Lab 1 Appendix

7/27/2019 Lab 1 Appendix

1/16

1 BIOL 3050U 2013 Lab Manual Appendix

APPENDIX:

TheCompoundMicroscope:1.Magnification,ResolutionandNumericalAperture:

Magnification:

Whenyoulookintoamicroscope,youarenotdirectlylookingatthespecimen;youareviewingan

imageofthespecimenthathasbeenincreasedinsizeduetothepresenceoflenses.

Magnificationistheratioofthesizeoftheviewedimagetotheactualsizeoftheobject.A

compoundlightmicroscope,suchastheoneyouwilluseinthelab,hastwomagnifyinglenses:

1)theocularlens(theeyepiece) 2)theobjectivelens(theoneclosesttotheobject)

Theocularlensofourmicroscopeshasamagnificationof10X.Objectivelenseshavedifferent

magnificationsforexample,4X,10X,and40Xand100X.Themagnificationofeachlensisetchedontothesideofthatlens.

Thetotalmagnificationofthemicroscopeiscalculatedasfollows:

Forinstance,amicroscopewitha4Xobjectivelensanda10Xocularlenswillhaveatotal

magnificationof40X.Magnificationcanbeincreasedvirtuallywithoutlimit,butbeyondacertainlimitthequalityoftheimagebecomesblurred.

ResolutionandNumericalAperture:Althoughmagnificationcanbeincreasedvirtuallywithoutlimit,resolutionisthefactorthatlimits

theeffectivenessofamicroscope.Resolutionisthe

abilitytodistinguishtwoadjacentpointsas

separate.Thetheoreticalmaximumresolutionofa

lightmicroscopeis0.2m.Thereforeitiscapableof

differentiatingindividualobjectsthataremorethan

0.2mapartasbeingdistinct.However,itcannotresolvetwoindividualobjectsthatarelessthan0.2

mapart.Anincreaseinmagnificationwillnot

allowadditionaldetailtoberesolvedandtwoobjectsthatarelessthan0.2mapartwillstill

appearasone.Thisphenomenoniscalledemptymagnification.Differentmicroscopeshavedifferentresolutionvalues.Thesmallertheresolutionvalue,thebetterthemicroscope.

TotalMagnification=MagnificationofocularlensxMagnificationofobjectivelens


2/16


Resolution(R)dependsonthewavelengthofthelightsourceused(forvisiblelightisbetween

380750nm)andonthelightgatheringabilityoftheobjectivelenswhichisquantitatively

expressedintermsofthenumericalaperture(N.A.).Resolutioncanbedeterminedusingthefollowingformula:

Where:isthewavelengthoflight(innm)

N.A.isthenumericalaperture

Theresolutionvaluecanbedecreasedby: 1.Usinglightwithashorterwavelength 2.Increasingthenumericalapertureoftheobjectivelens

(seetutorial:NumericalApertureandImageResolution).

Whenlightraysfromthespecimencrosstheair,theymaybedeflectedoutsidetheperimeterof

theobjectivelens.Asaresult,lesslightfromthespecimenreachestheobjectivelens.Thisresults

inapoorimage.Increasingthenumericalapertureoftheobjectiveallowsincreasinglyoblique

raystoentertheobjectivelens,producingamoreresolvedimage.

Notethatanelectronmicroscopehasaresolutionvalueof0.2nm,whichis1000Xsmallerthan

thatofalightmicroscope.Thisisbecausetheelectronbeamusedhasamuchshorterwavelength

thanthewavelengthoflight.

