OPTIKA MICROSCOPES B-500DK User manual

OPTIKA MICROSCOPES - ITALY
www.optikamicroscopes.com - [email protected]
Ver. 3.0.0
B-500DK
B-500DK-R
OPERATION MANUAL
MANUAL DE INSTRUCCIONES

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INDEX
1.0 B-500DK DARKFIELD MICROSCOPE page 3
2.0 PRINCIPLES OF IMMERSION MICROSCOPY page 3
3.0 PRINCIPLES OF DARKFIELD ILLUMINATION page 6
 3.1DarkeldMicroscopyatHighMagnications   
4.0 B-500DK DARKFIELD MICROSCOPE CONFIGURATION page 10
 4.1Troubleshooting
5.0 TECHNICAL TIPS FOR OIL IMMERSION MICROSCOPY page 20
6.0 ELECTRICS page 21
7.0 USING THE RECHARGEABLE BATTERIES page 22
8.0 RECYCLING AND RECOVERY page 26

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1.0 B-500DK DARKFIELD MICROSCOPE
B-500DKisadarkeldsystemspecicforbloodanalysiswitha1.36-1.25N.A.specialextraef-
cientdarkeldcondenseranda100Xplan-achromaticobjectivewithadjustableirisdiaphragm.
TheX-LEDilluminationensuresthehighleveloflightintensitytypicallyneededinhighmagnication
darkeldtechniques.
Inordertocorrectlyusethismicroscope,onehastogainsomefamiliaritywitha)oilimmersionte-
chniqueandb)darkeldtechnique.
Inthefollowingmanualwepresentthebasicsofthesemethods(chapters2and3)andthenwegive
astep-by-stepguidetocongurationofB-500DK(chapter4).Generaltipsforimmersionmicroscopy
arealsogiven(chapter5).
Theabilityofamicroscopeobjectivetocapturedeviatedlightraysfromaspecimenisdependent
uponboththenumericalapertureandthemediumthroughwhichthelighttravels.
Anobjective’snumericalapertureisdirectlyproportionaltotherefractiveindexoftheimagingme-
diumbetweenthecoverslipandthefrontlens,andalsotothesinofone-halftheangularaperture
oftheobjective.Becausesincannotbegreaterthan90degrees,themaximumpossiblenumerical
apertureisdeterminedbytherefractiveindexoftheimmersionmedium.Mostmicroscopeobjectives
useairasthemediumthroughwhichlightraysmustpassbetweenthecoverslipprotectingthesam-
pleandfrontlensoftheobjective.Objectivesofthistypearereferredtoasdryobjectivesbecause
theyareusedwithoutliquidimagingmedia.Airhasarefractiveindexof1.0003,veryclosetothatof
avacuumandconsiderablylowerthanmostliquids,includingwater(n=1.33),glycerin(n=1.470)
andcommonmicroscopeimmersionoils(averagen=1.515).Inpractice,
themaximumnumericalapertureofadryobjectivesystemislimitedto0.95,andgreatervaluescan
onlybeachievedusingopticsdesignedforimmersionmedia.
2.0 PRINCIPLES OF IMMERSION MICROSCOPY

