Use of VarmagniFnder lof rocusng meter Useot exposure (EMM,7) .tlse ol buill-nexposure meier ,'i f -' E+ r needed for photomicrography Equipment : st checkthe charl to seeif lhe equipment -:.uned ior pholomicrography hasbeencom :'.iely assembld.fihe lypeslistedbeloware -€ mostbasicattachmenlsneededfor taking roto6 ol stainedspecimens inlransmitt€d lighl. . quip49!! !99!ie! !!!!9pgEP! (m r :ocusiigmagnifier I :ocusingTelescope ! 35mmcameraback . iLrlomalicaeosure body . t | , :onneciingcord lrgeformal cameraback :dapter for largeJormalcamera tulomatic exposlrecontrollnil -rff df^ P" U- B _slruclion manlals t :crm lor recordingdata rr leaningutensilsi blower,cleaningiquid, ?1SCeaning lissle oOQ ooa G 6") t n U' Suitable locations for settingupthemicroscope Ressonsfor unsuitablelocalionsand corectivemeasures Unsultabl€room Cons€qugnces .Localedtoocloseto mechanical appliarices or machinerythatcancauseexlernalMbralions Blurredimageas a rcsullof vibralions .P aces in whichthe vibralon ol persons walkingpasl can be transmitled Ga T|Ealm6nl .Remove lhe microscopeJromthe soufce of vibralions .Use a slurdylableas suppod .Use a vibralion proollable Brlghllightlromthewndowprevents neara wall correci .Sei up lhe microscop€ .Positionlhe microscope locusrng in sucha way lhatihe overhead lightiallsin slightty in froni oi the microscope. .Place whereroomlighl entersthe eyepiece ljghtor flaresare rcproduced on the .Cover ihe eyepieceswith caps .Shui oul siraylighlgetlingntotheeyepiece photo or thefocusing lelescope by changing lhe oplicaPalhseleclor .Use of lhe microscope neara windo/ .sel lhe microscope .A dusryand dlrly room up in anotherroom B ackspolsare reproduced on lhe specimen.Cover .Placenea.a winclow the wholemicroscopewith a dusf wheredustcanenier image fromlhe oulside Exampleol a suitable room lor photomlcrography L suitable andphotoeyepieces Objectives for photomicrography Obieciives :.' pholomicrography cbjechigh'resolulion la€s wilh flahessall the way10the periphery r - visualfieldare requircd.Oi the LB(ong:a"e ) objectives,seies S PlanApochromal, a >an Achromai,and D PlanAchromal, ._c oi lhe short-barrel objeclives,Plan ,rccchromaland PlanAchromaltypesare Cor€ct comblnalions ol obioctiYeand ey€pl€c€ with FK/P Combination Combination with NFK tlpto eyepleces -e pholoeyepieceis opticallycoanpensated E .ermil the objectivelo deliverils full r€.'cmance on the film plane.ll musl be :..'eclly malchedwilh the objectives. s PlanApochromat eeri€s Shod.bargl Achromal s€ties Differences in the peripheral images depending on objective design Accordng lo lhe lype 01objectve the per pheryoi both lhe observedimaqeand the pholographed rnagernayappearoul ol locus This efiecl s causedby lhe pe.formance characlerslicsoj Achromallype oblecives Us ng Pan Achromaloblectves however. wi res! t . a sharpand Tlatmageexlendng a lhe way 10the perpheryof the Jeld iL y! rhe rierd ,rarnessor a D Pan Achromai objeclive eiiend nq a th€ way lo the pefprrery s superof to rhe one orovidedbv a D ach/omal 0b _.civa Difference in resolution depending on the typeof objective :- .: -: on n lhe centerof the lieldoi borh _- :cserved andthepholoqraphed images : _. : according to lhe type of objectve. _ - -:c cassobjeclive series,S Pan Apo:-:1s. as wel the S PanAch.omal - :: crovidesuperorresolulion. Sill'9oT.--tl? : ; i- Apochromat 40x.NFK33XLD ligss-ffi IU*H Gives srrarp ,esorutor .r lh€ whoe mase. allowino obseflation oi mlnul€ dela s Somehintsaboutthe objective It ls importanl lo choos€ en obi€ctiye sultable ior your sp€cltlc purpo66 For all objectives,properusedependson lhe specilicpurpose,sometypesrequiringsome adjuslments.ln orderto tulv rJtiiizethe objective,you shouldknowthe meaningoi the variousnumbersand letiersengraved on lhe objeclive barrel. 1. Obi€ctlvewlth con€ction collsr (N.A.) Method ot adiustnent In objeclives witha numeicalapenure above0.6- €xcludingoifimmersionl}pes(1)Setthe scalelo lhe 0.17mmposition, lhe thicknessoi the coverglassslrongly ailecls ihe imagequalily.Coverglasslhick(2)Rotate ihe coreclioncollar2 or 3 gradua. nessis theoretically designaled al 0.l7mm, llon marks(0.02{.o3mm)in the direction althoughin actualpraciicethis may vary by oi 2, and refocus.lf lhe imageis sharper +0.3rnm-By oplically compensaling ior this thanunder(1),rotatethe collaranolher thickness devation, the correction collar 2 or 3 graduation maks in the same assuresthe best image. direclionand tocusagain. (3)lt you are not satisfiedwilh the image quality,try rotatingthecollarin the opposite direction tor 1-2gradualion marks,r+ focusing andcomparing the image. (4)Gradually reducethe amountoi rolalionot the correclioncollar,andtry to iind the op iimumcondilionby repealinqsteps(2H3). r-Ihe abo,/eadjustmenlproceduresmusl be repeatedeveryiime lhe specimen is changed. objFljw typ€ and hagntfication Num€icarapenu,e(N.4.) 2. Obieclhr€wlth lds dlaphragm Someobiectivesabo\€,lOXareequippedwith prevenls an irisdiaphragm. Thisdiaphragm direcl lightfrornenteing the objectivein clarKieldor lransmittedlightlluorescence observalion.By watchingbothconlrastand resolutionof the image,lhe apenurecan be adjusledto the optimumposilion. la -r at d aa raa aJ al I I q -a D Pranf{X, S PlanADo100X,Nc s Pt.n ADo100x -r s malk indicaresrhat no cov€r : \o-cov€r (NC) objeclive ::cmens wilhoui coverglasssuch as :-: objectvesbearinqthe mark NC : :over glassattectsthe imasec arity, .!h ch is padicuarlyobvous in lhe case :.tves with a largenumerca aperlure. ':s!1, when observlngspecmens nol : :C by a cover glassat hqh maqnl : specia NC oblecliveis used. . ewingufcoveredspecmensthfough : .bjecllves, on y poor images w th 'ares and insuficieniresoluloncan 'i,,' '-, ) \]ruSi 3 ;+:'\)/ \ )'. d" ' .,.-. Li I' 'l )'r /;l- ( sp@imenw ihouicoverolass NcsPanApoloox -ous LB s€riesno cov€fobjeclvesi : rn 40X,NC D P.n FL 60x NC S Pran : : ! a ^ d N CS P a n r m x d r y SDecimen wlhoul cove,s ass S PlanApo r00X 4. Nocovor lmx dry objeclive High.maqn l calionI00Xobleclivesare uslaly oj lhe oll mmersiontype bul ior the nocover l00X objeclve a dry type is avalabe Using this objectivelogelherwth other non. imme6on objeclves,e.g. nGcovef40X and 60X.obviateslhe needlo pul mmersion o on llre specmen slide The l00X dry type lens can aso be usedfor pholomc fography. bLrlior achevng oplimumpiclurequaily, the lse oi an oil'immersonobjeclve s Obletlve lypeand m.gnific.tlon Numdi€r aperluc (N.A-) Mehanial llb€ l€nglh ofthenlcD6cop€ 5. Oil.immersionobjectlv€ Useof the oil-innercionobiective Oiljmmersion objeclives havea numerical apenureabovel-0 and use manulacturerspeciiiedoi belweenthe lront lensol the (Oilmmersiof objeclive andlhe specimen. objeciivescarry lhe mark "oii .) In orderio makefull lse ol the resolvingpowerof the objeclive,il is preterableto useoil also betweenlhe condenseffront lensandthe How to apply the oil (l)Focuson lhe specimen, wilhthe 10X, or 40Xobiective andbringlhe desired specimendetailin ihe lietd of view ' Tnlckns ot lie cdor Olas (2)Rolalelhe revolvingnosepieceso lhal lhe oilimmersionobjectiveis pointingtowards you,andapplyoii io theirontlensol hg 'Use only oll specilied by the manutacluret li youus€old Cargilloil or cedaroll,lhe obieclivecannotdisplayils lull potentiat, sincetheir difiaclion coetficienlsdiffer irom lhe nominalvalue. li theoil is llnted.it affecls lhe quallyof colorrcndilion in colorphotos. Theretore,il is advisableto useonlymanufac. lurerspecifiedoil.In parlicularfor fluorescence examinalion, useonlylhe iluorescenceiree oil provided wilhthelluorescence microscopes. clicksinlothe lighipalh.Makesurelhai lhe objectivelronl lensis tully immersed ntotheoil on lhe speclmen slide. (4)lftheirnageis notin vieq slowyrolalelhe linetocusing knoblill it comesintofocus. I Makesurethattheobjectivefronllensdoes notgel 1oocloselo the specimen, since lhe imagewilldeteriorale il air bubbtes gel inlolhe oil.ll a hazeseemsto coverparls of lhe image,evenlhoughit is in focus, swng the nosepiece1 or 2 timesfrornlhe cljckslopin orderto removebe air bubbles. (3)Applyoi lo the specimensurfaceand rotatelhe nosepiece untillheoii.immersiorl objective,ils tip likewisecovercdwilh oil, lf the lmagestill doesnol improve,remove the eyepieceas illuslraledabove,check ior air bubbles by viewing the backtens of lhe objective,wip€oii the oil and reapplyoil. - - 1..j .i t.,,,. t. cte.n an a I. I l}t all -rl rb al - ObjeclivBtyp. .nd magnltrcatlon NlmsdcalaperllB {N.AJ Mehanicallub. l.ngth ol lh. mlcrosope Thickn6s ol th€ cd.r glss (2)Rotale 5. Oiljmm€r$on objeclhr€ the revovingnosepiece so thallhe Useof the oil-imnercionobiective objecliveis pointingtowalds oil-immercion you,and appy o I lo lhe lronl lensof Oijmmerslon objeclives havea numerical lhe objeclive. aperlure above1.0andusemanuiaclurer, speciliedoil belweenthe iront lensoi the (Ollimmersion objective andthe specimen. objectivescarry the mark 'o l ,) In orderto makelull lse oi th€ resolvingpowerol the objeciive,I is preferableio useoil also belween lhe condenser lrontlensandlhe ob., How to apply the oll (l)Focuson the sp€cimen, wilhthe 1OX, or 40Xoblectiveandbing the desired specimen delailin the fieldol view ' 'Us€ only oil sp€cifiedby th6 manulaciuler ll youuseold Cargilloil or cedaroil,the objeclivecannoldisplayils fu I polential, sincelheir diffraciioncoeflicienlsditfer lrcm thenominal value.lf lheoil s tinled,ii aitecls the qualityof colorrenditionn colorpholos. Thereiore,il s acfuisable to useonlyman!1ac. turer-specifed oil,Inpadcularforlluorescence examination, useonlythe fluorescenceiree oil provicled wilhthefluorescence micrGcopes. clicksinlothe lightpalh.Makesurelhai lhe objectivelronl ens is fully immerced lntotheoil on lhe specirnen slide. (4)lftheirnageis notin vieq slowyrctatelhe linelocusing knoblll it comesintofocus. I Makesurcthaltheobjectivefronltensdoes notgel too closelo the specimen, since the imagewilldeteriorale get il air bubbtes inlo the oil. lf a hazeseemsto coverparls of lhe