2.TheCondenser:

Withabrightfieldmicroscope,visiblelightfromanincandescentsourceisaimedtowardathird

lensbeneaththestagecalledthecondenser.Thecondenserfocuseslightontothespecimen

throughanopeninginthestage.Afterpassingthroughthespecimen,thelightistransmitted

throughtwomorelenses(theobjectiveandocularlenses)thatareplacedatbothendsofalight

tighttube.So,themicroscopyyouwillbeusinghasthreelensesintotal:twothatproducethe

magnifiedimageofthespecimenandonethatfocusesthelightontothespecimen.(Seetutorial:

TransmittedLightMicroscopyOpticalPathways)

Notethat

the

image

is

inverted

(upside

down

and

reversed)

and

magnified.Brightfieldmicroscopyisbestsuitedtoviewingstainedornaturallypigmentedspecimenssuch

asstainedpreparedslidesoftissuesectionsorlivingphotosyntheticorganisms.Unstained

specimensaredifficulttoseewithabrightfieldmicroscope.Increasedcontrastcanbeachieved

byreducingilluminationand/oradjustingthecondenserdiaphragm.Viewingunstained

specimenscanalsobedramaticallyimprovedbyusingtechniquessuchasdarkfield,phase

contrast,anddifferentialinterferencecontrast.


3/16


3.FieldofView(FOV):Thefieldofviewistheviewthatyouseewhenyoulookintothemicroscope.Itshouldappearas

onelargecirculararea.Itisimportanttonotethatasyouincreasemagnificationwiththe

objectivelens(from4Xto100X)thefieldofviewbecomessmaller.Ifyouarefocusedona

specimenatlowpowerthatisnotinthecentreofthefieldofview,itwillseemtodisappearwhen

youswitchtoahigherobjectivebecausethefieldofviewgetssmaller.Inactualfact,thespecimen

isstillinthesameposition.

4.DepthofField:Depthoffieldreferstothethicknessoftheplaneoffocus.Inotherwords,thedepthoffieldisthe

distancealongtheverticalaxisinwhichthespecimencanmoveandstillremainsharptotheeye.

Thedepthoffielddependsontheresolutionofthemicroscope.Imagethatonyourmicroscope

slideyouhavethreecellsonatoptheother.Ifyouhavealargedepthoffield,allthreecellswillbe

infocusatthesametime.Ifyouhaveashortdepthoffield,youwillonlybeabletoseeoncellin

focus,therestwillbeoutoffocus.

5.Parts

of

the

Microscope:

Thepartsofthemicroscopeandtheirfunctions,areoutlinedinAppendixA,pleasereviewbefore

attendingyourlab.

TheStereomicroscope(DissectingMicroscope)Stereomicroscopeshavetwoocularlensesandproduceathreedimensionalimage.Theyare

usefulforviewingspecimensthataretoolargeortoothicktoviewwiththecompound

microscope.Theyarealsousefulforviewingsurfacefeaturesofspecimens.Stereomicroscopy

typicallyusesreflectedlight(fromthesurfaceofthespecimen)ratherthanlightthatis

transmittedthroughthespecimen.However,thestereomicroscopecanalsomakeuseof

transmittedlight.Inthiscasetheilluminationisfrombelowandisreflectedfromamirrorbeneaththestageonwhichthespecimenisplaced.Reflectedlightallowsviewingofinternal

featuresoftranslucentspecimens.

4Xobjective 10Xobjective 40Xobjective 100Xobjective

Specimen

FOV FOV FOV FOV


4/16


APPENDIXA:

CX31OLYMPUSMICROSCOPE

Partsof

the

microscope


5/16


PARTSOFTHEMICROSCOPE:

Identifyandfamiliarizeyourselfwiththefollowingpartsofthemicroscopeandtheir

functions:

Powerswitchturnsthelamponandoff.

Lightadjustmentknobcontrolsthebrightnessofthelamp.Theintensityofthelightcan

beadjustedbyturningtheknob.Highmagnificationsrequirehigherilluminationintensity

whilelowmagnificationsrequirelowerintensities.Inanycase,adjustilluminationsothat

thefieldisbrightwithouthurtingtheeyes.Illuminatorconsistsofatungstenhalogenlampthatemitsacontinuousspectrumoflight

whichisthenpassedthroughacollectorandfieldlensbeforebeingreflectedthroughthe

fielddiaphragmandintothesubstagecondenser.Thefielddiaphragm,whichhasaniris

thatcontrolsthediameteroftheilluminatingbeamoflight,servesasavirtualsourceof

lightforthemicroscopeanditsimageisfocusedbythecondenserontothespecimenplane(SeeAppendixB).Properadjustmentofthefielddiaphragm(i.e.,centeredintheoptical

pathandopenedsoastoliejustoutsideofthefieldofview)isimportanttopreventglare

whichcanreducecontrastintheobservedimage.Theeliminationofexcesslightis

particularlyimportantwhenattemptingtovisualizesampleswithlowcontrast.Whenthe

fielddiaphragmisopenedtoofar,scatteredlightoriginatingfromthespecimenandlight

reflectedatobliqueanglesfromopticalsurfacescanacttodegradeimagequality.