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Figure 1 Oil immersion and numerical aperture
TheprincipleofoilimmersionisdemonstratedinFigure1whereindividuallightraysaretraced
throughthespecimenandeitherpassintotheobjectiveorarerefractedinotherdirections.Figure1
(a)illustratesthecaseofadryobjectivewithverays(labeled1through5)shownpassingthrough
asamplethatiscoveredwithacoverslip.Theseraysarerefractedatthecoverslip-airinterfaceand
onlythetworaysclosesttotheopticalaxis(rays1and2)ofthemicroscopehavetheappropriate
angletoentertheobjectivefrontlens.Thethirdrayisrefractedatanangleofabout30degreesto
thecoverslipanddoesnotentertheobjective.Thelasttworays(4and5)areinternallyreected
backthroughthecoverslipand,alongwiththethirdray,contributetointernalreectionsoflightat
glasssurfacesthattenddegradeimageresolution.Whenairisreplacedbyoilofthesamerefractive
indexasglass,showninFigure1(b),thelightraysnowpassstraightthroughtheglass-oilinterface
withoutdeviationduetorefraction.Thenumericalapertureisthusincreasedbythefactorofn,the
refractiveindexofoil.
Microscopeobjectivesdesignedforusewithimmersionoilhaveanumberofadvantagesoverthose
thatareuseddry.Immersionobjectivesaretypicallyofhighercorrection(eitheruoriteorapochro-
matic)andcanhaveworkingnumericalaperturesupto1.40whenusedwithimmersionoilhaving
theproperdispersionandviscosity.Theseobjectivesallowthesubstagecondenserdiaphragmto
beopenedtoagreaterdegree,thusextendingtheilluminationofthespecimenandtakingadvanta-
geoftheincreasednumericalaperture.
Afactorthatiscommonlyoverlookedwhenusingoilimmersionobjectivesofincreasednumerical
apertureislimitationsplacedonthesystembythesubstagecondenser.Inasituationwhereanoil
objectiveofNA=1.40isbeingusedtoimageaspecimenwithasubstagecondenserofsmaller
numericalaperture(1.0forexample),thelowernumericalapertureofthecondenseroverridesthat
oftheobjectiveandthetotalNAofthesystemislimitedto1.0,thenumericalapertureofthecon-
denser.
2.0 PRINCIPLES OF IMMERSION MICROSCOPY

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2.0 PRINCIPLES OF IMMERSION MICROSCOPY
Modernsubstagecondensersoftenhaveahighdegreeofcorrectionwithnumericalaperturevalues
rangingbetween1.0and1.40.Inordertoeffectivelyutilizeallthebenetsofoilimmersion,thein-
terfacebetweenthesubstagecondenserfrontlensandtheundersideofthemicroscopeslidecon-
tainingthespecimenshouldbealsobeimmersedinoil.Anidealsystemisschematicallydiagramed
inFigure2,whereimmersionoilhasbeenplacedattheinterfacesbetweentheobjectivefrontlens
andthespecimenslideandalsobetweenthefrontlensofthecondenserandtheundersideofthe
specimenslide.
ThissystemhasbeentermedaHomogeneousImmersionSystemanditistheidealsituationto
achievemaximumnumericalapertureandresolutioninanopticalmicroscope.
Figure 2 Homogeneous immersion system
Inthiscase,therefractiveindexanddispersionoftheobjectivefrontlens,immersionoil,substage
condenserfrontlens,andthemountingmediumareequalorverynearequal.
Inthisidealsystem,anobliquelightraycanpassthroughthecondenserlensandcompletelythrou-
ghthemicroscopeslide,immersionoil,andmountingmediumundeviatedbyrefractionatoil-glass
ormountingmedium-glassinterfaces.
Whenusinghigh-powerachromatoilimmersionobjectives,itissometimespermissibletoomitthe
stepofoilingthecondensertoplens.Thisisbecausethecondenseraperturediaphragmmustoften
bereducedwithlesser-correctedobjectivestoeliminateartifactsandprovideoptimumimaging.The
reductionindiaphragmsizereducesthepotentialincreaseinnumericalaperture(providedbyoiling
thecondenserlens)sothelossinimagequalityundertheseconditionsisusuallynegligible.