image,eventhoughit is in focus, siwingthe nosepiece1 or 2 limesl.om lhe clickslopin orderto removetheair bubbtes. (3)Apply oil10the specimen surfaceand rolatethenosepiece untillheoiljmrnerson objective,ils lip likewiseco\€redwilh oi, li lhe image slill does not improve, remove lhe eyepiece as illlstrated above, check for air blbb es by viewinglhe back lens ol the objeclive, wipe off the oil and reapplyoil. .i . ,). I i. ./.;. .i. oii. --t.: ,. a..e 32 l :ta ! - !ta q 'I Selection of a condenser :rrpcse ol lhe condensers to eficenlly 'le ighl emanalng lrom the lighl ! on lhe specjnren.1rrcreale ightn! i :.is malchng the oblecliveand lhls r. de a be11er image Dependng on the : . a a b e . P a d c u l a r yw i l h L r l r r oa w m a g .n obleclvessuch as lX 2X and 4X. : n s i k e u n e v e ni u m i n a l o na n d i n s u l f i c e n ta m o ! n lo f r g h l a 1 l h ep e r p h e r ya r e key 1oocclr Thereloremake slre lo lse lheseobjeclles n combnalon wtir lhe ! tra ow magnificatoncondenser n orde. 1r]oblan betterphotoswtir the S Pan FL 2X objectve.lse oi tire Lrtra ow nragnilicalion condenserBH2ULC s :nbination of BH2 series condensersand LA obiectives Condenser :.r2aAc :-2 SC :-2 CA :-2 ULC procedures Observation Now we are Ina y gettng to observalion bul firsl makesure that no dust or d ri s on ihe obleclive,eyepiece,and specimen. lvake t a habt lo check ior dirt beforeyoLl Lrsethe mcroscope sinced rt prevenls locusingand resulls n poor imagequaily ol filters 1 Placement Turf on lhe ma n swtch and adluslthe voilagelo poston Pholo(ca 9V) t Adiustmentof interyupillary r,- ob . C tt'@ I 3 r4 dislance h^ a =-1. (3)Placethe specimenon the stageand focus wth a lox objective @_ I ( 1 ) P a c el h e i g h tb a a n c i n gf i l l e r( L B D . 2 N ) of the lighl ext wlndow. (4)Adl!sllhe nterplpilarydslance |]nl boU. leil and righl vew fieds mefge nio one. (2)Pacean ND i ter in the slot close1olhe amp housng Dependlng on the objectve r.agniiicationand the densly oi the specmen.Lrselhis fltef so thal t provides enoughbrghlnessfor easyexa..inaton. { Condensercentering Diopteradlusiment rc Jsllhe dioplerlo sul lhe observels -,.esighl. Ihe melhodd flersaccording 10 I rnlessthe diopieris adjusted,parfocalily .r I nol be maintained' hen lhe objeclive the : r;_enusingWHKlox eyepieces, .,:-s is adjusled withlhe focusng knobs .. ^-€e observinglhrcughthe .ighl eyepEce aiopleradjuslmeniing 6) on the letl 'd): s thenadjustedlor maximum image ;'f lor the lefteye. I I (8)Alterlocuslng on lhe specimen withlhe (7)Alrhattime,adjusllhe irnageso lhalthe I 0X objective,rotatethe lie d slop A in crossinesn lhecenterof lhelramemask lhe d rectionof lhe arrowandreducethe as twoseparaie areclearydslinguished fieldirisdiaphragm d ameterio a minimum. ines.Thenadjuslthe focusby roiating Then move the condenser lromlop slowly cross knob so that the linefocuslng the helghl to bonomby usingthe condenser imagearein Tocus Inesandlhe specimen posilion knob and stop at a adjuslnrenl ghl_eye I mullaneously. Alter compleiing s wheretherieldiis diaph€gmir.age alsoadjusllhe diopterlor lhe adlustmeni, is sharplydeiined. (5). as n by rolating secllon lefl eye A mage1olhe I Movethefieldirisdiaphragm cenierof thevsuallied wilhlhe con' knobsc . densercenlerlng @@a I S ncethe \,pe of the lindereyepieceditlers (g)Open accoding lo lhe sizeoJlhe film used, thelieldiis diaphragm imagelntil chooselhe typesuitablefor yolr patticular it almosllouchesthe peripheryol thevislal iield,andmakesomelinalcenlering Fornormalobservation adjuslmenls. make conditions, thediaphlagm slighly EwlrKlox largerlhanlhe visualfield. E wFrhlox I lfthefieldirisdiaphragm cannotbe sharply A'pholomicrG us ng iinder eyepiece tor a focused,checkthe thicknessof the spec| a E wHkiox4 1 / 4 x 3 1 / 4 " P oa r o i d o is madeby rotaling menslide,Useslideswilh a lhickness t=.y adjuslment @wNrro{ -: :r B.of lheeyepiece al therighteye. belween 0.9and1.2mm. I -a Handling of specimens Make I a habLltoc eantirespecmenreg! ary bolh beforeand afler obsetualionJlsl as wlh lenses 1is mosl mportanllo work wilh a c ean specmen Nlakesure thal no dlsl pairces stck lo the specimenwhen you store I and do not toLrchthe gass slrlaces H o l dl h e A l a s ss l i d e a l l h e c o r n e r o ny , o r h o d l h € g a s s s l i d €b e l w e e ny o u r r i n 0 e . s , / F i n o e @ a n l s o rnh e . o v e rg a s s t , Noteson specimenplacementon ihe stage when you pace a sp€cimenon the staqe the specmen irolderis openedwide and i t s re easedrapdly.i1wl h t the edgeoj the slrdeand damagethe g ass.Aner coni rm ng lhatlhe sp€cimenhasbe€nlirmlyput n pace llre sprng loadedspecmen holde...u$ be retractedsowLyso thal il genly lolches lhe n casethe s de is damagedcarefuy renrovethe tny glassffagmenls.I fragments are eil on lhe stage.lheymay calse inilres. or the specimens de may be paced n a t t€d poston on lhe stage.causng one sde o i t h e v i s l a i e l d t o b e o L roi i ' c c ! s i . T h e i m a a ei s b u i i e d a o n s t h e l r a c € s o l y o u . I n9elpnnls o n es d e o i r h ev s u a r { i e ri ds b Useof the fieldirisdiaphragm (with35mmJilm) ljsing the Plvll0AD tunet masklocusingtelescope . ' e d l r l sd a p h r a g m s e v e s1 r ra o l u stlh e - "ated areaon the specmendependn! '',e obleclveoowe. lh s daohragmpays r . a r o e d u rn 9 D h o i o nc r o l r a p h ya n d i ._:. ode thannecessaryr uninal n! gh1 . ecred..d scalteredrre_o! a ly on the : ren eslLlrngin a ossoi mageconlrast : : i g d o w nt h el e d d l a p n r a g 1 m0l ! s 1 :1d the irame rel c e areav/i resu1 n : rlreph c mageswih rmprovedconlrasi s stopDe.jdo!,/n :re ted rs dapl-rra_om : c ose io the irame retc e tire ohoto :!red ma_oe may be cLrtai tfrecorners ! d a p h r a g n , s h o Li hr de r e T o b r ee o o e n € d trly morelhan 1l.ere:c e slro\ls ' ' . . r - ." , .l I l. ' ,.. : ' :,1 :i:i; 'r I ' e r d , 3 d a p h f a g mh 3 s b e e n Useof the aperture irisdiaphragm andits effeits The diaphragmrnounledon the condenser s ca ed aperturers diaphragmTheiunclion oi this diaphragmis lo maintainopl mlm condilionsoi image resolulon.conlrasland ioca deplhby adluslingthe nurrericalapenure of the lLrmnalionsyslemdependng on lhe nur.ericalaperlLreof the objectivein use. For most specimensopllmur. irnagequajly s achrevedil lhe apenuredaplrragm s adjustedlo between60% and 80% ol lhe How lo adjusl the aperlure ids diaphragm Thereare lwo methodsoi adlushent: Pu oul lhe eyepecewith lhe specmen n locus and then adlusllhe daphfagm!,vhlle watchng lhe rs al the rear ioca planeoi the oblecliv€as n pholo(1):usethe gradLra ton markson tire condenser.as in (2) 0)O r€cl viewng wilh lhe eyepi€ce - s nis darh€ap€r1ure Reducino prragmlo 6os0%ot lh€ numeica apenureor lhe obleclve. (2)Usnqlhe oraduaroi ma*s on rhe Samp e Us nOaloxoblecrivewilh a numercalap€duf€ ol 0.25andreducingll to 3 0 % , i h a q G d uoanr m a l k on lhe connenser should b es e ra r0 2 ( = 0 . 2 s ' 0 3 ) . I Example Whsn uslng S Plan Apo 20)( NFK 2.5X " *" "*n"*iris draphrasm I -*o F€ge resolLilion if lhe aperture deleriorales I I I I I I I I I I I I I .: d aphraom is sloppeddownloo much r'h lheexceplion ol specialyslainedlhn .ec rnenslhe diaphragm shouldnotbe sDped doBnlowerlhan lo 60% ol lhe nrencal apedLire ol Iheobieclive. I Ar effeclsrmrl lo stoppngdownthe rDenLrfe rrisdiaphragm canbe achieved :t movinglhe condenser clownward bul :rislendstointerferewith lhebasicillumina' :o1 iunclionof lhe condenser andresults _ uneven ilumination. Thusalwaysuselhe : aphragmandclonot rnovelhe condenser. $#ffiffi Fully op€nedposlilon \i{ =t &'/" Conn4l b enhanced. Detailsarealso cr€anyvblble,.nd localdepthls incr6ased, r€sullingin an oprimum g./o Re.oluriond.r.rioralesas a resull I Basicfocusingmethods Focusing lor obseruation A lOX obleclve s usedas lhe slandardfor locusng.thef the objecrlves changedlrom r(]X lo 4X and rrom rOXto 40X and i!(her lo 100XDo nolchangeabrupilyiromiowmag n ficaton (2X and 4X)to h gh (40Xand 100X) As a resLr t of lhe imled eye oi the obsetuer and the largeioca deplhoi low magnilicatan objectves(2X and 4X) lhe ironl mounll)1 the h gh magniticat on objectvecouldtouch the specrrnensurtacewhen the revovng Focusing lor pholomicrography Focusng dlring photographys done ether throughthe locusinglelescopeof the photo graphlcaltachmenlor lhrolgh the eyeoieces of the binocLr ar tube.When locusngthroLrgh lhe eyepieces.a jnder eyepiecemLrstbe used Pror to pholomicrography the fnder eyepecehas 10be foclsed by meansoTi1s f o c u s n gl r o n tl e n s .t o m a k ec e a r l yv i s b e doLrbe cross linesas 1wopara el nes ln lfre cenlerot the lramingrelice 1 l g iI I - b AJleriocusng on the specmen $,th lOX obieclivesel the upper mt ol the coarse adllslrnentexcursonwillr the preioclsing Movethe specmendetai 1{)be examlnedto the centerof lhe vsua leld and increase maqnilicatonby rolalingthe nos€pece. 1 2 --)F l-. i-rl *r+ /n a' "i]". I Focusing lelescope ol lhe photographic For pholoqraphy the specimenlocus mLrsl be adllsled wth the same eye wilh wh ch lle .ross ines were foclsed { Er Finder eyepieceol lhe binocular tube Focusngthroughthe binocLr ar tube s possbrewlh microscopesVanor BHS.BFT and BtsTU I l 3 s; 1. Focusing when using 1x,4 and 4x obieclives Foclsingerrorsocc!r qu le irequentywth low nragnflcaton oblectves Tierelorelse a l o c u s n gm a g n j e ra n d a d l u $t h e i o c L r s by iolow ng the procedureslsled beow shoud Bui beiorethat the pholographer adjustdioptera1the cross nes oTthe J o c u s n gi e e s c o p eo r t h e f n d e re y e p e c e 4r! 2. Focusing when using 10X and 20X rs achievedby adjustnglhe FocLrsrng c r o s sI n e s s o l h a tt h e ya r e c e a r y v i s b l e . and then rolatng tire lineJocusngknob Lrnt both lhe cross nes and lhe mage 0 1t h e s p e cm e na r e c l e a r yv i s b e s m ! laneousy.By sLghly mov ng yolr eyes oi lhe to a sdes. see i the posnLons cross inesand the specmen mage'1onol s h i l f t i r i s s t h e c a s el h e P c 1 ! r es n focLrs t they do sh it. the tocls rs not l,^,* rff) 'rage is s ghlly oul oi iocls aner . : o j m a g n l c a l i o nl s e o ny t h e f n e _j knobior refocLrsingn partcLrar rse the coarselocusngknob for _! a h gh-magnlflcat on objectve. s nce . a dangerlhal the obleclve w rLn - specmen ana uer oamageo fs p r o p e r ya g n e da n d m l s l w tl-rtire lne jocusn9 knob 3. Focusing when using ,lox and 10{x Adl!