Olympus Bx51 User manual

OlympusBX51Microscope

(May2014)

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BX51Microscope

Brightfieldhalogenlamp;reflected(top)ortransmitted(bottom)illumination

Fluorescencemercurylamp

Filterpositions1.HQ:RDil(red)

2.DAPI(340‐380Ex;400dichroic;435‐485Em)

3.DIC

4.brightfield

5.darkfield

6.QdothxCM(red/blue)



Objective MagnificationImmersionN.A.WorkingDistanceFilters

MPlanFluorite 2.5X Dry 0.08  10.7 brightfieldonly(w/polarizer)

UMPlanFluorite 5X Dry 0.15  12.0  all

UMPlanFluorite 10X Dry 0.30  11.0  all

LMPlanFluorite 20X Dry 0.40  12.0  all

LMPlanFluorite 50X Dry 0.50  10.6  all

LMPlanFluorite 100X Dry 0.80  3.4  all(availableuponrequest)

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Filters

Wheel Position1–HQ:Rdilred

Position2–DAPI,fluorescencefilterforfluorophores,nuclearstain

Position3–DICNomarskiDifferentialContrastfilter

Position4–BF,brightfield(forallwhitelight)

Position5–DF,darkfieldfilter

Position6–QdothxCMred/blue

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CookeSensiCAMCamera

BlackandWhitecamera

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ImageProPlusSoftware

Capturesingleimages(8‐bitor12‐bit)ormovies(.avifiles)

Imageadjustments(gamma,saturation,backgroundsubtraction,etc.)

Measuredistances

Addlabelsorscalebars

Coloradjustmentsandfalsecolor

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TableofContents

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QuickGuide…………………………………………………………………………………………………page3

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Diagram Where’sthatknob?………………………………………………………………..4

What’sthatknobdo?...............................................................5

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Brightfield(transmittedlight)………..……………………………………………………………… 5

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Brightfield(reflectedlight)………..…………………………..…………………………………….. 6

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Fluorescence………………………………………………………………………………………………… 7

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Imageacquisitionandmeasurements………………………………………….……………..8

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Troubleshooting……………………………………………………………………………………………..11

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ShutdownSteps

Alwaysturnoffthecamera.

Alwaysturnoffthelamps.

Swingthe2.5Xobjectiveinplace.

Transferyourimages.

QUICKGUIDE

(Boldnumbersareshownonthemicroscopediagram).

1. LogintoCORAL.

2. Turnonthehalogenlamp(1).

3. Selecttransmitted(bottom)light(4)fortransparentsamplesorreflected(top)light.

4. Turnonthecameraforpicturesormeasurements(switchatopthebluecamera).

5. ONLYIFusingfluorescence,turnonthemercurylamp(18).

6. Setthelightpathforthehalogenormercurylamp(30).

7. Choosethefilterwheelsetting(17)(NOTBFforUVlightoryouwilldamageyoureyes).

8. Choosetheobjective(6).

9. Loadsampleonthestage.

10. Focus(10)andadjustillumination(3).

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Iftransmittingfluorescence

Slidethefilterwheeltoposition2.Theposition4(BF)filterwillexposeyoureyestoUVlight.

Usethe25%NDfilter(31)toreducetheUVintensity.

Closetheshutter(20)toprotectsamplefromexcessUVlight.

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Forpictures(Seepage9fordetails).

Sendlighttothecamerawiththecamera/eyepiecesplitterknob(2).

OpenImageProPlussoftwarewiththedesktopicon.

Openanacquisitionwindow.

SelecttheSensiCAMCookeDriverv3.6.

Setbitacquisitiondepth.

Setfilesavingoptions.

Setsingle,intervalortimelapsecapturemodes.

Setspatialcalibrationtomatchtheobjectivethatisbeingused.

Setexposuretimeoruseautoexposure.

Openalivepreviewwindow.

Focus,adjustexposuretimeorlighting,adjuststageposition. 

Stopthepreviewandcapturetheimage(ormovie).

Savetheimage.

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Formeasurements

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Verifythatthespatialcalibrationmatchestheobjective.

Measurethedistanceorfeature.