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3.0 PRINCIPLES OF DARKFIELD ILLUMINATION
Darkeldmicroscopyisaspecializedilluminationtechniquethatcapitalizesonobliqueillumination
toenhancecontrastinspecimensthatarenotimagedwellundernormalbrighteldilluminationcon-
ditions.
Allofusarequitefamiliarwiththeappearanceandvisibilityofstarsonadarknight,thisdespitetheir
enormousdistancesfromtheearth.Starscanbeseenbecauseofthestarkcontrastbetweentheir
faintlightandtheblacksky.
This principle is applied in darkeld (also called darkground) microscopy, a simple and popular
methodformakingunstainedobjectsclearlyvisible.Suchobjectsareoftenhaverefractiveindices
verycloseinvaluetothatoftheirsurroundingsandaredifculttoimageinconventionalbrighteld
microscopy.Forinstance,manysmallaquaticorganismshavearefractiveindexrangingfrom1.2
to1.4,resultinginanegligibleopticaldifferencefromthesurroundingaqueousmedium.Theseare
idealcandidatesfordarkeldillumination.
Darkeldilluminationrequiresblockingoutofthecentrallightwhichordinarilypassesthroughand
around(surrounding)thespecimen,allowingonlyobliqueraysfromeveryazimuthto“strike”the
specimenmountedonthemicroscopeslide.ThetoplensofasimpleAbbedarkeldcondenseris
sphericallyconcave,allowinglightraysemergingfromthesurfaceinallazimuthstoformaninverted
hollowconeoflightwithanapexcenteredinthespecimenplane.Ifnospecimenispresentandthe
numericalapertureofthecondenserisgreaterthanthatoftheobjective,theobliquerayscrossand
allsuchrayswillmissenteringtheobjectivebecauseoftheirobliquity.Theeldofviewwillappear
dark.
Thedarkeldcondenser/objective pairillustrated in Figure3is ahigh-numericalaperture arran-
gementthatrepresentsdarkeldmicroscopyinitsmostsophisticatedconguration,whichwillbe
discussedindetailbelow.Theobjectivecontainsaninternalirisdiaphragmthatservestoreducethe
numericalapertureoftheobjectivetoavaluebelowthatoftheinvertedhollowlightconeemittedby
thecondenser.Thecardioidcondenserisareectingdarkelddesignthatreliesoninternalmirrors
toprojectanaberration-freeconeoflightontothespecimenplane.
Whenaspecimenisplacedontheslide,especiallyanunstained,non-lightabsorbingspecimen,the
obliquerayscrossthespecimenandarediffracted,reected,and/orrefractedbyopticaldiscontinui-
ties(suchasthecellmembrane,nucleus,andinternalorganelles)allowingthesefaintraystoenter
theobjective.Thespecimencanthenbeseenbrightonanotherwiseblackbackground.Intermsof
Fourieroptics,darkeldilluminationremovesthezerothorder(unscatteredlight)fromthediffraction
patternformedattherearfocalplaneoftheobjective.Thisresultsinanimageformedexclusively
fromhigherorderdiffractionintensitiesscatteredbythespecimen.

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Figure 3 Cardiod darkeld condenser
Idealcandidatesfordarkeldilluminationincludeminutelivingaquaticorganisms,diatoms,smallin-
sects,bone,bers,hair,unstainedbacteria,yeast,andprotozoa.Non-biologicalspecimensinclude
mineralandchemicalcrystals,colloidalparticles,dust-countspecimens,andthinsectionsofpoly-
mersandceramicscontainingsmallinclusions,porositydifferences,orrefractiveindexgradients.
Careshouldbetakenwhenpreparingspecimensfordarkeldmicroscopybecausefeaturesthat
lieaboveandbelowtheplaneoffocuscanalsoscatterlightandcontributetoimagedegradation.
Specimenthicknessandmicroscopeslidethicknessarealsoveryimportantand,ingeneral,athin
specimenisdesirabletoeliminatethepossibilityofdiffractionartifactsthatcaninterferewithimage
formation.
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Formorepreciseworkandblackerbackgrounds,youmaychooseacondenserdesignedespecially
fordarkeld,i.e.totransmitonlyobliquerays.Thereareseveralvarieties:“dry”darkeldcondensers
withairbetweenthetopofthecondenserandtheundersideoftheslide–andimmersiondarkeld
condenserswhichrequiretheuseofadropofimmersionoil(somearedesignedtousewaterin-
stead)establishingcontactbetweenthetopofthecondenserandtheundersideofthespecimen
slide.Theimmersiondarkeldcondenserhasinternalmirroredsurfacesandpassesraysofgreat
obliquityandfreeofchromaticaberration,producingthebestresultsandblackestbackground.
Perhapsthemostwidelyuseddarkeldcondenseristheparaboloid,consistingofasolidpieceof
glassgroundveryaccuratelyintotheshapeofaparaboloid.
Asdiscussedabove,thedrydarkeldcondenserisusefulforobjectiveswithnumericalapertures
below0.75,whiletheparaboloidandcardioidimmersioncondensers(Figure3)canbeusedwith
objectivesofveryhighnumericalaperture(upto1.4).Objectiveswithanumericalapertureabove
1.2willrequiresomereductionoftheirworkingaperturesincetheirmaximumnumericalaperture
mayexceedthenumericalapertureofthecondenser,thusallowingdirectlighttoentertheobjecti-
ve.
Forthisreason,manyhighnumericalapertureobjectivesdesignedforusewithdarkeldaswellas
brighteldilluminationaremadewithabuilt-inadjustableirisdiaphragmthatactsasanaperture
stop.
Thisreductioninnumericalaperturealsolimitstheresolvingpoweroftheobjectiveaswellasthe
intensityoflightin theimage.Specializedobjectivesdesigned exclusivelyfordarkeldwork are
producedwithamaximumnumericalapertureclosetothelowerlimitofthenumericalapertureof
thedarkeldcondenser.Theydonothaveinternalirisdiaphragms,howeverthelensmountdiame-
tersareadjustedsoatleastoneinternallenshastheoptimumdiametertoperformasanaperture
stop.
The cardioid condenser is very sensitive to alignment and must be carefully positioned to take
advantageoftheverysharpconeofillumination,makingitthemostdifcultdarkeldcondenserto
use.Inaddition,thecondenserproducesasignicantamountofglare,evenfromthemostminute
dustparticles,andtheshortfocallengthmayresultinpoorilluminationonobjectsthatexceedafew
micronsinsizeorthickness.Whenchoosingmicroscopeslidesforquantitativehigh-magnication
darkeldmicroscopy,makecertaintoselectslidesmadefromaglassmixturethatisfreeofuore-
scentimpurities.
3.1 DARKFIELD MICROSCOPY AT HIGH MAGNIFICATIONS