$ the cross nes so tfral lrreyare c eary v sibe. llren s owy adlustihe locLrs ot the speclmenimageu71hlhe l ne locusng knob lnli cross Ines and lmage oi tl-respec..en are c eary vsDle a o c a tn 9 : : ' e y e p l e c ei s o r o v d e d w t h : . n must be firmy nse(ed nto the r ' t h e e y e p i e csee e v e f l h e p n l s _-_t rnserted.correcliocLrsngbecomes : e a n dl h e m a g e w b e o L t o i f o c l s . Camp the iocusingma!nlfleron the 'oclsing leescope.afd side the top sec the tocls ron n or olr therebyreadluslLng al lhe cross lnes. FocLrsis co(ecl t bolh the cross lnes and lhe specmen m a g ea r e c e a r y v s i b e s i m u l a n e o u s y Basicfocusingmethods Focusing lor observalion A lOX obleclve s usedas lhe standardior locusng.then the objecllves chanled from rOXlo 4X and irom 10Xlo.10Xand ilnher lo I 00X Do noi changeabrlpl y iromlow mag nirical0n(2Xand lxl to hrgh{40Xand t00X) As a res! t 01llre lmled eye oi lhe observer and the argeioca depthof ow magnificaton obleclives(2X and 4X] lhe Tronlmountoi the highmagniiicalionobleclivecoud louch lhe specimensLrrlacewhen lhe revolvng 2 1 3 I I -''b , ,l Focusing lor pholomicrography Foclsrngdlrng pholographys doneeilher throughthe iocusng leescopeoi the pholograprrc attachmenlor tlrrouqhthe eyepeces o t t h e b n o c u a rl u b e W h e nl o c u s f g t h r o l q h l h e e y e p e c e sa. f n d e re y e p e c em u s tb e used Priorlo photomcrographylhe inder eyepecehas 10 be focLrsedby meansof ls focls ng iront ens to rnakecteartyvrsbte d o u b ec r o s sl i n e sa s l w o p a r a l e l n e s n l h e c e n l e ro l t h e l r a m i n gr e l c e After ioclsing on lhe specimenwith l0X oblectve.set the lpper mil oJ lhe coarse adjustmenlexclrson wth lhe preiocusn9 fvlovelhe specr.fendela to be examned 10 lfre cenlero1the vsLra fied and ncrease maqnlcalion by rolalng the nosepece 1 2 -)E Ir-' la l**r+ il1 : : lr ( ---- i Focusing t€lescope ol the photographic For pholography the specimenfocusmLrsl b e a d l l s l e dw l h t h e s a m ee y ew l h w h i c h l"e .ross lnes were foclsed Finder eyepieceol ihe binocular rube Focusnglhroughthe binoc! ar lube s possbrevilh microscopesVanox.BHS.BNT ard BrITU , fs 1. Focusing when using 1X, 2X, and 4X 2. Focusing when using 10X and 20x F o c u s n ge r r o r so c c u rq ! i l e j r e q u e n t y w i l h ow magni cal on obieclivesTheretoteuse a focLrsng magnilierand adlu$ the iocls by fo owingthe procedlres sled beow should Blt bejorethal llre pirolographer adlusldopler al the cross Lnesof the tocls ng leescopeor lhe fnder eyepece Focusng s ach evedby adlustnglhe c r o s sl n e ss o t h a tt h e ya r e c € a r y v s b e . and lhen ro1alingthe fne ioclsng knob unt borhthe cross ines afd the mage o t l h e s p e c l m ea n r ec e a r y v s b l e s i m u taneousy By s ghlly moMngyoLrreyes to a sides see I the posilbnsor the cross lnesand llre specimenlmagedo noi shil li th s s lhe case the Pclure s n iocls li they do shjl. the iocus is nol -')I :,:,'"Hll"l lfn : male is slghty oul oi jocus after t e 0 1m a g n i c a l l o nl ,s e o n y t h e l n e : -! knobior reiocusingIn pancuar : lse the coarselocusng knob for . r g a h g l r . m a gfnc a t o no b l e c t v es. n c e s a dangerthal the oblecl&elvr rln : specmen and get damaged - qt*i-j fs b-t properya gn€dand musl be readlusled w t h l h e f n e i o c l s i n gk n a b 3. Focusing when using 4OXand 10ox Adilsi the cross nes so thal lhey are c eary v s b e thenslowy adlustlhe locLs ol the specirnenimagewlh the r ne jocusng knob un1 cross lnes and image o f t h e s p e cm e na r e c l e a r Yv s b l e \ r: r eyep€ce s provicied w lh a localing _. pln musl be I rm y inserledinto tl-re ::r y nserled.cotrectlocls ngbecomes : o e . a n d l h em a g e w i b e o l t o f i o c u s Camp llre iocls ng magnifieron lhe IocLrsngieescope and s de the lop sec lon n or o!1.ih€rebyreadjustngthe tocLrs at lire cross lnes Focus s correcl 1 boththe cross nes and the specmen i m a g ea r e c e a r l yv i s b e s m L r l a n e o L r s l y . Howto spotdirtandspecksof dustin the opticalsystemsof the microscope and the photomicrographic attachment D ( and dLrstpanc es are sor.el mes noted dur ng observaliofor phologrpahy. but pin pontingthe r exact localionnraybe diiiicuit Whenphotographing imponantSpecmens which cannolbe photographed again11can b e r e a l yi r u s l r a l j nigd u s tp a d i c e sa r ev s i b e on tlre pclLrre.An eliectivemethod s therefore requred thal w hep you deiecl I S i n c e dr l a t o c a t o n sn o r _ a n di m a y c o m e irom paniciesoj lhe n m lisei (oadng. j m transpo().regularchecksard necessary I lf the aperlLrre daphragmis.edlced below 30%. dirl and dusl Specksbecornecearly Obser. grapny \frf' oescrirrion Dlst speckson the pr sm insde the obser' valionlube.on lhe insideof the objeciive and on lhe jnsde oi the rn croscopebase cannob t e w p € d o J le a s y . i c e a n n g becomesnecessaryconlactyourOympls Melhod ol verilication Rernove theadapterior large{orrnal cameras and check ior dirt by peerng ln lhrouglrthe lop li you spot dilr unsc.ewand removellre re ay ens Rotalelhe lop lens eement as you observelhe z 199!li9 :91 Cameraprism a Selto,Timemode,openlhe shulterandpeerin Eitherremovephotoeyepleceand checkior dusl rotate q"lig919l r".9""phgto9lepecein prace, t andcheckfor movngdustparlces. -_ r Oplicalpath O Tubeenglh correcton O objectve cha^ge rfe op_icalpa f 4- le alernarelyooqetu g lhroughlhe foclsing leescopeor lhe fnder a a Remove lhe observation tubeiromthe mcroscope bodyandchecklhe prisrnsu.faceior fngerprnts or conlaminalion - checkt lor d rt or conlaminalon. Specmen _O !0 Condenser ^ - ; ; * .' ce ovF the obectNp roa l\e nos6prale d o Observelhe specmen and move lt in lhe fied. li dust is on lhe specmen,il wi also move. Removelhe condenserlrom the mlcrcscopeand look for dlir and oi deposits. Checkthe iilef after removingit lrom the m.rG scooe oase Swilchon the lluminalion andexamnelhe lens l? Lghl exil 8 gtass Frosted j.i Co lecior lefs a a Bulb ^ ' -' check lhe irosledg ass Aemovelhe amp houslngand checkrhe enses in lhe colleclorassembly. Bemovethe bulb trom its sockeland check sgns of backenng fingerprnts.dirr etc. frame Howto cleanthe microscope :ii _son the rnicro€cope lramearelirsl Mslerials us€d lor th€ mlcroscooe ..€d wltha pieceol clolhwetedwitha i: amounlof neutraldelergent,andthen ..€a cleanwltha pieceof clolhlhal has )ir- ,mmerced ln luke-warm water Butmake L-. roi io louchlhe lenssection,rvhile :er_ ng the microscope ifame. \. : usingorgancsolvenls whichmay --:ge paslicparls. ::r duslconlamnationthaladheres 10 = _ - pansand is difficultto remove,wipe r: . pieceof clothor soll tissuepaperthat -3: :een soakedin a mixlureoi 7 partsether r: I parlsalcohol, Keeplhe m xtureaway +:- claslicpanslo prevenldamage. E E E Howto cleantheopticalsystem 'p6ora . 6 oor.ot . ran . pa. o dt. . s. o..o r e r . o r . -o " surtacet canformay berenroved wth a bower Yo! shoLr d irowever.make a habl oi coveringthe mcroscopewth a dlstprool cover after eacn lse ,- ll:e i\ Cleaning melhod Tduslspolsonoptca glassessuchas lenses prisms and i ters are e11unallended.the dLSrbecomesd ficLri ro renrole and may calse mold By alwayskeepngoptca glass s!rfaces clean.yo! avojdma nlenanceprob ems ard prolonglhe ie of yolr mcroscope Cleanng oj lhe ens suriacesapp es ony 10 exposedareasoi obleclives. eyepeces lirlers and condensersIf lnlerna or malorcean ng becomesnecessary.pease conlactyoLrr O ympusN4icroscope deaer ls 2 | I +'. 'il 'fl 'n .s.b |i To preventscratcheson coalingsand oplical ! ass removedin and dlsl lhat stcks 10 the rsuriaceswth an ar gunor b owerbrush Wrap lhe lens lssLrearoLnda woodenor bambooslck as ustraled. )t 5 Requiredlools - e , - t ..ii-' '€ i A r gLrnor bower brlsh 2 c e a n n g m x l u r eo t 7 p a r l se l h e ra n d 3 pai(s acohol.or efs cleannq f! d 3 Ol ps wood sl ck a Sojl qauze.lens 1ss!e ! Magnryng glass An €yepiececan aso be lsed n pace ot the magnf e t -------"'- '" __ Whencleanng largeg ass slr{aceson both sdes ot an accessorysuch as a j t€r fod two or three ayersof lens lssle soakedn t h e c e a nn g m x t u r e h o d t h e a c c e s s o rayl ils edges and wipe lrom the cenierlowards llre perpheryas yo! s owly rotale I Whenc eanng tl-resuriacesoi the condens: and oJ the iighl exil gass hod a pieceoi y o l r m d d e a n di n d e r l e n sl s s u e b e t w e e n ingers lod I afd $/rapi1arolnd yoLr ind: ringer Thenhod lhe lissuedownwth yo! lhlmb wlrie vr'rpnqllre lens sudacescea. tL, -=t \ w -- v .:, - YJ :.:. lhe lens by pulilnga smallamounlol ? : .eaning ilud or cleaningmi*ure on the . :'a lenstissue.Dlscardeach ens tissue wipe Whencleaning a largelenssurface, if fromthecenlerlowardslhe periplrery T, *-'l ti ]- : eanng,examnethelensslrfacewlth . -.:t. [rng glass.]i colorrcfectedirom t- -,_ssurfaceooksuneven, it is an ind. - :. :nattherearesl I duslspecksand . " . ewngihrough thebonomoi an eye ,oLrcanuseit as a magntying gass i., Qtt' Whencleaning a argelenswilhlenstissue afoundyourllnger,yoLrshouldwipe wrapped in a Iromlhe centertowardslhe periphery Aways,usea cean pornori circuarrnolion. of the ensilssueas yoLrrotaieyourindex Aftercleanng,breatheghllyon the ens surlaceunli lhe wholesurfacehaslurned whle,thencheckwhelherihe hazedis appearsunlormly.Spotswherethe haze d sappearsony slowlyare not yet wiped Howto cleanan oil-immersion objective w pe rhe llonl rens oi llre oblecl've r\1 Cl€an the oil-immersionobieclive dunng Aner in sh ns obseryalon wth an oi mmersonobjectve.wel a