Condensercontainsasetoflenseslocatedimmediatelyunderthestagethatfocusthe

lightonthespecimen.Thecondenserhasthefollowingcomponents:

Focusingknobmovesthecondenserupanddowntoadjustthefocusofthelight

onthespecimen.Centeringknobsareusedtocentertheirisdiaphragmimageinthefieldofview.

Frontlens,theglasssurfaceclosesttothespecimen.Becarefulnottotouchthis

surfaceasitiseasilyscratched.

Irisdiaphragmwithalevertoopenandclosethediaphragm.Theapertureofthe

irisdiaphragmdeterminesthenumericalapertureoftheilluminationsystem.

Matchingthenumericalapertureoftheilluminationsystemwiththatofthe

objectiveyouareusingprovidesbetterimageresolutionandcontrast.

Filterholder,locatedatthebaseofthecondenser,allowsinsertionofafilter

(usuallyblue)toadjustthecoloroftheilluminatinglight.

Note:Eachtimeamicroscopeisturnedon,makesurethatallopticalcomponentsofthecondenserareproperlyalignedandthatthelightbeamiscenteredontoyourspecimen.ProperadjustmentsofthecondenserandfielddiaphragmproduceKhlerilluminationwhichallowsyoutoobtainanimageofhighquality.

Stagehorizontalsurfaceonwhichtheslideisheldinplacebyspringloadedclips.A

mechanicalstage(suchastheoneonyourmicroscope)isequippedwithtranslation


6/16


controlknobsthatallowtheslidetobemovedalongtwoaxes:longitudinalandlateral (See

AppendixC).

Revolvingnosepieceholdsseveralobjectivelensesthatcanberotatedintopositiontochangethelens.

Objectivelensesgatherthelightfromthespecimen.Anobjectivelenscreatesamagnifiedimageofthespecimenandprojectsthemagnifiedimageintotheobservation

tube.Yourmicroscopeisequippedwiththreelenses:

4Xlensisusedtogetanoverviewofthestructurespresentinasectionandtofindareas

formoredetailedobservation

10Xlensisthemostusefulmagnificationtoidentifytissues.

40Xlensisusedtoseethedetailsofcellandtissueorganization.

Typically,theinformationetchedonalensisasfollows:

Focusingcontrolsusedtoraiseandlowerthespecimenstagetofocustheimageofthespecimen.Thecoarsefocusisinitiallyusedtofocusthespecimenat4Xand10X.Thefinefocusisthenusedtofinetunethefocus.Thefinefocusisusedtofocusthespecimenat40X,butonlyafterinitiallyfocusingatlowermagnification.Highermagnificationlensesarephysicallyclosertothespecimenitself,whichposestheriskofjammingtheobjectiveinto

thespecimen.

Themicroscopeisparfocal.Thismeansthatonceyourspecimenisfocusedat10X,the

specimenremainsinfocusornearlyinfocuswhenyouswitchtothe40Xobjectives,soyou

onlyhavetoadjustthefinefocuswhenswitchingtothe40Xobjective.

Observationtubehollowtubethroughwhichlighttravelsfromtheobjectivetotheocularlens.Itcontainsaprismatthebaseofthetubethatbendsthelightrayssotheycan

entertheinclinedtube.Ocularlens(eyepiece)magnifies(typically10X)androtatestheprimaryimageproducedbytheobjectivelensandformsanimagethatcanbevisualizedbytheeyeora

camera.Theocularlensisattachedtothemicroscopewithaneyepiecetube.Inabinocular

microscope,thedistancebetweenthetwoeyepiecetubescanbeadjustedtofitthedistance

betweentheobserverseyes.