Page9
3.1 DARKFIELD MICROSCOPY AT HIGH MAGNIFICATIONS
Carefulattentionshouldbepaidtothedetailsofoilingahighnumericalaperturecondensertothe
bottomofthespecimenslide.Itisverydifculttoavoidintroductionoftinyairbubblesintothearea
betweenthecondensertoplensandthebottomofthemicroscopeslide,andthistechniqueshould
bepracticedtoperfection.Airbubbleswillcauseimageareanddistortion,leadingtoalossofcon-
trastandoverallimagedegradation.
Problemsarealsoencounteredwhenusingmicroscopeslidesthatareeithertoothickortoothin.
Manydarkeldcondenserscontaintherangeofusableslidethicknessinscribeddirectlyonthecon-
densermount.Iftheslideistoothick,itisoftendifculttofocusthecondenserwithoutresortingtoa
higherviscosityimmersionoil.Ontheotherhand,slidesthataretoothinhaveatendencytobreak
theoilbondbetweenthecondenserandtheslide.Itisagoodideatopurchaseprecisionmicroscope
slidesofthecorrectthicknesstoavoidanyoftheproblemsmentionedabove.
Highnumericalaperturecondensers,whetherintendedforusedryorwithoil,mustbeaccurately
centeredintheopticalpathofthemicroscopetorealizeoptimumperformance.
Toachievethis,manydarkeldcondensersarebuiltwithasmallcircleengravedontotheuppersur-
facetoaidincenteringthecondenser.Centeringisperformedwithalowpower(10x-20x)objective
byimagingtheengravedcircleandusingthecondensercenteringscrewstoensurethecircle(and
condenser)arecorrectlycenteredintheopticalpath.

Page 10
4.0 B-500DK DARKFIELD MICROSCOPE CONFIGURATION
• Inamannersimilartothatwithlow-powerdarkeldmicroscopeconguration,placeaspeci-
menonthestageandfocusitusingthe10xobjective(refertoB-500generalmanualforthe
descriptionofthestandardbrighteldtechnique).
• Usethecondenserrackknobtolowerandremovethebrighteldsubstagecondenser.
• Insertthereectedlighthighnumericalaperturecondenserintotheholder,andsecureitwith
thelockscrew.Slowlyraisethecondenseruntilitislessthan2millimetersfromtheunderside
ofthespecimenslide.