Toburnthenotationsintotheimage,selectthecameraiconandsavethenewfile.

Toburnascalebarintotheimage,usethecaliperbuttonandthe‘Marker’button.Savethenewfile.

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 

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 



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WHERE’STHATKNOB?

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WHATDOESTHATKNOBDO?

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(1)White(halogen)lightpowerswitch–turnsonbothtransmittedandreflectedlight.

(2)Eyepiece/Cameralightpathselector–sendslighttoeyepieces,cameraorboth.

(3)Brightnessadjustment–controlsintensityoftransmittedandreflectedlight.

(4)Lightintensityandtoggleswitches–Thetoggleselectsthelower(transmitted)orupper(reflected)

lightsource.The‘PRESET’buttongivesmaximumillumination.

(5)Eyepieces–canadjusttheinterpupillarydistanceandfocus.Thelefteyepiecehasamicrometer.

(6)Objectives

(7)Stagewithsteelplatesformagneticprobearmsforfluidicdevices.Removetheglassstagetouse

magneticprobes.

(8)Stageclip.AskBethtoputiton.

(9)Stagecontrols(actuallyonleftsideofstage).

(10)FineandCoarsefocus.Eachunitis1microninstageheightadjustment.

(11)Diopterring–forfocusingtheocular

(12)Rotatingnosepiece–forselectingobjectives

(13)Fieldirisdiaphragmfortransmittedlight–forestablishingKoehlerillumination

(14)Condenserheightadjustment‐forestablishingKoehlerillumination

(15)Condensercenteringscrews‐forestablishingKoehlerillumination

(16)Condenserapertureirisdiaphragm‐forestablishingKoehlerillumination

(17)Filterwheelholdingmirrorsandfiltersforreflectedlight

(18)Mercurylamppowerswitch–toturnthefluorescencelightonandoff.

(19)Mercurylamppowerindicator–redwhenthemercurybulbisON.

(20)Shutterknob–shutterforreflectedlight(whiteorfluorescent)toprotectsamplefromexcessUVlight.

(21)Fieldirisdiaphragm–restrictsthediameterofthebeamoflightenteringtheobjective(excludes

extraneouslightandimprovesimagecontrast).Alsoslowsfadinginotherpartsofthesample.

(22)Apertureirisdiaphragm–adjuststhebrightnessoftheobservedimageandimprovescontrast.

Usetofine‐tuneafterusingtheNDfilters.

(23)Notonthismodel

(24top)‐condenserscrew.Notonthismodel

(24bottom)–reflectedlightanalyzer.Notonthismodel.

(25)Polarizer

(26)DICprismslidefilter–adjuststhecontrastwhenusingDIC

(30)Lightpathselector–selectshalogenlampandormercurylampforreflectedlight.

(31)Buttonsforbuilt‐infilters

ND6decreasesexcitationlightintensityto6%(fluorescenceapplications)

ND25 decreasesexcitationlightintensityto25%(fluorescenceapplications)

LBDcolorconversionfiltertosimulatedaylight(BF,imagecapture)

OPdunno

(32)Slidefiltersforreflectedlight

U‐25FR,frostslidefilter‐toeliminateunevenillumination

U‐25ND25,NDslidefilter‐toreducetheexcitationlightintensity.

(33)Rearhalogenlamphousing–sourceofthereflectedwhitelight.

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BRIGHTFIELDTransmittedlight

Whitelighttransmitsthroughatransparentsamplefromunderneath.

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1.Turnonthewhite(halogen)lightswitch(1)fortransmittedlightto“│”(ON).

2.Engagethebrightfield(BF)mirrorunit(17).

3.Adjustthelightpath.Thelightpathselectorknob(2)shouldbepushedinalltheway.

4.Selecttransmittedlight(lowerposition)ontheswitchbythebrightnessindicator(4).

5.Adjustthelightintensityusingthebrightnessadjustmentknob(3).Thenumeralsbythelampvoltage

indicator(4)indicatethevoltage.Deselectthe“PRESET”button(notilluminatedgreen)toavoid

maximumillumination.