pad oj cottonwoo or a pece oi ens lissuewilh a sma amoLrnt of cean ng mxlure conlanrng7 pans ether and 3 pans acohol,to femov€ol adherng lo the objectve S nce an oil 1 m w I oilen adhere1olhe obieclivefronl ens t sholld be wipedclean l\/ice after eaclrJSe .) ! ) llse only lactory recommendedimmercion oil and r€moveit atter examination i the o femainson the objectveior a ong p e r o do i t r n e . t w l l h a d e n ( e g c e d a r o ) makng t diiiicLrllto removeeven if yoLr wipe the ens repeatedy Tlre lens may be l n l h e p r o c e s sU s eo n y s p e c i € d damaged oi. and afler]]se wlpelhe o irom the mmef sion surlaceof the objectve.keepng t lj you lrequentyuse an oll rlrme|sonobleclive olmay contaminatethe sLrrjaceoi a dry t/pe oblecl ve *hen you changeoblectves To preventlhe o irom adherngto llre obleciivecareiullyrolalethe nosepieceafter lowerng lhe slage.so thal the o does nol I lhe mage of a dry lr gh-magnifica|on obleclve appearsiuzzy.check for o thal nriglrthaveadhefed10lhe 1ipoTthe \ Lower rhe slaoe slowly so lhal no oi l louch lhe rronl rens oi the dry objecr ' I 6 I r- ,Lt a t Alle. owernslhe sla0e carerury.ola nosepiece and crickrh€ dry obleclNe Howto cleanspecimens r,:ke il a habl lo cean eachsp€cimen bolll :r'cre andaflerobservation. OlheMise, dirt -r dusiihal youlailedto noticedlrng riervalon mghtappearon llre pholo. ::- cleaning thespeclmen a sonc oth,gauze, :, seceo1lenslissuemaybe usedwiihoul :.aing liqrid.Bll ii ihe conlaminal on s ,-.Lrll lo clean,brealheon the specmen ri:re wipng il.Whenceaninglhespecimen, *,nts to note duringcleaning ' iemovalof oi as wellas roulnecleaning :an b€ donemorceasilyil lhe specimen ; removedlrom the stage. : .ihenusingceaningmxturcor lens : eaningillid, usea moistened clolhor :?aniaglissue.Becerlainnotto apply :.aessivefluid,as il mayseepunderneath -. coverglassanddarnage lhespecimen, --:re areMo lypesoi sp€cimens: thosewiltr a ::.ergassandlhosewlhoulacovergass, rJ:- as blood smeaG .b + cleanlncsp€im€ns wilh coverqlass Cleanlng specimsnswith coverglassos ?swhencleaning lefses,wpe ol1lhe :'3 dirlwilha pieceof lenslssue ighlly .'enedwilhcleaning mixlure.Because : cannolbe complelelyremovedwith roe repealwipingunlillheol film Cle6ningspcimens wilhoutcover tElss6s ::-eing to uncovered speclmens cannol a 3edofl. Youcan, however,removelhe :. Tmelsnglhe specimen for 5 to 10 -.s n a xylenebath.Therearecontaners :'::. horizonlal andverticairnmerson, :..o€r selecllon of whichdepends on t/t cleaninsspecmenswrthoutcov€rorass @ Section2 Whentaking piclurcs in photomicrography and OperatlnoInstructlonsfor modds PM-1oAD (with36mmcameraback). . . . . . . . . . .36-37 PM-1oADS . . . . . . . . . . . . .38 Magnification of photographlc equipment Dlfferencesin re6olutionaccordingto the combinations of objectivesand ey€pieces. . . . . . . . . . . . . . , . . . . . . . 36 Settingof photographicmagnlllcation(effective magnification)..................................39 Framiog of the specimen.... - . . . . . . . . . . . . . . . . . . . . . 39 . . . . . . .,O-41 Checking ot the linishedphotomicrographs. E x p o s uar d e j u s t m et snct h n i q u e. .s. . . . . . . . . . , . , . 4 2 - 4 3 . . . . ., ., .44-45 L,seot theAElock failure... . . . . . . . .,16 Compensating for a film'sreclproclty , , , , . , . . . . . . . . . . . . . 47 Ocoforfilm .............. ',|8 aTyp€offilters on the a Dlfferences In colorrendition dgpendlng of film andlightsource. .. .. . . . . . . . .. .,$ comblnatlon a Dlfferencesin color reproductlondepending .. . . . . .. . . . . . ., .50 on differences in colortemDerature a Diff€rencesin color r€nditiondependingon the type ot tilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51 O Purchas€ of colorfilm. . .. . . .. .. .- . . . . . . . .. . . . . .... .52 [Jseof colorrompensating(Cc)filt€rs whentakino color photos a T e s tp h o t o g r a p h y . . . . . . . . . . . . . . . 53 .,...........54 ...........56 a B l a c k € n d - wfhi li m te a c o m o a r i s oonf d i f f e r efnl ltmb r a n d s. . . . . . . . . . . . . . . . . 5 6 . . . . . . . . . . .57 a Filters. . . . . . . . . . . P h o t o g r awp iht yhP o l a r of i |dm . . . . . . . . . . . . . . . . . . . . . . 5 8 tD Basicintormation in photomicrography formodelsPM-1OAD Instructions Operating (with35mmcameraback) andPM-10ADS Loadihe film propody 1 Setingthe lilm lormat 2 Plsh lhe buton ior 35mm camem. Loadthe ii m nto the cameraand makesure rhat the end ol the iim does nol protrLrde beyondthe spoo groove OlheMSe tire Jramespacingmay not be Lrnlorm. 7 Centerthe specimento be , photographedin the visuallield andadjustthe apeiure R v Fgcus msotapnragm . r l thewinding ortnerirm 3 confirm f Wlnd the fim onlo rlre emptyrakeup spoo and advancei110irame m. I To conflrm that rhe fim s winding be cedain thal the f i m f e w n d n g c r a nsk f o l a t n gt l s n o t . rolale the crank 2 or 3 tmes n the d recton oj lhe dotledarow lo pick up lhe slackol the I m nsde the car.era. and aga n check lhe rewndng crank to coniirmthe wndng of lhe i rn V Adjustshullsr speed X Movelhe area ol llre specmenyo! wanl 10 pholographinlo the cenler of the visua ield wilh lhe stagecontros.WhenyoLruse mode PM-1oADS. cenler rhe area10be pholo. graphed n the spot mete.ingseciionoTlhe Focuson lhe specmen and adluslthe condefrserapenureirls diaphragmso lhal sutabe cofirasl s achievedThe aperllre .laphragm s ordinary sel at 60% lo 80% or the objecuvenLrmercaapedure Checklh€ shunerspeed.Adluslllre speed to between001 and 05 secondsby using ND i itels 4 Color phologEPhy Set lhe colorlemperalureto malchthe type of color film used.Measurelhe temperature at a blankareanot coveredt'v the specimen. I!,De of fflm Daylbhi film Tumsi.n lllm S€rlhespeedfor lhe lih Lrsed. Sel lhe characleristicsior reciprocityfailurc for lhe filmused. tldrl Dlal pG! helarclng thn CIn ffllor t.BD-2N I-BT D T Thevollageposilionwhennot usingthe color temperaturemetercTF is: 8.5-9.5V.for modelsBHSand 4 or 6V lor modelsBHI and BHTU Elackdnd'white photography Sel the voltageabove6V Normally,a gleen lilteris used. (Fat enphaslzing padicular colors rcler ta page57) Focusis adjustedeitherthroughthe tocuslng or the oi the PMI0AD/PM_I0ADS telescope binoculartubeof the microscq*,(rcfer to pages2627) oADS/ I Thefocusingtelescopeof lhe PM_1 PM-1oADincludesdifferentphololrames lor diflerentlypesof film. I l}le lype oJJindereyepiece\?tes wilh iilm sizeand eachtype of nnderelepiece incluclesseveralpholoframesindicating oi NFKphoto diile€nl magnilicalions Presslhe shutlerreleasebutlon.After complelingexposure,checkior the sound of ilm wnding. aesure adjustmentis basedon the lolal lo ion oi the specirnen.freler qt Magnification equipment of thephotographic Photomagnlcalon on the flm surfacevaries wth the prolectonlenglhoi lhe photographc eqLrpmentand lhe type of pholoeyeoi-oce lsed ln a casespirolorna_on fcaton s c o m p u t eb d y m u t p y n ! t h e o b j e c t v em a g n I cat on andtheprrotoeyepiece magniTicat on v r t h t h e c o e i i ce n t s l e di n t h e t a b e o n rne flgnr errotogaptricequipment Photoevepiece FKNFK{ype P.type PM.lOAD P]\,4.1OADS 0 5x P[.410M 0.5x PN,l,6 09x BH2 PM 6 08x 0 4x Whef usnq modelsPf.'ll0ADor PNrl]OADSrOXoblecliveand 2.5XNFKpholoeyep€ce P h o t om a n1 c a lo n = r 0 x 2 . 5= 2 5 Differences to the in resolution according combinations andeyepieces of objectives Eventholglrlvhenlhe overalpholo magni ca Example:Pholo magnilicalion ol 100X l o n s l h e s a m e .r e s oL r l 0 n! a r e s d e p e n d n g on the combnalon of obleclveand pholo l n o n t h e1 m p a n e e y e p e c eT h er e s o u o s mprovedj lhe magnflcaton oi the photo eyepece s ow and thal oi the objeclve h glr tl [ . As the photoshows.a combnalon of oblec. l a Uve40X and NFK2 5X provdesa c ear dspay of evenminLle magedela Foca deplh.lrolrever.becomesslra ow - at' t , t. a ' 't . t t tt i ' ,'l? ' o ' ;" ?1 'tr'-t .,...1 lt a I i l.. 1r ' z l{ :'' .''l . a r t .e ..tt... t- .."tr ,1'.''. ' .o ', 1 ". .,.a\ I ^1 Oble.l ve S Pan 20X and prrolo eyep ece NFK 5x Obrecrve s Pran40X and pholo eyepece NFK 25r ,"- --.' Settingof photographic magnification (effective magnification) n'en takingpctures,youhaveto Jirstdecide ar rhedesiredenlarqemenl ralio.Themag. r_calionon ihe film paneis generaly :E:erminedby objectivemagnilicalionlimes 9oro eyepiecemagniiicaiion.But the eliec *Theetfeclvemagnilicalion llve magnilicallon ol the piclurechanges is basedon the wilh the nume cal apenureol the objective, assumpllonthal ihe picturcis viewedwilhin and ls normallybasedon lhe lollowingrealhe closesldistanceatlordingdislinclvision, The values istedbelowdo not applyllten tionship,which mustbe lakenintoaccolnl when enlarginglhe pholomicrcgraph. tryingto proiecl35mrnlormal slides. [email protected].<M<1qnN.A. Si6ctiYe .agnilication tF ol ai.tclive t-4" EHive atnilication 't0x tx N.A =nune,iczta\dup at th. obta.iE M mx 'tmx 40x S Plan S Plan S Plan S Plan S Plan S Plan S Plan S Plan S Plan S Plan 0.13 0.16 0.30 0.40 0.46 o.7D 0.70 0.95 '|.25 1.30 65- 130 80- 160 150-3m 200-4m 230-Affi 350- 700 350-700 475- 950 625- 1250 650- r 300 Framing of thespecimen -! pholography,lhe specimen ollen operationot lhe microscopebecomesmorc .rollil inlolhe ohotoframe.Thisoroblem d ficull.Herc,theiramingof the specimen is carriedout the p€ferrcd way,by rotating !e overcome by rotaling the camera the slage. in relalion lo the specimen, though 'nE of the finishedphotomicrographs Checking Phobrnicrography inuol!€sscientjficpt|otc gradry,which makesit impolatn/ethal lhe dDl€rapher accuralelyrecor(bhis fndings on film.Addliooally,lhe pholographshorid conv€ya stror€ estrlelicimpressimto lhe vis/ver Valuablerecordsshdid mt be documeitedwith runof-theflill dtolo6. Tofurlher inpro/e your rcsultsin photc micrography. lt h importantlhal you aler4/s checkyo|lr o n pholo6.By relerdngto the follo/vingchecl@inF,liou c€n pinpoinlany problenEconcemingliour photo6.Tll€n prc c€edlo the secondhalf of this section lilled Photomicrcgraphy Techniques. 