Page 11
• Selectadarkeldspecimenandplaceitontothemicroscopestagebetweentheobjective
andthecondenser.Thecondenserwillprojectaspotoflightontothesamplethatcanbe
usedforcenteringtheopticalpath.Usethecondensercenteringscrewstomovetheringof
lightintothecenterofthevieweld.Itcouldbeusefultovarytheheightofthecondenserin
ordertoviewthespot.
Figure 5 Centering darkeld condenser
4.0 B-500DK DARKFIELD MICROSCOPE CONFIGURATION
LED
CENTERING
SCREWS
LOCK SCREW
CONDENSER
CENTERING SCREWS

Page 12
4.0 B-500DK DARKFIELD MICROSCOPE CONFIGURATION
• Itisoftenadvantageoustousealowpower10xobjectivewhencenteringhighnumerical
aperturedarkeldcondensers.Whenviewingaspecimenwiththe10xobjectivewhileslowly
raisingandloweringthecondenser,apointwillbereachedwhereabrightspotwillappearin
theeldofviewasillustratedinFigure5(a).Asthecondenserisslightlyraisedorlowered,
adarkspotsimilartotheoneshowninFigure5(b)willappear,ifthecondenserisproperly
centered.Incaseswherethecondenserisnotproperlyalignedandcentered,atypicaleld
ofviewmightlooklikethatshowninFigure5(c).Theidealandcorrectpositioningofthe
condenserisillustratedinFigure5(a),andthecondensershouldbeadjusteduntiltheeldof
viewappearsinthismanner,withthecondensercenteringscrews.
• Toapplyoiltotheimmersioncondenser,removethemicroscopeslideandslowlyrackthe
condenserdownbelowthemechanical stageandplaceadropofoil directlyonthefront
lens.
• Replacetheslide,thenslowlyraisethecondenseruntilcontactismadebetweentheoildro-
pletandthebottomsurfaceoftheslide.Carefullyobservetheoilcontactareatodetermineif
anyairbubbleshavebeenintroducedintothespacebetweenthecondensertoplensandthe
microscopeslide.Itshouldbenotedthatnomatterwhatthemagnicationoftheobjectiveis,
ifanoilimmersioncondenserisusedwithoutoil,lightwillnotemergefromthecondenser.
 Airbubbleswillscatterlightandshouldbeobservablebyremovingtheeyepieceandviewing
thebackfocalplaneoftheobjectivewithacenteringtelescope.
 Ifnobubblesarepresent,proceedontothenextstep.Whenbubblesaredetected,remove
alltracesofoilfromboththeslideandthecondensertoplensandrepeattheprocedure.
Makecertainbothsurfacesarecleanandcompletelyfreeofoil,dust,dirtandothercontami-
natingartifactsbeforere-oilingthecondenser.

Page 13
4.0 B-500DK DARKFIELD MICROSCOPE CONFIGURATION
• Swingtheprovidedspecial100Ximmersionobjectivetoapositioninthenosepiecewhereit
willbethenextobjectiverotatedintotheopticalpathwaydirectlyabovethespecimen.
• Positiontheareatobeimagedinthecenterofthemicroscope’sopticalpathusinglower
powerobjective(e.g.10xor40x).Next,theoilimmersionobjective(withoutoil)isplaced
intotheopticalpathandthespecimenareaofinterestisroughlybroughtintocenterofthe
vieweldandfocused.Thispre-positionsallofthecomponentsofthesysteminpreparation
fortheadditionofoil.Swingtheimmersionobjectivetoanadjacentstoponthemicroscope
nosepieceandapplyoilbothtotheobjectivefrontlensandspecimenasdescribedinthenext
point.
• Useapieceoflint-freepaperoraneyedroppertoplaceasmalldropletofoildirectlyonthe
frontlensoftheimmersionobjective.Next,placeanotherdropletofoilonthecoverglass
immediatelyabovetheareatobeimaged.
• Next,quicklyrotatetheoiledobjectiveintopositionabovethespecimenmergingthetwooil
droplets(oneonthesampleandoneontheobjective)intoasinglepool.Abrightashshould
occur(fromscatteredlight)theinstantoilonthefrontlensoftheobjectivecomesintocontact
withthemicroscopeslide.
• Checkforairbubblesbyobservingthebackfocalplaneoftheobjectivewiththeprovided
centeringtelescope.Ifbubblesarepresent,swingtheobjectivetothenextdetentstoponthe
microscopenosepieceandwipeoffexcessoilwithapieceoflint-freelenstissue.Re-apply
oiltotheobjectivefrontlensandtryagain.
• Withthecenteringtelescope,andthe100xobjectiveinserted,checkthatthecondenseris
wellcentered(asymmetricalringoflightmustbeseen).Finelycenterusingthecondenser
centeringscrews.