7/16


CAREOFTHEMICROSCOPE:Amicroscopeisaprecisioninstrument.So,HANDLEITWITHCARE.

EVERYTHINGonagoodqualitymicroscopeisunbelievablyexpensive,sobecareful.

Whenmovingthemicroscope,carefullycarryitwithonehandunderthebaseandtheotherhandinthehandleontherearofthearm.Neverholdthemicroscopebythestage,X/Yaxisknob,binocularsectionoftheobservationtube,etc.

Holdtheplug(notthecable)whenunpluggingtheilluminator. Sincebulbsareexpensive,andhavealimitedlife,turntheilluminatoroffwhenyou

aredone.

Alwaysmakesurethestageandlensesarecleanbeforeputtingthemicroscopeaway.

NEVERuseanythingbutgoodqualitylenstissueandappropriatelenscleanersolutiononanyopticalsurfacetopreventdamageofthelenselementsorcoatings. Covertheinstrumentwithadustjacketwhennotinuse.

Focussmoothly;don'ttrytospeedthroughthefocusingprocessorforceanything.CleaningthemicroscopeItisimportanttokeepyourmicroscopeclean:

LensesshouldbecleanedONLYwithlenspaperthathasbeenmoistenedwithwater(toremovegeneraldirt)orethylether(toremoveoil).

Useabsorbentpaper,suchasKimwipes,toremovedirtandoilfromthestageandothermechanicalpartsofthemicroscope.

TheDo'sandDonts:

Washyourhandsbeforehandlingslidestoavoidtransferringfingergreaseontothe

slides.

Toremoveoilfromanobjective,moistenapieceoflenspaperwithlenscleaningsolution

(ethanol)andslowlydrawthewetpaperacrossthefrontsurfaceofthelens.

Toremovedirtfromaneyepiece,breatheonthelensandGENTLYwipeoffthe

condensedwaterwithlenspaper.

Gethelpifthesestepsdonotsolveyourproblem.

Nevertouchalenswithanythingbutlenspaper.

Nevertouchalenswithdrylenspaper.

Neverrubalens.

Donotwearmascarawhenusingamicroscope.


8/16


Appendix B

Preparation of Wet Mount for Biological Specimens

Visit the following website for an animation of the preparation of a wet mount.

http://www.wonderhowto.com/how-to/video/how-to-make-a-wet-mount-for-microscope-

sample-viewing-259931/view/


9/16


MICROSCOPY:The microscope is one of the most important tools available to the developmentalbiologist. There are many different types of microscopes that serve specific needs,

including both compound and dissecting light microscopes. Primarily, microscopes areuseful for viewing objects that are too small to see clearly without magnification.Learning how to use them properly not only will allow you to achieve the greatestresolution your microscope can give, but will also avoid costly damage. Although youshare your microscopes with students in other laboratory sections, you are responsiblefor seeing that the microscope is properly cleaned and covered. The following briefexercise is designed to familiarize you with the use of a compound light microscope anda binocular dissecting microscope. Remember that only lens tissues should be used toclean microscope lenses (do not use regular tissues; it will damage the lens).

Binocular Dissecting Microscope:

Binocular dissecting microscopes are useful for viewing material that is too large to beviewed by compound light microscopes. In addition they are invaluable for dissectingand manipulating small materials/organisms. The magnification of these microscopestypically ranges from 8X to 40X. What is the range of your dissecting microscope?Dissecting microscopes have two ocular lenses and produce a three-dimensional image.Binocular microscopes have two adjustable ocular lenses. This enables you to adjust theviewing for each of your eyes so that you do not need to wear your glasses. You arewelcome to use your glasses if you wish, but they might get scratched.

Your dissecting microscope can be fitted with either a light source from above or frombelow. Be sure you know how to change back and forth. You will find that some slidesor organisms are better viewed with light from above and some from below. Inaddition, light intensity required for good visual images differs from specimen tospecimen. You should try different settings so you are comfortable changing betweenthe settings. Your dissecting microscope also has a zoom adjustment on the right sideof the microscope.