6.Adjusttheinterpupillarydistance.Whilelookingthroughtheeyepieces,adjusttheeyepieces(5)untilthe

leftandrightfieldsofviewcoincide.

7.Swingthe2.5xobjective(6)inplace.Mountthespecimenonthestage(7).

8.Findthespecimenusingthestagecontrols(9).

9.Focusonthespecimenusingfine/coursefocusingknobs(10).

10.Adjustthediopter:

‐Closeyourlefteyeandfocusonthespecimenusingthefinefocusknob.

‐Closeyourrighteyeandfocusonthespecimenusingthediopterring(11)ontheleftocular.

‐Openbotheyestoseeifthefocusiscorrect.

11.Ifneeded,switchtothenextobjectivebyrotatingthenosepiece(12)andfocus.

Continuetothedesiredmagnification.

12.EstablishKoehlerillumination:

 ‐Closethefieldirisdiaphragm(13)untilyoucanseetheedges.

 ‐Focustheimageofthefieldirisdiaphragmbyraisingorloweringthecondenserusingthecondenser

heightadjustmentknob(14).

 ‐Checkwhetherthelightiscenteredinthefieldofview.Ifnot,usethecondensercenteringscrews

(15)tomovethefieldirisdiaphragmimagetothecenterofthefieldofview.

 ‐Openthefieldirisdiaphragmuntilitsimagecircumscribesthefieldofview.

 ‐Matchtheopeningofthecondenserapertureirisdiaphragm(16)withtheN.A.oftheobjectivein

usetoachievetheoptimumobjectiveperformance.

13.Examinespecimenandtakeapicture,ifneeded(Seepage9fordetails).

14.Whenfinished:

‐Lowerthestagebyturningthefocusknob(10)andremovethespecimen.

‐Turnthenosepiece(12)untilthe2.5xobjectiveisintoplace.

‐Lowerthelightintensitytozerousingthebrightnessadjustmentknob(3)

‐Turnthehalogenlightswitch(1)to“○”(OFF).

‐Returnanyslidefilters(26)(31)(32)totheirdisengagedpositions.



BRIGHTFIELDReflectedlight

Whitelightisshoneontoareflectivesamplefromabove.



1.Turnthehalogenbulbswitch(1)forreflectedlightto“│”(ON).Engagethebrightfield(BF)filter(17).



2.Adjustthelightpath.

‐Thelightpathselectorknob(2)shouldbepushedinalltheway.

 ‐Theknobonthereflectedlightsplitter(30)shouldbepushedinalltheway.

‐Theshutterknob(20)shouldbeslidtothepositionmarked“○”(OPEN).



3.Selectreflectedlight(upperposition)ontheswitchbythebrightnessindicator(4).

4.Adjustthelightintensityusingthebrightnessadjustmentknob(3).Thelightintensityscale(4)indicatesthe

lampvoltage.Deselectthe“PRESET”button(notilluminatedgreen)toavoidmaximumillumination.

5.Adjusttheinterpupillarydistance.Whilelookingthroughtheeyepieces,adjusttheeyepieces(5)untilthe

leftandrightfieldsofviewcoincide.

6.Swingthe2.5xobjective(6)inplace.

7.Placethespecimenonthestage(7).

8.Findthespecimenusingthestagecontrols(9).

9.Focusonthespecimenusingfine/coursefocusingknobs(10).

10.Adjustthediopter:

‐Closeyourlefteyeandfocusonthespecimenusingthefinefocusknob.

‐Closeyourrighteyeandfocusonthespecimenusingthediopterring(11)onthelefteyepiece.

‐Openbotheyesandconfirmthatitisinfocus.

11.Switchtothenextobjectivebyrotatingthenosepiece(12)andfocus.Continueuntilyoureachthedesired

magnification.

12.EstablishKoehlerillumination:

 ‐Closethefieldirisdiaphragm(13)untilyoucanseetheedges.

 ‐Focustheimageofthefieldirisdiaphragmbyraisingorloweringthecondenserusingthecondenser

heightadjustmentknob(14).

 ‐Checkwhetherthelightiscenteredinthefieldofview.Ifnot,usethecondensercenteringscrews

(15)tomovethefieldirisdiaphragmimagetothecenterofthefieldofview.