Page 14
• The100Xobjectivehasaninternalirisdiaphragmdesignedtoallowadjustmentofthenume-
ricalaperture.Itshouldbeadjustedatthistime.
 Removetheeyepieceandplacethecenteringtelescopeintotheeyetubetoobservetherear
focalplaneoftheobjective.Ifabrightringoflightisseenaroundacentraldarkregion,the
apertureoftheobjectiveistoogreatandmustbereducedusingtheinternalirisdiaphragm.
4.0 B-500DK DARKFIELD MICROSCOPE CONFIGURATION
OIL
(SLIDE-OBJECTIVE)
OIL
(CONDENSER – SLIDE)
ROTATE THIS TO
ADJUST THE
INTERNAL IRIS

Page 15
4.0 B-500DK DARKFIELD MICROSCOPE CONFIGURATION
Adjusttheobjective’sirisdiaphragmuntiltheringoflightfallsoutofthevieweld.
CENTERING TELESCOPE
VIEW INSIDE THE TELESCOPE
WITH A WELL CENTERED
CONDENSER

Page16
4.0 B-500DK DARKFIELD MICROSCOPE CONFIGURATION
Ifacrescent-shapedarcoflightisobserved,thenthecondenseris not properly alignedincenter
ofthemicroscopeopticalaxis.UsetheLEDcenteringscrewstonealignthecondensersothatthe
arcdisappearsorbecomesacompletecircleoflight.
• Replacetheeyepieceandfocusthespecimen,whichshouldnowappearbrightlyandevenly
illuminateduponadarkbackground.
Carefulattentionshouldalwaysbegiventomicroscopealignment,irrespectiveofwhethertheillumi-
nationmodeisbrighteld,darkeld,phasecontrastorsomeothercontrastenhancementtechnique.
Timespentinthisendeavorwillberepaidinexcellentperformanceofthemicroscopebothforrou-
tineobservationandcriticalphotomicrography.
VIEW INSIDE THE TELESCOPE
WHEN THE LIGHT RING IS DARKE-
NED BY CLOSING THE OBJECTI-
VE’S IRIS

Page17
4.1 TROUBLESHOOTING
Therearenumerouscommonproblemsassociatedwithdarkeldmicroscopyandphotomicrography.
Theserangefrominsufcientilluminationandcondensermisalignmenttousingaeldstopofincor-
rectsize.Mostdarkeldilluminationproblemsareassociatedwiththesubstagecondenser,andthis
shouldbetherstsuspectwhenthingsdonotworkproperly.
Thefollowingproblemsandsolutionsshouldbeusedasaguidewhenimagingspecimensusing
thistechnique.
ProblemThereisinsufcientilluminationtomakethespecimenvisible,orthespecimenisvisible
butveryfaint.
SolutionCheckthesubstagecondensertoensurethatitispositionedcorrectly.Also,checktheli-
ghtpathforcolorand/orneutraldensitylters,polarizers,retardationplates,oranyother
componentsthatmightreduceilluminationintensity.
Checktomakecertainthatthecondensertoplensiscorrectlyoiledtothebottomofthe
microscopeslide.Iftheimmersioncontacthasbeenbroken,re-applytheoilandrack
upthecondenseruntilthetoplensiscompletelyimmersedinoilandincontactwiththe
bottomoftheslide.
ProblemThevieweldhasadarkspotinthecenterreducingilluminationintensity,butobjectsinthe
peripheryarewell-illuminatedandappearnormal.
SolutionThesubstagecondenserisincorrectlypositioned.Carefullyrackthecondenserupand
downwhileobservingthespecimeninthevieweld.Checktomakecertainthatboththe
condenserapertureandelddiaphragmsaresettothewideopenposition.
Occasionallythisproblemariseswhenmicroscopeslidesofexcessivethickness(greater
thanthestandardonemillimeter)areused.Useamicrometerorasetofdialcalipersto
determinethethicknessoftheslideandtheslide/coverslipcombination.
ProblemTheimagehasabrighteldperipherywithorwithoutdarkregionsinthecenter.
SolutionThisproblemistypicalwhenthesubstagecondenserisincorrectlycentered.
 Reverttobrighteldandre-establishtheconditionsofKöhlerillumination,thenrepeatthe
darkeldprocedure,makingsurethecondenseriscentered.
Ifthereisaninternalirisdiaphragmintheobjective,reducetheirisopeningsizetodeter-
mineiftoomuchobliqueilluminationisenteringthefrontlensoftheobjective.