3. Bfackrndwhlb photogt $if teler to 1. Ouosdonar€lallng to all phoiography pages 5557) (l)ls lhe imagein focw f/eter to pagesb-27) (])Do€sthe tinishedphotographsfrori, and lhe e&o6ureprcperlyadjusted? gradedblackard whiletoneswilhoul (refet to pages 4246) (aHave din or dust speckson the sp€cimsn sivelydark(solidblack)shadowareas washed{ur(corndelely$/hite)highlights? foundtheirway onlo the photograph? pages to 2&33) telet (3)lslhe specimenproperlystained? (4)lsthereunevenilluminalion? ffetef fo pages225) Z Coforphologr.phy frefer to pages 47.54) (1)lsthe baclloround(emptyspace)whiteor ol lightgrcy lone? (4ls lhe color of he specimenaccuralely reprcduced? 25 nrmKodachrome 1984Dec.l Purpos€ 1 a a 3 6 7 8 I 10 tl 12 13 t4 11 0 1 77 lq 18 18 rs 19 ry 21 i 4 a a_ n. a 4 a I 31 3 .v 5 Photomicrography techniques techniques Exposure adjustment Whentakng pholomcrcgraphs,exposure compensalon is necessary depending on ihe dislibUlonof the specimen in lhe iield. Butii the specimen is evenlydistrbuted ZP at al or spoimeieingrcnge, withinthe nlegrated is requred(incaseoi 1X). no compensalion 1. PM.lOAD (60% Averagemeierlng wlth mod€l PM.lOAD) $ $ # Z PM.IOADS rMod€lPM.1oADS can b€ u3€dtor inleg6ted melering ol 3ool"anctspol melenng ot 1oh.\ Measuringalaa Soecimencondlllon wlthin the l% meledngar€a dotted Brightf ieldbackground wilhfairlydensesp€cimens 0.25X con' ieldbackground Brighlf specrmens lainingscattered 0.5x d slribLned Specimenis evenLy area. wlhin lhe I % metering 1X Sellhereclpro lilmbelngused. Aboulhalf(50%)01the wiihln specimen is distribuled the da background *t Abouionefourlh(250lo)of lhe specimen ls distributed n the darkbackground Thedarkbackgrcundis dotled the lSo/ASA dial sensitivily Useof theAE lock By prcssingthis buttonduringthe automatic AE lock operetion exposurernode,you can adjustlhe exposure P -10lDSfnr6grat€dmetoringg,%, lime indicaledon the displaypanet(expected spot msteringl%) exposurelime, actualexposuretime). _lhe Wlen pressing AE 'ock hJtton,the wuning light abo/e it nashe6to indicate AE lock mo(b. The unil is oow readyfor ptDlog€phirE.Tocancelthis tunctton pressthe AE lock buttonagain.This sl|itcheslhe lightoft andthe de\ricere verlsto nonnalautornatic oocure anode. gimlnaling density yadatlooeby AE lock 1. Takingpanoramlcpictues uslng ih€ AE lock For panoramapictureswheieany numberoi copiesis lakenfiom differenlseclionsoi the samespecimenor whereseveralpholosare patchedlogeiher,pertectpanoramicphotc graphswilh uniformdensitycan be lakenby locking-ina lixed exposurefor all the pholo PanoEmaphotography Plr.loAD (averlge mei.riig d}%) Aller poGiliorng lhe specimenlo be photogmphed and pressingthe AE lock button,lhe $/amirulightabo/e it tlashes to indicateAE lock mode.Then,after laling the frst photograph. eecr.ire lirne is locked.in. Tocancelthjstunclionpr€6slheAEbck butlonagain.Thisswitchesthe waming lightoff, and lhe devicerevertslo the norml automallce4osure mo(b. z Erampleol uslng lhe AE lock on model PM.'IOADS 4)ln conbination with spot neteing ' specirnenareasrequiringspoi metering of faminqprob arenol cenlered because in the cenler €ms,lheyarefirslposilioned andthe AE lock is activated.Thenthe soecimens are movedbacklo lheirorlginal aosilionand pholographed. 1 Th€sperm€ndetaills al th6 locallon bul whereit is to be pholographed, exposurecannolbe m€suGd 2 Aller movhqth€ spocinendelailto areaand aclival,ng the measuring the aE lock,lhesp€clm€nd€tailcan now be movedbacklo iis o.loinal postllonand rh6 pholooraphs lak€n 2)!!9!9gEP!!!9vt!!i! lJ to naximum exposurctime ' exposure displayls c oselo the maxrmum duringfluores_ eroosure lime,lor example the pholographic :ence photomicrography, an underexposure mighldisplay eouipment Thiscan exposure. ^?rningduringautomalic by usingtheAE lock,resulting .€ prevented . ongrme exposure. Th€sAFEry lampcomerro r€d .nd an audlblewarningsounds. Compensating fora film's reciprocity failure W:thnormallyusedphol€raphicemutsions Exrnd.: Dai. ior rhe afirracl€r&llcs ol r€clprodty law teilu|e {Kodrkcotortflm DKD-|/|1) thereis a rule(the reciprocilylaw)thal determinesthe luminanceof he lightstriking the lilm surface-Accordingto this rute,lhe tolal amountof e&osure is delinedas the produclof the lumimnceand lhe exposure time. For example,the anoont of oocure wita 1,60sec. o@surc at E is the iame as lor I B0 sec,at f11. &Jtfor longerexpoGure limesthis ruleno longerapplies,leadingto unclerexposureand changesin color re producljon,Thisphenomenon is knq n as reciprocilylaw lailure_Bul sjnce,in d|oto exposure.lJnevencolor reprcdrctionmuat compensation by lilter are necessary.For mrcrography, be compensat€d expoGure compensation for with a CCflter. cannot 'll|e additiomldah on recipnccitylaw tailure be carriedout via the aperlufediaphraqm, abore chan [sts lhe compensation data characterislics conlactthe lilm manufacturcr exposuretime is lenglhenedor shonened lor rcciprocitylawfailurecharacterislics when lo obtaina suitableexpmurelevel.lt the usingKodachrome 25 ilm lor geneElptrolo reciprocitydial is set on rnodetsPM-1OADS micrography. The chan showslhat for tong and PMIoAD, compensaiion is carried expo6uretimesexceeding1 sec.,bottl oul automatically, resullingin pfoper exDo6ure time @rnoensation and color Ex8mpleol comp€rcallon(u,t|€nUstngFulhhromeROl00) No corn. p€nselion Compeft sallon Colorphotomicrography Colorfilm 'i.v differenttypesoi coor i m are oiiered :.e markettoday eavnu tfre Lrser2t a :: rs 1')lrhich brandto use tor photomrcro :rhy Normaly. day ghl typ '- an ISO/ASAspeedof 50.100 s lsed e microscopesreq! re a lghl baancing ' i r L B D - 22. N ) EKachrorne 64 EKachrame 100 Dayighttyp€ Agfachror.e CT18 Agiachrome 50Type S Agiachrome 100 Fulchrome 50D Fujichronre r00D t4 g Eldachrome50 Agfachrome50 lype.L ::quiremenls forthe selectionof colorlilm I m w t h h q h r e s o l v f gp o w e rs. f c e p h o l o _ .rograplryrequres disoiayoi dela ed -_! d i s p a ym u s tb e a b e t o d s c r m n a t e :.lween the f ne coor dilferentalonscon. : .i e d i n a s n ! e s o e c m e n . : : l h l L rr e p r o d u c t ooni s p e c m e nc o o r s inoul backgrolndd scooraiion l. vew or theselactors the iolowng condi tons can be sel ior pholomcrolraohy ! l!!qq1! (2)Good colorcontrasl l?lqlgqJ9!9r!9!!Iro! For noma photomcrographyn lrans. m l r e d l g hbr r g h l f i e d u m i n a l i ocno l o ri m wth an lSo/ASAsoeedbelween50and 100wi ass!re salisiaclorvqla i1y.For specal cases s J c ha s p r n t e dp i r bc a t o no r e n l a r g e m e n i s <cd:chro'.e fim oijers lood qla ly ng dark specmens(phase r.,/.e. oholograprr conlrasl poarz€d ght. 1l!orescence) exmsurelime ncreasesAtiroLrghtirere .nay be ercessve gra n for cases reqLrr n-o iasl shullersoeedslse flm willr a h gh rSO/ASAspeed Typesof filters Fill€rs Seectionoi filers lor usein phoiomicrc graphyis basedon lhe tlpe ot I m used. lor daylight UseLBD-2N lilmsandLBTI lters lor tungsten typelilm Forchanging lighl inlensity, useof a neutraldensiiyiiler (ND) is recommended. Thenumberon the lllter rirngiveslhe transmssion valle In case oi iillerND6,94% of rherotaliluminaling lighiis absofbed andony 6% of lhe ighl is lrafsmiired. Typesof lilter and their lunctions F tersior lse n coor pholomicrography comprise ihe io lowingmaintypes: I Lighl balancinglilter Toconverllhe colorlemperalure oi ihe mlcro scopelghl sorirceto the lih beingused. eND (neuiraldensity)lilter .F a To feduce the lighl inlens ly without atiecllng color lempelalure,when llluminalionis too b.ighl. 3 CC (colorcompensaling) liltor suchas slghldi Forcolorrendlonproblems pholos. lerencesin co or hueor ladingin developed a4-Didymiumlilter (markedFF on Olynpusmod6ls) € Heatabsobinglilter Toeriphaszecolor,i.e.wirentrying1ocompensate tor the insullicient inlinsicredcolorrendition oi a llm or lo enhance theredcolorofthe specimen or lhe coiorcontraslol Polaroid coiorfim. To absorbheal raysemanatingtrom lhe jghl sourceof lhe m croscopelo preventdamageto or deslruction oi livespecimers. Sincelhisfiler lransmils a smalqlanlty of bluelighi,I may becomenecessarylo usea color.compensal ng lilter (CCl0Y or CCl0M)\rheniaklngco or photos. depending in colorrendition Differences of filmandlightsource on thecombination -.F are tro types0i coor i n's d.y 9r1 sm .r _vrh.n !s n,othe llngslen lype I m - 1 b ra n c e dt o r s l n g r i l : n d i L r r l s l e f o l t c o o r sa b ! s i r . l ' o t o qa t t s o b t a l ^ e o The ta! I r Lrclrc:ses s ta Lrr. 10lse : . 