10/16



11/16


Usingacompoundlightmicroscope

Foroptimalviewingwithamicroscopeyoumustadjust:

o Eyepieces(interpupillarydistanceanddiopter)o Khlerilluminationo Focus

Mostmicroscopedamageisduetocarelesstransport.Itisimportantthatyoucarrythemicroscopesecurely,withtwohands,andinanuprightposition.Rememberthatyouarehandlingprecisioninstrumentation.

1.Carryyourmicroscopebyplacingonehandunderthebase(1)andtheotherhandholdingthehandleontherearofthearm(2)asshown.

2.Pluginyourmicroscopeandturniton(1).Makesuretoadjustthelightintensitytoacomfortablelevel

(2).

Makesurethatthecondenserapertureisopen(slidethecondenserdiaphragmleverbackandforthtocheck).

3. Place a slide on the stage. Ensure that it is locked in place with the slide holder.

PositionthespecimenunderthelightpathusingtheXandYaxisknobs.


12/16


4.Initialfocusingmustbedoneusingthe10X(or4X)objectivelens.Slowlyrotatethecoarseadjustmentknobuntilthespecimenisinfocus(turnclockwisetomove

thestagedown/counterclockwisetomovethestageup).Fineadjustmentismadeusing

thefineadjustmentknob.Allsubsequentfocusingshouldbe

donewiththefineadjustmentknobonly.

5.Toreduceeyestrain,adjusttheeyepiecestoyourinterpupillarydistance(distancebetweenyoureyes)andadjust

thediopter.

a.Interpupillarydistancemovetheeyetubesuntilyouonlyseeoneuniformfieldofview.Thisdistancevarieswitheachindividual.

b.Diopterusingthe10Xobjective,closeyourlefteyeandfocusonthespecimenwithyourrighteyeusingthefocusknobsuntilyouobtainasharp

image.Next,closeyourrighteyeandadjustthefocusofthelefteye

byrotatingthediopteradjustingringlocatedonthelefteyepiece.

Donotreadjustthefocusofthelefteyewiththecoarseorfineadjustmentsofthemicroscope.Step5needonlybeperformedonceatthebeginningofyourlab.Itmay,ofcourse,becheckedperiodicallyifdesired,andwillneedtobereadjustedifsomeoneelseusesyourmicroscope.


13/16


6.AdjusttheKhlerilluminationforoptimalillumination. Makesurethatthespecimenontheslideisinfocus. Usethe10Xobjectivetocompletethefollowingsteps:

Positionthecondenser: i.Closetheirisbyturningthefieldirisdiaphragmring(1)counterclockwise.

ii.Turnthecondenserheightadjustmentknob(2)tobringthefieldirisdiaphragmimageintofocus.Theiris

diaphragmisinfocuswhenyoucanclearlyseethehexagonal

shapeoftheiriswhichisusuallysurroundedwithablue

halo.

Centerthefieldirisdiaphragm: iii.Rotatethetwocondensercenteringknobs(3)sothatthefieldirisdiaphragmimageiscenteredinthe

eyepiecefieldofview.

iv.Openthefielddiaphragmuntilitsedgetouchestheperimeterofthefieldofview.

Matchthenumericalaperatures. v.Matchthenumericalaperture(1)ofthecondenserwiththenumericalaperatureoftheobjectivetoobtainabetter

imageresolutionandcontrast.

Neverusethecondenserapertureforcontroloflightintensity.Controloflightintensityisthepurposeofthevariablerheostat(dimmerswitch,orvoltageregulator)onthelightsource.7.Checkthefocusat10Xagain,centertheobjectyouwishtoview,androtatethe

nosepiecetothenexthighestmagnification(40X).Notethatthefieldofviewissmaller

athighermagnification.Usethefinefocusknobonlytoobtainthesharpestimage.

DONOTUSETHECOARSEADJUSTMENTKNOBWHENUSINGTHE40Xor100XLENS.