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ProblemTheperipheryofthevieweldisbrightononesideonly.
SolutionCheckthenosepiecetoensuretheobjectiveiscorrectlypositionedintheopticalaxisof
themicroscope.Thereisadetentstopthatshouldproduceapositive“click”when the
objectiveisproperlypositioned.
ProblemImagesofthespecimenarenotclearandlackinginsufcientcontrastanddetail.
SolutionThespecimenmightnotbesuitablefordarkeldmicroscopy.Manystainedspecimensdo
notscatterenoughlighttobeclearlyimagedunderdarkeldconditions.
 Specimenthicknessmayalsobeaproblem,becauseverythinspecimensareoftennot
imagedwellwiththeobliquelightraysemittedfromdarkeldcondensers.Changetobri-
ghteldorphasecontrasttodetermineifthisimprovesspecimencontrast.
ProblemBrightareasthatareoutoffocusobstructviewingand/orphotomicrographyofdarkeld
specimens.
Solution Thisisprobablyduetodust,hair,bers,and/ordirtcontaminationofanopticalsurface
somewhereabovethecondenser.Thoroughlycleanthespecimenslidewithoptical-grade
tissueorcotton.Occasionally,thisproblemarisesduetocontaminationontheobjective
frontlens.Carefullyloosentheobjectivefromitsseatinthenosepieceandslowlyturn
whileobservingthespecimenthroughtheeyepieces.Iftheobstructionrotatesalongwith
theobjective,thenitisprobablyduetodustonthefrontlens.Removetheobjectiveand
moistenthefrontlensbygentlyexhalingonit,thencleanwithlenstissueoracottonswab
wrappedonalongwoodenrod.
Replacethe objective andcheck to makecertain the obstruction has beencompletely
removed.
ProblemTheimageofthelampcondenserpartiallyllsthevieweld,obscuringdetailsofthespe-
cimen.
SolutionTheslidemaybetoothinorthecondensermaybeadjustedtoohigh.Measurethethi-
cknessofthemicroscopeslideandchecktomakecertaintheoilcontactisnotbroken.If
difcultyisencounteredkeepingthebottomoftheslideoiled,tryusingathickerslide.
4.1 TROUBLESHOOTING