1 b a a n c e cI o ' a r t i c l a g h 1 ). h es e e c lo n :he pr..e- I r. .r r:lcf .he ,ori sc! cF . . i d n g u p o ni h c L l l n ls o ] ' c e V r l l a y . s e r sh a v ee r p e r e n c eich ea s a c p o n l m c n i r e r o h o t o sa p p e a r . ! ? l c e s s l e y r e d _ ' ! . l h e yl s e d d a y Slr_ m lnder n.andes lo or rendilion dependingon the combination ol lilm and lighl source Daylishl.lype(sunlighl) I Tungsten.type(adificial lighl) incolorreproduction depending Differences in colortemperature on differences Color l€mp€ralure Dillerenc€s in color reproduclion d€pendingon dillerencesin colorlempefature Coor temperaturedesignatesthe propenles of the ight source.A blackbodyradator. whenhealed.emanalesighl of dfferenlcoor dependrng on the temperatureThe properlies oflhe ightsourcecan be nd caledby reiering to lhe lemperalLrre oi the backbodyat a fxed ter.peratureevel.Ttiis ieature s ca ed coor tenrperatLrre the numericaLrnlbeingex pressedether rn absoute lemperalureor in degreesKevin (K) C o o ri l m f o r u s e n p h o l o m c r o q r a p h sy normaiydaylghl lype bul sincethe ight sourceoi lhe mcroscopeis a tungslenlype ls coo. temperatures low (2800.3400rc and as such il is unsuilableior daylighlfllril wlh ts color lemperalureoi 550C6000K In orderto achlevethe propercoor rendilion a ight baanc ng i ler is used,and llre lungslen ghl s convened10dayight l j , ',. ' s h qh l exhbilsa'blue s lli colortemperalur". s low lhe photoshowsa redsh11 and .doi renoeralure E ' > in colorrendition depending Differences on the typeof film Colorr€r'dilionfor diflerenttypesot film f.om lhs sam€manufacfurcrlendsto vary,ard evenditferentorcduclionlots of lhe same typeshov/sljghtdtferences. Whenyou slan to lake color photo6,lirD drouldflrst d6l6mlnelhe oFffmumcondilibns to thenlmthrought€stphotogkphs,matchlng flem wih boh micrGcooeand soecimen (Fot the methoclol taking t$t photos rctet to page 54) Offl€r€nce3In color davalopftGit &pddlr! dr lh. m. ol lllm I}re 6lx ptroloGon the rlghtweretak€nwllh flms ol dlfler€it brancls,and wflfi thE €xc€p tbn of lhe ISOASAspeedall drotographic conditions wereidentical.lhis €vampledearly *lo € that propertiessuchas color rerditior! contrasl,cladtyof baclgrerd, etc.are alldlfftrcnt becauseof lhe variousbrards of tilm. Photognphlc condldort BHS, PM.ioAD, LBD,{ (color tompontu|l 5500f0,LBT (color tampor.ture 3,mr+ duii.. 3pe€d0.1+l s€c D.tlglrt tyF llrngd€.r lpe Purchase of colorfilm 1. Whatlo walchlorwh€nbuylngcolorlilm I Coorlilm is a hghy sensitive subslance, andenvircnmenta laclorssuchas heal andhumdityeasilycausechangesn film speedandcoor rendition. Whenbuyng coor film,avoidcamerasioresin which the sheves slorng color t m are exposed to sunlghl. I Choose onlycobr r m wiiha sufficienly longpeiod beforethe explraiion dare as markedon lhe package. I Coor fenditionoi lhe samelilm tlpe may diiier,ii il cornprises dltierenlprodLrction lots.li youlrequently lse coor film,you photographic canuseil underideniical conditions andavod varaiionsin thecoor ol the t lrn if youbuy argequanlilies irom the sameproduction lol. EE: ;:! 1602/54 sg: im of Yalldlry(ddEnon dal€) 3. Poinlscalllngtor sp€clalatt€ntlon 2. How to slore lilm whenusinglhe lllm Forthe basicpinciplesol composlon and colorrendition checkthe lechnicai literalure I Donotleaveihe Jimin ihe cameralonger on lhe subjecl.As hasbeenpoinledout, perforrnance changes according 1othe con. dilionsunderwhichthe f m is slored, ColorI m is normallykept n therelrigerator lo protectit irorn the etlecls ol heatand humidjty. Removelhe lilmtromlhereirgeralor t hourbeforelse andalor t 10reachroom lempe€lure. Do nol remove ihe fim lrcm lhe rc immediatey aftertakinqit out ol the /-f--\ *, [ (,r , /-F\ I Developexposedf m as soonas possible. @M z % o reirigeralor lo prevenlcondensalion on lhe I I ;:E ii I Eventhougholherpholograph c condilions colorrendilion mayvary maybe idenlical, wlh lowor highambient temperalures. I Do nol usethe fim in a gaseous en(suchas lorman),sincecolor vironment rcndilion wil be aclversely aflecled. (CC)filters Useof color-compensating whentakingcolorphotos liltersarehighly S:ncecolorcompensaiing sensilive lo heai,do nol usethemclose :c lhe lighl source. ICC filter is the abbrevialionfor colorfiler,andlhe coloroi ihe compensaling parllcularJilteris markedin capita letters on its rim.Sixdillerenlcolorsare available, eachcolor beingofferedin six dilferenl oi densily. No.5 andNo.10 are degrees mostwidelyusedin pholomicrography. lcolor fltersare madeby severananu'lacturers. CheckyourOlwpus dealeror camerasloreior avaiiability. a repuiable Color to b€ r€duc€d BLUE CYAN GNEEN Color€mp€n6ating lllt.r rcqulr€d ccY Red Magenla YELLOW Blue RED CYan MAGENTA Green ccB ccM ccB CCG H Testphotography 1. The need lor lest pholography equp' Evenii you |]seaulomatcpholographic menl and set bothlhe ISO/ASAspeedand lhe rec proclly aw iaiure characteislcs, you cannolbe sure that yo! wil always ior everytype ach eve the perfeclexposLrre ol specimenThe reasonior llrls s that phoiograplrlc equpmenl s mafufactu.edto malch dstfbLrtonstate,coor and exposure slandardoi averagespeclmens.ln orderto obralnbelter resulls,you shold I rst lake tesl pholographslo match lhe conditionsoi iilers etc the sp€cimen,fim, nricroscope. 2. Method lor test photography Charl2: Datacharl (when using nadels BHS and PM-12AD Color Colorcom. and reversal filn) lemperelure Exposure (1)Prepare one rol oi illm (36 exposlres)and 1 a irequentyusedspecmen 0.8x No com' (Z)Setlhe lest condilions(seechan 1) and 2 1X pensar on preparea data chait (seecharl 2). Listng 1.25X a the condtons gven n charl 1 wil resLrll in a dala chan 2. TakeyoLr photosbased 0.8x_ 1 bgooK on this chair and delermnelhe optmum 1X 5 o r CC5G pholograph c conditionson the basjsol lhe 10v resulisoblaned on lhe developedI m. a 1.25X it 7 8 9 cc5M 0.8x lx 1,?sI J9 ssoox sv 1' 0.8x 'lx '| 25X Charl l: Testconditions 170mired,180m red, lmmred, 5900K 5500K 5260K Volase 10V,9V 8V 0.8x, 1.25X Measuingwilh CTR No CTR ng I lter Color compensatlon Coorcompensal 3. Evaluatlnglhe test photos observalon oi reversali m transparenc es can be caried oui eillrerwilh a lighl box or a slideprojeclor,bLrtihe dispiayof cotor vares wde y wilh lhe colortemperalureot lheselighisourcesAs a slandardior evauaf ing the pfopef coor Lrsea ghl box wilh a color 'Tl]e lemperalurecloseto 5000K. io lowinglp€s ol lghi boxesare avaiableon lhe market C Fuji film color box 5000 O Dlrotesl ColorClasse.50 O GeneralEecrric Chroma50 4. Organizingphotographingdata [.4akeI a habil1o recordal dala rearing io bollr lesl phoiographyand reguarphotc graphy.lf the pholographiccondilionslhat resulledin goodpholosare relaned on i e. lrolbeshoolng and correclon of probems can be caried oul quicklyand efiiclenly. Cc5Gnocorapensat on,c,cs\,tr 5. Slodng developd film Whenstorng I m ior a ong perod oi tme. the most imporlantprecaulon s proteclion frornlight Siice mold is prevalentin hot anc humd paces, t is advlsablelo keeprhe iim liglrty seaed, togetherwilh a desccant We recamnend storing film in an envnon. nentwith a relativehunidiry of 1U0% and an ambient tenperaturebelow 21oC(71'f ). (fron Kodak Colot OKP 141instructions). photography Black.and-white film Black-and-white : nce the condtons diier ior plrotomcrcr phoiography. i1 s : aphyand generaL :cessaryto seecl lhe properiim Joreach '.oe oi pholographyIn photomc rogfaphy. ! h c o n l r a sjl m w t h r n e g r an i s u s e di n -.der to documentm nule slrircluresol : oo! ca specmensand to achievesharp :_ololraphic reproduclon.Examplesoi lhs n typeare KodakPanatomc X. Agiapan25 .rd Pan F. and Fll NeopanF I I € ,:jl r# Jse ol black-and.whileliln ':re are manylypesof blackand.whle ilm j the keytoobla ninggoodpholographses r e e c t n gt h e r i g h tf l m l o r a p a r l c u l ajro b Specinren Normaor hgh conkasl ior tlreslanedsecllonoi generapathoogical Agfapan25 KodakTechnicalPan palno09 ca speclmens. 2415 as we as thoseol other Agfaonho25 Wlren shape s areas. more mpo(anl llran Agiapan 400 Dark specmens.and when ong srrltter speed l l o r d H P 5 SO/ASA 32 50 25 32 on the coor 01lhe 1 Srce the conlrastoi the film is very lrigh,the rangeor Pro 64 per exposureis very narrcw expos!reis sel wlhln 100 Uriless +l/3 slep oi lhe opi murn 25 lhe pholow I be ether over 32 *r,"nvo, X33 u""t eseryp"" 400 want10shol(enexposlretme ef flm glan 5 G 1 6 0 0a 1 l h ee x p e n s o 400 Comparison film brands of different Typesoi fim orovdinga wde gradalon iom shadowro h 9lr _ohts and featurnggood conlrastare Fll NeopanF and Kodak PanalomcX. TechnicaPan oiiers s ghty h gher contrasl wh le N4n Ci]pyerimnales NeopanSS and Tr X on lrre olher hand. havefanly o!\rconliast Kodak Techncar Pan 24t5 t Filters Ellects ot verlous conlrasl lillers Us€ ot contrast lillors Conirastfllers are usedio conlrollhe conlrast pholos. or b ack.and'white green Forblackandwhilepholomicrography, i tersare normally used,bul choceoi lhe mosleffective flter depends on lhe tlpe of Filterclhatenhafcethe colors specimen. andconlrastof the specimen are: Color ol lh6 Colorol rh€ tllt r Green Blue Yellow/orange Oranqe Blue usea Ulleroi lhesarne Forreducing conlrasi, .oloras lhe specmen. Reasonsfor using a gr€€n lill,ar -iere aretwo reasonswhyconlrastimproves shef a greenllter is used. T)Snce objectiveabe(ationsare rnosl effectivelycompensalednearlhe green wavelenglh, lossoi imagec arityduelo chromaticabefialionis avenedby a green I ller. 2)Dyessuchas hematoxyln andeosin in higher absorbgreenlighlwell,resulting conlrast whena greenlllleris used. the red po.lion is €nhanced lhs r€d Porlionis reduced Photography with Polaroid@ film Oneof the acl€ntagesof Polarod litm is thal I canbe quick]yvlewedas a iinished pholo, bul il is seldompossible10oblaincotor reproduclloncomparableto thal oi 35mm ol lllm us€d ln r.Pny t 1E' xS th' (actuelslzo73crnx 9.5cn) I k I a n I Type 107 67 tso/AsArackcontainin! Tlpe spe€o 8 photos 3m0 3000 o o ISO/ASA speed 52 Sheel 4(J0 o 30m o rack coolaining I photos o 4m 665 (PdnU I negalave) 50 108 80 80 I ,l'x5" (actualda gdnx11.5cm) 668 669 14'x5' o 55 (P nU negative) o o o 80 50 58 59 80 559 80 80 2. Photogrrphlc t€chnlqu€s l1JBIackand -whi te photogh phy Usea greenfilter to obtaingoodcontrast. (2)Colotphotogftphy Sincelheoverallcolorhuetendsto be either ighlgreenor blue,useCC10-20Mor CC10-20Ylor compensation. ll youwanl io enhance colorconlrast, usethe FFiiller availabletromyou. Olympusdealer. SincePolaroidiilm is morc easityatfected by reciprocilylaw Jailurethan normaliilm, exposurclime shouldbe adjusted10belween 0-05and0.5seconds. o o o o filmhoLclers dilleraccording to lhe typeot titm sh@i lllm-:(s typei m hotd€r Packedfr/m-550 typ€ tllm hotdef by'haPora|o]dcor@ralioi'camb.ido€.MlEs'U's.A' 4500K-LBE!2Nolus FF tllto. cororlemoeraiure SectionS Howto obtalngoodprinis . . @-61 H o wt o o b t a i no o o dc o l o rp r i n t s. . . . . . . . . . . . aPreparingblacLand-whitebrlnts. . . . . . . . . . . . .. . . 