14/16


Evenifyouwishtoviewyourspecimenathigherpower,alwaysbeginfocusingwiththe10Xmagnification.Theobjectivelensesareparfocalandparacentric,whichmeansthatoncethe10Xlensisfocusedandcentered,thehighermagnifyinglenses(40Xand100X)arealsoinfocusandcentered.Onlyfineadjustmentsareneededtoobtainasharpimagethatiscenteredinthefieldofview.Whileusingthemicroscope,onehandshouldremainonthefinefocusasconstantreadjustmentwillbecalledfor.Usetheotherhandtomanipulatestagemovements.

MeasurementsandSize(Scale)Bars

Measuringyourspecimenusingtheocularmicrometer

Thesizeofthespecimencanbeaccuratelydeterminedbyusinganocularmicrometer.Theocularmicrometerisagraduatedscalethatislocatedinoneoftheeyepiecesofyour

microscope.Thisscaledoesnothaveanyunitsandmustbecalibratedagainstafixedand

knownruler,thestagemicrometerorgreticule.Wecanmeasurethediameterofthefieldofvieworacellusingastagemicrometerand

eyepiecegraticule.Youshouldrememberhowtodothis,buthereisareminder(figure2).

Figure 2. Method for calculating the size of a specimen.

Ingraticuledivisions,thecellillustratedaboveis41divisionsinlength.41divisionsonthe

eyepiecegraticuleisequivalenttoapproximately10X0.01mmdivisionsonthestage

micrometer.Therefore,thiscellis100minlength.Thismeansthat

ONEOCULARMICROMETERDIVISION=LENGTHOFSTAGEMICROMETER#OFDIVISIONSONOCULARMICROMETER

ONEOCULARMICROMETERDIVISION=10x0.01mm=2.43m 41CalibratetheocularmicrometerandshowthevaluesinTableAbelowtoyourTA:


15/16


TableA:ConversionTablefortheOcularMicrometerObjectivelensmagnification Ocularmicrometercalibration(inm/oculardivision)

10X

40X

Onceyouhavethecalibrationvaluesyouarereadytomeasureaspecimenunderthe

microscope.Useyourocularmicrometerasyouwouldarulerandmeasurethelengthof

theobject,thenmultiplythisbythecalibrationvaluetogettheactualsizeofthespecimen.

Remembertonotetheobjectivelensandusethecorrectcalibrationvalue!ScaleBars:Whendrawingacellviewedunderthemicroscope,itisimportanttoalsoindicate,onyour

drawing,theactualsizeofthecell.Thisiscommonlydonebyinsertingascalebarinthe

rightbottomcornerofthedrawingthatshowstherelationshipbetweenthesizeofthe

drawingandtheactualsizeofthespecimen.

Calculatingyourscalebars

Youmaycalculateyourscalebarbyusingthefollowingratio:

Remember:the

size

of

the

drawing

and

the

actual

size

of

the

specimen

must

havethesameunits(i.e.bothmustbeincm,mmorm)oryourcalculationwillbeincorrect.Theseunitsmustcancelout.

ActualSizeofSpecimen =MicrometermeasurementxCalibrationvalue

=Sizeofdrawing(units)Sizerepresentedbyscalebar(units)

Actuallengthofscalebar(units)Actualsizeofspecimen(units)


16/16


ExampleA:

Youhavemeasuredyourspecimentobe300mlongandhavedrawnadiagramthatis

5cminlength.Youwantyourscalebardrawingtobe1cminlengthi.e.thiscorresponds

to1cmofyourdrawing.Whatisthesizerepresentedbythat1cmlongscalebar?

Using the ratio above, the size of the drawing must have the same units as the actual size ofthe specimen, so convert cm to m: 5 cm = 50,000 m. Now you are ready to plug yourvalues in the ratio:

Them units cancel so the size represented by the scale bar = (300)(1cm)/50,000= 0.006 cmWe know that since our original specimen was very small (measured in m) then mwould be better units to use. So 0.006 cm converts to 60 m.Now you are ready to draw the scale-bar on your drawing. For example:

60 m

=0,000 m 1 cmSize represented by scale bar00 m

You draw the bar 1 cm long

You write, above the bar, the actual sizeit represents.

Documents

Lab 1 Appendix