Page19
4.1 TROUBLESHOOTING
ProblemWhenviewingaquaticorganismsinaaqueousmount,specimensdriftconstantlyinasin-
gledirection,makingobservationandphotomicrographydifcult.
SolutionConvectioncurrentsarebeingcreatedduetoslowevaporationattheedgesofthecover-
slip.PlaceabeadofpetroleumjellyorCytosealmountingmediumaroundtheperipheryof
thecoversliptosealitrmlyontothemicroscopeslide.Commercialproductsareavailable
thatcanbeaddedtothewatertoslowthemovementoftheseminuteorganisms,making
themeasiertophotograph.
ProblemWhenviewingthespecimenthrougha10xobjective,thereisaoblongspotoflightinthe
centerofthevieweld.
SolutionThelampcondensermaybeincorrectlyfocused.Re-focusthecondenser.
ProblemThevieweldhasbrightspotsandthereareproblemswithachievingsharpfocusofthe
specimen.
SolutionSmallairbubblesmaybetrappedintheoilbetweenthetopofthecondenserandthe
bottomofthemicroscopeslide.Removetheeyepiecetoobservethebackfocalplaneof
theobjective,wherethebubbleswillbevisible.Toremedy,breaktheoilcontactandremo-
veanyresidualbubbleswithacottonswab.Cleanexcessoilfromthecondenserfrontlens
andthemicroscopeslide,thenapplyacleandropofoiltothelens.Iftheproblempersists,
thoroughlycleanallopticalsurfacesbeforere-applyingtheoil.
ProblemAthighmagnications,colloidalparticlesdisplayincompleteNewtonringsandappearto
beunevenlyilluminated.
SolutionThecondenserisprobablyoff-center.Changetoalowerpowerobjectiveandre-center
thecondenser.

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5.0 TECHNICAL TIPS FOR OIL IMMERSION MICROSCOPY
Therstruleinmicroscopyistokeeptheopticalelementscompletelyfreeofdust,dirt,oil,
solvents,andanyothercontaminants.Themicroscopeshouldbekeptinalowvibration
smoke-freeroomthatiscleanaspossibleandhasminimaldisturbanceofthecirculatedair.
Useadustcoveronthemicroscopewhennotinuseandkeepallaccessoriesinair-tight
containers.Avoidusingcorrosivesolventstocleananypartofthemicroscope,anduseonly
dilutedsoapywatertocleannon-opticalsurfaces.Oilshouldbeusedkeepingthefollowing
techniquesandprecautionsinmind:
• Therststepistolocatethesamplewithalow-powerobjectiveandpositionthemicroscope
slidesothattheareaofinterestissquarelyinthecentralportionofthestageopening.Itis
oftendesirabletorotatetheimmersionobjective(withoutoil)intoplacetoensurethecorrect
locationofthespecimen.Toachieveoptimumresultswhenusingtheoilimmersiontechni-
queitisimportanttoalsoapplyoiltotheundersideofthemicroscopeslideandtothetop
lensofthesubstagecondensertoformanoilbeadbetweenthetwo,asdescribedbelow.
Forapplyingoiltothefrontlensofthesubstagecondenser,usethefollowingconsiderations:
• Makecertainthatthemechanicalstageofthemicroscopehasanopeningofsufcientsize
toallowscanningoftheoiledspecimenwithoutspillingoilontothebottomofthestage.
Manyadvancedresearchmicroscopeshaveastageinsertthatisremovableforusewithoil.
Removethespecimenslidefromthemicroscopestagebeforeapplyingoiltothecondenser
toplens.
• Inamannersimilartooilinganobjective,placeadropofoilonthebottomofthemicrosco-
peslideandapplyasimilardroptothefrontlensofthesubstagecondenser.
 Makesurethatthecondenserisslightlyloweredinitsrackbeforereplacingthemicroscope
slideonthestage.Aftertheslidehasbeenplacedonthestage,rackthecondenserback
totheproperpositionwhilecarefullyobservingtheoildropletsonthecondenserlensand
microscopeslide.Thesedropletsshouldmergeintooneasthecondenserisbroughtinto
properposition.
• Swingtheimmersionobjectivetoapositioninthenosepiecewhereitwillbethenextobjec-
tiverotatedintotheopticalpathwaydirectlyabovethespecimen.
• Positiontheareatobeimagedinthecenterofthemicroscope’sopticalpathusinglower
powerobjectives.Next,theoilimmersionobjective(withoutoil)isplacedintotheoptical
pathandthespecimenareaofinterestisroughlybroughtintocenterofthevieweldandfo-
cused.Thispre-positionsallofthecomponentsofthesysteminpreparationfortheaddition
ofoil.Swingtheimmersionobjectivetoanadjacentstoponthemicroscopenosepieceand
applyoilbothtotheobjectivefrontlensandspecimenasdescribedinthenextpoint.