62 Maiks on the Dhoto. . . . . . . . . . . . . . . , . . . . . . . . . . . . . . .63 ... ......64-65 Howto avoidmarksduringdevelopment Howto obtaingoodcolorprints ThelollowlngdiagEmshowshow to get good color pdnts {positiyepdnb). Color negalivelilm ln pholomlcrography. lhe colorreprodlction oi lin shedpr nts takenwllh negalve fi m differsirom the observedcolor-a lact many usersare probaby very muchawa.eof. reasons forthisarethalthetechnician Possible who madelhe pinis d d notviewthe speci mencolorlhrougha microscope andthus didnol knowlhe actualcolor,oathe pholc gapher madea mistakewhenhe lsed a f ller andlailedto noticeihal lhe iilm quality deteriorated. In ordeflo gel beter resulls, you shouldlirst photographlhe specime. tromwhichyouwanl lo get prinlson reversa R6wtsalfllm photoas cojor llm, andatlachlhe iinished reterence sample1othe negative lilm.Pinls canalsobe madelromcolorrcvercal {ilm, bul lhe processdoesnot matchthe pint qualityas obtainedfrom negativelilm. a Color pdnl with Poor color r€produclion Color pdnt with good color reprcduclion Preparing prints black-and-white Therearemanytechnicat pubtications on ihe markelllrat exptan in delaithowto devetop iilm. li you planto do yourowr iitm pro cessrng,uselhesebooksas reference. Butsinceconlrastin photomlcrog.aphy is owerthanwithnormalphoiographlc subjecls, youshorildpaypanicular attention lo under. developmenl. ll youmakea positive print iromanunderdeveloped, /ow-density negalive, an evengrealerreduction in contraslwitl occur,badnglo a poorresuti. 1. How to obtain a good negative (])ljse a devetoper thalmalcheslhe iitm (2)Usethedeveloper allhe lemperalure specilied by the manuiacturer (3)Pdherelo the speciiieddeveopmentiime lhal malchesllm sensilvity (4)Agilate genllyandfreqlenllyto eliminaie unevenoeveopmeni i5)Makesurelhal lhereis neltherloo much nor loo Itl e it ng (6)Followthe paperspecificaliolswhen washing the prinl carelutty andpfolect OHandleihe negative 1 lromfingerp|nts, scraiches anddusl 2. Selectionof phorogEphlc pap€r Whenmakingpints, youcanvarytlte contrasl dependng on lhe t 9€ of pinting paper. selectthe lpe that bestsuitsbolhthe densityof thenegativeandsp€ctmencontrast. Theexamples on ihe fightshowprinlsmade iromlhe samenegative whenusingdifierent tlpes 01pinling paper. Gradeno ol photographlcpap€r t€ Grade3 nomalcontrast Markson the photo 1.Ma*s as a esult ol negaliv€d€veloFnent 2 Ma*s occuriog dudngprinl devglopment Markson the printtendto slandoul more prominenlly in pholomicrography thanon nomal pholosol p€opleand landscapes. empoyslilm with Sincepholomicrcgraphy faiiy highconlrasl, eventhe rosl minutedif ferencesin brighlnessshowup in the piclure. MarksresuI ng from improperdevelopment Thissecliondeals arealsoveryconspicuous. wth marksresulting lromdeveloprnenl. Bothuppd and lowerponbns of lho photoare bnohr* thanlhe centralsection. Markswith iregularb qhlness.howup. How to avoid creating ma*s (1)Confirm ol lhe thedegreeol exhaustion processorto develop It youusean aLnomatic lhe prinlingpaperno markswillocclr,but slncemar!€caneasirysho/ up when deveopingin a t€y, rhe io lowingpoinls Carelully noteproducl on daleandlre. quencyol useof the deveLoper (2)Agilale y beioreLrse. How to avoidcrcatingna*s thedeveloper lhorough (1)Agilale lhoroughly. lhe develop€r (3)Usethe developer al lhe specified (2)Keepthe lemperalureof lhe developerat 20'C.or as speciiied by the manuiaclurer. (4)Donot workwlh an exposirelhat resllts markscaneasilyoccurif deveor ina developmenl limeof Less lhan5 mlnules (3)Since (lheresullwil be exlremely clesensitized menttime s too short,set the exposure of lheenargeral sucha evelthatdeveloP developmenl). menttirnes betweenI min30 secand (s)Agilale duringdeveoF lhebaihthoroughly 2 min. (4)Agitate thebalhthoroughly duringdevelotr (6)Mainiain lhe speciiied lixng lme. y. f)Washlhe filmthorough (s)Conl nueto agilalelhorclghyevenwhile andiixing. stopping (6)Wash thepinl lhorcughly in runnlng waler. paper 30€0 min Regularbase paper-4.5 min Resin-coaled Howto avoidmarksduringdevelopment DoYslopmentol tllm 1 2 lmmerselhe ree in lhe developer,and aiter turningii 2'3 limes,lap il anolher2 3 limes agansl lhe botlomol thelankto remove a r bubbles sllcknglo the fim surface. Complete thisprocess wilhn 56 seconds. and Nowremove the reellromlhe developer immerse il again.Aflerrcpealng mmedialey iurnlhe ree immersed in lhs procedure, 23 times,thenpausefor lhe developer 30 seconds. Sncema.kson lhe lilm occureasilywhen uslnga ree-tlpelank,developmeni should be donein lhe io lowingmaanern the Development ot pdntlngpaper t, '@@@ .l 2 I Thisseclon dealswithdevelopmenl using X a tlay. / --- - - - -\- Whie holdlnglhe edgeol the exposedpr nling lAs shoM in lhe piclure,do noi immerse paperlighllywilh a pair of longs,quickly lhe prinlng paperinlo the developer parallelto lhe developersurface,bU till place il inio the developer. il nlo the deve oper balh. lollowlhe Alter completingdevelopmenl, iixlng,andwashng sequence of stopping, nol b€sut Thodeveloperwill ficientlyaoilaredsimplyby tuning lhet@|,andtheresull will be sporson ihe lilm, lh€ rcelfromlhe dev6lop.r,lhe By @movlng d€v€lopscanreachlh€ spac€sbetweentheroll6d p€vontlno lilm, lhe oon€nlingofspols. nlervals I I the compielon Pedormlhe operalon shownin piclure2 of ihe leftin 3o.second work. ol developmenl paper I Makesurethallhe photograplric doesnol float!p lo the surfaceof lhe ro agilale the phologGphic paper, ho d il at a cornerwlh a pair oi longs and move r back and lorlh whlle I is submergedn lhe aeveloper.Repeatthis operalion in 15-second ntervas unilldeveopmentworkis compe1ed. workon lhe Aftercomplelifg development prntingpaper,qucky pe.lormthe sequence fixing,andwashng. ol stopping, sheelsot prifilng I Makesurelhatseparale paperdo nol sticktogether if thefxer I Do nol usethe samepar of iongsduring andlixinglhalyourseddu.ng slopping developmenl work. thedeveloP Makesurenolto codaminale ef wth e lherslopbaihor I xer qud. Section4 Trouble-shooling P|obloms in linished pholos and lheir con€ction Poorcolorreproduction . . . . . . . . .. .. . . . . . . . . . .. .68-7'1 B l u r r ei m d age ..,,........72-73 Theimageis in focusbut not sharp.. . . . . . . . .. . . . 74-79 Objectsother than the specimenimageappeared on the tilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80-81 U n e v ebnr i g h t n e s s ........82-83 Prcblemsin finishedphotosand theil conection -Poor colorreproduction 1. Th€ beckgroundis coloEd (.€dibhF) Caul6 Cor'rctlon Rama*s Rel€r lo Daq€ The color temDeralu@of the lliuminatlon ls not malched to that of the fikn. .Lioht balanclngfllter specified by manulacturerls not us€d. .ljse flltels sp€clfled by the manu' lacturer. .Daylisht type: LBI!2N, 5,5mK, D settlng .Tungst€n type: LBT,34mK T setting Page48/ page 50 Lamp \oltage ls too low (loo high). .Raise (low60 lamp voltag€ .BHS above&5V .BHT and BHTUat 5-6V .lf you have reachedthe desired color temp€ature, do not chanoe the voltage position. lf you want to chango lrieBity, u6€ an ND fllt6r. .!!b recommendthat for iest photograplrythe voltage should be set lo the standad Dosition +1V whgnfi6t detednining lhE ootimum color temDeEturc. Pag65(y page 54 6v-rcltege ls t@ low {Bd hu6). l1v-rcllaoo l. too hloh{bluohu€). Blackenlngof lamp due to pre longed Uae .Replace wlth a now lamp @ 2. The backgroundis color€d (gr€€dmagenta) R€marks R€f€rto Deq€ Caus6 Con€ction .A iilm dilferentfrom yourusual film lype was used. .Since colorrenditionvarieswith differentfilm typesevenfromthe samemaker,choosea lilm with coloringsuitablefor yourtype .A iilm of the sametypebutwith difterent emulsion number was used, .li possible buyfilmwiththe numb€r in laryo sameemulsion quantities. .ln orderto maintainthe performancequalityof the film, slore it in a relfigoralor and remove it one ho0rpriorto us6,allowing it to reach room lemperature, .Even for film of the sametype therewill be slightvariationsin colof reproduction dependingon labofatory,developmentcondllions, and stainingof the specimen. Pag652 .l.Jsea color€ompensating (CC) lilter 'Color€ompensatin0 (CC)tllters Buyfromyourpholodealer, .Too muchgr€€n UseCcM {mag€nta) .Toomuchpink UseCCG(gr€en) Page53 a a t a rt a I oC O ta t t l O - t t t t ar I e aB t l a + t ra rt cc05M-optlmumcompensallon a a O rt :i. r.l r a a * .a cc10M-ov6r{ompensared Page 51 usecc Forphotomicrography, filters0+10.ForlurlhercompensaandCClologether tionuseCC05 3. Inconeclcolor rendilion Caus€ Con€ctlon Remarks Relerlo pag€ .An excessively longexposure time hasbeenusedandthe characieristicsof reciprocitylaw failureleadio incoftectcolor rendition. .Set shutterspe€dat 0.01{.05sec. Adjustshutterspeed uniJormly as fal aspossible byusingan ND{ilter. .When usinglongexposurelimes (above0.5sec)wilh models PIV'1oAD and PM'1oADS, set the characterislics numberof the film usedon ihe dial for reciprocity law failure, .With longexposuretimes,even wiih the exposurctime compensated,thecolorfenditionchanges as a rcsullof film properties. Usea colorcompensaling filter speciiiedby the film manu' .Automaticexposurehas been usedwithouladiustment. .When usingmodelsP[,]-1oADS/ PMJoADset the exposureadiustmentdiallo: 0.8-0.25X for bighl background specimens andto 1.2t4Xfordark backgroundspecimens. .Carry out exposu€adjustments Brlghtbackgrou nd:ISO/ASA 100to 50 Darkbackground: ISO/ASA 100to 200 .ll you changethe magnification Page4Z of the objeclive,the lightdistribu- page 43 lion withinthe visualfield will atsocnange. .Aclual and nominalsensitivity ol lhe iilrn differ .Vary the ISO/ASAspeedsetling. .Nominal speedmay diiJerlrom actuallight sensitivityas much as 1/3to 1/2aperture. Page 216 Page 42 4. Poorcolorol a prinl enlarg€dlrom a negative .The printingtechnicianin the laboraloryis nol sureof lhe subiect'scorreclcolor. .Add a lransparency of the same specimenwith correctcoloras a sample. .Better coior is achievedwhen with shootinga transparcncy reversalcolorfilm (butthe cost of pints goesup and quality 1 5. Poorcolorol Polaroidlilm Cau3e colortemperature. .Abnormalcolor characieristics. .Term of validilyof the lllm has .ll roomtemperature is abruptly lowered(raised), sensitivityis alfectedand printcolorturns towardsblueted). Coneclion Remarks R€ferto .The film is a daylighttype,but becauseof the film characteristics colortemperature shoutdbe set lowerthan for 35mmfilm. .lf you s€t colortemperature b+ tween2rc00K and 5000Kand use an OlympusFF filter,color contraslwill b€ enhanced. Page58 .lf the abnormalilies are within the adjusimentrangelor color temperature and withinthe operalionalrangeol the colorcompensatlng lilter,determine ihe conditionsbytest photography .Use film whoseterm of validity has not yet expired. .Since Polaroidfilm is easily Page54 aftected by adversestorage page58 conditionsas far as colorcharacleristicsare concemed,protectit irom heatandhumiditybystoring it in a refrigerator. .For developmeni time and color reproduction, referto the film instructions, Blurredimage l. The ov€rall locus ol the oicturc is blun€d Cau3€ .Finder 6yepisceor focusingt6lescopeis nol prope y adjusted, Conlction .Adjust thediopteruntilthedouble crosslinesare clearlyvisible. Remarks .Slnce most peopledo not have the samevisualacuityln both eyes,d€l€mlnewhicheyeyou alwaysuse for focusing. page Pages 2e27 lt ll .Blur anddrittresulting from vibrations. .Uss a vlbration.proof table. dF lm.o..fi6t.d by vlbEttons .Uss an ND fllt€r and lncrease shutter speed (1/2sec to 1 sec). .Use a standfor the photographlc equipmentto separatephotographlcequipmenl andmicroscope. Page12 paqe 48 2. Focuslngenor occurs wh€n you u96 a low.magnlcallonobiectiveol less than 4X Remafts Ceus€ Coar€cllon .lf magni{ication is low,tocal depthat the lllm planebecomes shalloq easilycausingerors in focusing. .Mouni a locuslngmagnifierto lhef Indereyepiece or thefocusing lelescope,and afterfocusingon the doublecrosslines,fine focus the specimen. .lncreasingthe magniticatlon eliminate6tocusingerors, Reiar pa96to Page27 3. The p€rlph€ryls unilomly blun€d .An Achromattype obj€ctive was us€o. l,r,i';.,r', 1.,iil;.' R6ma|ks Coreclion Cau3€ ausea PlanAchromat type obiective Reler to paq€ Pag€14 ;;1..i.'i:.1 ;i:'-:: l,i;"li ;1 /:::).\ .'- -::' .):tt. :: ::.:':itl:i: -r11.;'ij;:. : .ij, ':i ;';iiii,''''r,;ii'':.:r'::;ji liir'i'.1.t...'1,.: :',:,1:,11i,;i;,,.,. .;.,. ;t::'. . - . _ :J : - - i : . . ! : - : r . .: .i!:::.:'1:.:):.t . D Plan1oXNFK2-5X .Objective and photo eyepiece were usgd in th6 wrong combination. .LB series(longbanel)-NFK photo eyepiece ashort-barrel serles-FK photo eyep|ece Page13 Theimageis in focusbut notsharp 1. Inadequat€ resolvingpower .Use ol a combinationof lowmagnification obiectiveand highmagnification pholoeyepiece, .ln orderto obtainhigh resotuiion, usean objectivewith large numeicalapertureand a pholo eyepiecewith low magnification. -? ,1 .Stop downthe apertureiris diaphragmto 60€0% .Use of the condenserwith lhe apenureiis diaphragmfully opened. .The field iris diaphragm was fully opened, .Use of thick coverglass. straylight,slop downihe field iris diaphramto an areaonly slightly largerthanlhe .Use a coverglasswith a thick. nessof 0.17mm. ': ' -:-. ,, .'''. ' - : .= ' . .. '.t .ln orderto obtaina magni{ication ;i ioot i; tniirr. pti ", ,iJ- eitheran objectivezloxwith a photoeyepieceof 2.5Xor an objective20Xwith a pholoeye piece5X.To increaseresolving power,the combinationof obiective40Xand photoeyepiece2.5X i (Focaldepth, is pfeferabie. however, will b€comeshallow) .Vary the amountby wh;chyou reducetheaperlureirisdiaphragm accordingto the magnificaiion of the objectiveand the contrasi ol the specimen. .Do not reducethe diameteroflhe field kis diaphragmto such an extentthat it louchesthe frame reticlebecauselhe aclualarea photographed is alwaysslightly largerlhan the areawilhin the lramereticle. .Olympusobjectives lor biological specimenshavebeendesigned in sucha way ihai optimumresolvingpoweris obtaaned when a coverglasswith a lhickness of 0.17mmis used. Pase 38 Cause Cor€ction .The specimenslain is too weak, resultingin lack of conlrast. (Photographing a specimenthat cannotbe stained) .Use a denserstain, .Use a contrastlilter (fof black-and.whiie pholography only). .ll the specimencannolbe slained,usephasecontrast,differentialinteF iercncecontasl or darklieldto createconirasloDticallv. Beler lo pag6 Page57 gee G*,il _Effiil ST oiii€Gnlial inlorfercnce conlrasl Cause Remarks ConEctlon Colorphotography Ref€rto peg€ Page51 .Use of a low convasifilm, .l.Js€a high conlrastfllm. :', r-'d):.:')*.:;i; tt))b,;nite-:i i:);'.':ii;i;?;":i photography Black'and.whlte .Variationsin lhe so€clrals€nsitivityof the film ifiect ihe Page57 .Normally,a greenfilter is used, but if you want to emphasize a sp€cificportionof the sp€clm€n, use anothercontrastflller, .lf a filtercomplementing thecolor of a specimenis used,it will emphasizelhe contrast. 2. The image appearshazy Caus€ ConEctlon aTheconectioncollarof the obiectiveis not adlustedto the lhicknessof the cov€rglass, ,i , . r, 'Ja. 'rt . . j .:-1 'l: i ,' '-r',!! ba . ' . r . ' l r . .Adjust the conectioncollarwhile €xaminingthe specimenand set it at a positionprovidinga cl6ar image. R€marks .On the LB objectivea correction collaris mountedon the S Plan Apo 40X S Plan100Xdry and D PlanApo 60X. Rolorto pags Page16 Cause .An objectivenormallyusedwilh cover{lassedspecimenswas ussdon a specimenwithoutcover glass(orvice vefsa), Bema*s Con€cllon .Use a no{overobjective. ' ji;t, ) \.'1t -.J Relgl lo pags LB objectives .NC S Plan40X .NC D PlanFL mX .NC S PlanApo 1mX oil .NC S Plan100Xdry Page17 .lt you use dry 40Xor 60X objectivestogetherwiih an oil. ths oilon ihe immersion objective, specimenmay soil the fronl lensesol lhe dry objeciives. Pages 26- 32 '\ )/ '-. J .'1\)/' U) . . ^ \ J \ - ' ,. 4 )- ''(t -\ !.- !i - - - , ,- ',--'(. r ' - - \-v:' - 'J .Finqenrints,an oil lilm or dirl particlesin the opticalsyslem (objectivefront lens,photoeyepiece,prism,specimen,etc.) ..,t, . 1: ,', .' .-.t . .Clean lhe oplicalsystem. .Alwayscoverlhemicroscope with a dust coverwhen not in use. 3. No sharpimag6is obtainedwilh a 10OXolflmmerslonobiective Cau36 Conection Remarks .No immersionoil was used, .Use oil specifiedby the manufacturer, .Unsuitableoil was used, .Use specifiedoil, sincethe types andnomal usedfor fluorescence white light oftendiifer .The oil containedair bubbtes. .Apply th€ oil aft€ryou hav€ removedbubblesln the botlle. .Usingoil in a roomwithunsuitable (toohighor low) temperatures .lf roomtemperaiure is eithertoo highor ioo low,o. if the air is too humid,the diffiactionindsx changes,causingchangesin the imag€,Usethe oil at a room temperatureof 22-25"Cand at a humidilyof about56%. .The specimenis too thick. .lt is advisableto usea specimen with a thicknessof 2'3r. - l, r1S:. '.o'*. -j- p !.'l R€l6rto page Page18 Page18 .Removelhe eyepiecebefore examinalion andlookthroughthe Page18 Page12 lllm ls nol sharp 4. Entireroll ot black.and.whlle Cruse .Possiblecausesare:type of film, emursrcn, overexposure, overdevelopment, improperhandling or accidentsduringdevelopment, Con€ction Rema*s Relerlo oaQ6 Pages .Check lhesepossibililiesand B{- e,5 5.Th6linishgdpdnl app€arsgrainy Caus€ .A fllmwlthcoarseorainwasosed, R€mark5 Coreclion .Use a finegrainfilm. .Kodak Panatomic X .Kodak TechnicalPan2415 .Agfapan25 .lliord PanF .A slandarddevelooer was used. .lJse a linegraindevelopet. .Kodak Microdol D-23 D-25 .Fuji MicrcFine .Magniticationratiowastoo high. aL,sea largsformatfilm. .4" x5" sheetfilm. Referlo paq€ Page55 image Objects otherthanthespecimen appeared on thefilm '1.Shadow.llk€ lmag€ Caus€ Con€ction .Optical palh selectorot the pholographlc attachmgntor vinc cularlube was intenupledat .Engagethe opticalpathselector at its prop€rposllion, .The lield kis diaphragmwas stoppeddowntoo much. .Open the field kis diaphragma littlewiderthanthe photographed areaot the lindereyepieceof the focusing telescope, .Tlny bits of film, dirt,etc. stuck to the p sm of lhe photographic equipmentor to the largefomat .Ch€ck for, and remove,dirt from the prlsmot the photographic atlachmentwhilethe shutleris open(Iime setling). .Flemovethe largefomal relay lens and cleanit. Remarl(s Feterto Daqe Page23 .Periodicchecksare recommendedif a largenumberof photographs is taken, Pages 2931 I Cau3e .Dirl In the opticalsystem Corectlon alocate the dirt and removeit. Cause .When movingthe film or when unrollingthe backingpaperof the fllm, staticelectricitycauses spafis. Con€clion R6ma*s .Yo0 can locatethe dirt by moving and rotatingeachcheckpojnt, alt€natelylooklngthroughthe blnoculartube,the focusing telescopeof the photographic attachmsntand the lllm plane (by placlnga pieceof frosted glass in the camsra body.) R€lerlo Page PageI Pages 30-31 R€l6rlo page .Do not rewindlhe film too rapidly. .Keep humidityat zl5%minimum in the roomwhereyou handle the tilm. aMakesurethat the camen back and the darkroomars lree of dust, 3. Rellocllonol windowor roomilluminatlon Cause .Stray light gntsrs from the ey€" piecesor lhe focuslnglelescope. Coneclion .Move lhe oplical path selector of the tdnoculartub€io lhe Camera1m7oposilionandcover the locusingtelescopeof the photographic atlachmenlwilh a cap. aPut capson both eyepl€ces and the locusingtelescopeof the photographic atlachment. .Set up the mlcroscopein a dlllerentlocation. Referto Pag€ Page12 Uneven brightness 1. lJn€yenareas occu. on one sld6 of th6 fram€, in lhe c€nt€r,and under the pertoratlonof th€ film Cause R€ma*s Con€clion .Th€ microscopelight sourceIs not prope y centered. .Prope y adjustthe light source. .The field iris dlaphragmis off axis. .Clos€ th6 fleld his diaphragmso that it appearsin the visual field and adjust the conden6erto center the diaphragm. .The optical syslem is contaminated by dirt. .Clean the opticalsystem. .Dgvelopment problems(on blackand.whltepint) .Developthe film prcperly. @ R€ler paqelo Page21 Pages 29-31 .8e aware lhal a change In l6mpe€ture occurs betweenthe center and the pedpheryof the tank as a resultof hgatconductlon by th6 sl€€liank Pages 64-65 2. Ma*s on the nogative Caus€ .Fixing time was too shortor exhausted fixer was us€d. ConEction .lncrea6etixingtime or use a new lixet Roma*s RaLr lo paEe OLYMPUS Matc[ yout skil]s rrith ours. OI.YMPUS OPT|CALCO, LTD' OTYMPUS OPIICALCO{EUFIOPA) GMEIH OLYMP(IS CORPORATION P' nied ln Jap.n M132E.123,1I