 ‐Openthefieldirisdiaphragmuntilitsimagecircumscribesthefieldofview.

 ‐Matchtheopeningofthecondenserapertureirisdiaphragm(16)withtheN.A.oftheobjectivein

usetoachievetheoptimumobjectiveperformance.

13.Examinespecimenandtakeapicture,ifneeded.(Seepage9fordetails).

14.Whenfinished:

‐Lowerthestagebyturningthefocusknob(10)andremovethespecimen.

‐Turnthenosepiece(12)untilthe2.5xobjectiveisintoplace.

‐Lowerthelightintensitytozerousingthebrightnessadjustmentknob(3)

‐Turnthehalogenlightswitch(1)to“○”(OFF).

‐Returnanyslidefilters(26)(31)(32)totheirdisengagedpositions.



FLUORESCENCE(Transmittedlight)



NOTE:Themercurybulbisfragile.Itneedstobecoolfor30minutesbeforebeingturnedonagain.Itneedsto

stayonforatleast15minutes.



1.Switchonthemercurylampbysettingtheswitch(18)to“I”(ON).Theindicator(19)shouldlightup.

Thebulbwillstabilizein5‐10minutes.



2.Setthemicroscopeforbrightfieldimaging(‘Brightfieldreflectedlight’page2,steps2–12.)

Usethereflectedlightsplitterknob(30)toswitchbetweenwhitelightandUVlightlamps.



3.Turnthefilterwheel(17)toselectafiltercube:

 position#1.HQ:RDil(red)

   2.DAPI(340‐380Ex;400dichroic;435‐485Em)

   3.DIC

   4.Brightfield(BF)

   5.Darkfield

   6.QdothxCM(red/blue)



4.Whenusingthefiltercubes,turnthelightintensitydownalltheway(3).



5.Toavoidphotobleaching,closetheshutter(20)“●”(CLOSED)whennotacquiringanimage.



6.Toimproveimagecontrastandtopreventphotobleachingotherpartsofthespecimen,pulloutthefieldiris

diaphragmknob(21)sothattheimageofthefieldirisdiaphragmjustcircumscribesthefieldofview.



7.Toadjustthebrightnessandtoimprovecontrast,pullouttheapertureirisdiaphragmknob(22),the

diaphragmwillbesmaller.



8.Examinespecimenandtakeapicture,ifneeded.(Seepage9fordetails).



9.Whenfinished:

‐Slidetheshutterknob(20)to“●”(CLOSED).

‐Pushtheknobonthereflectedlightsplitter(30)inalltheway.

‐Ifmercurybulbhasburnedforatleast15minutes,turnitoffbysettingthemainswitch(18)to“O”.

‐Lowerthestagebyturningthefocusknob(10)andremovethespecimen.

‐Turnthenosepiece(12)backuntilthe10xobjectiveisintoplace.

‐Pushthefieldirisdiaphragmknob(21)andtheapertureirisdiaphragmknob(22)allthewayin.

‐Turnthefilterwheel(17)toBF(brightfield).

‐Returnanyslidefilters(26)(31)(32)totheirdisengagedpositions.





ImageacquisitionCookeSensiCAMB/Wcamera;driverv3.6&ImageProPlussoftware;v6.2



1.TurnontheblueSensiCAMcamera(switchatthetopofthecamera).Waituntiltheblinkinglightturnsgreen.



2.Slidethelightpathselectorknob(2)tosharetheimagewiththecamera(eyeandcameraposition).



3.OpenImageProPlusandadjustsettings. 

‐LogonthecomputerasCNFUSER(nopassword).

‐Double‐clickonthe“USEthisImageProPlus”desktopicon.

‐SelectAcquire:Video/Digital.

Ifawindowtoselectan“AnalogSimulationImageFile”appears,click“Cancel”

‐Awindowtitled‘AnalogSimulation’appears.UsetheCurrentDriverpulldownmenutoselectthe

“SensiCAMCookeDriver(3.6)”BESURETHEDRIVERREMAINSSELECTED.

‐SettheCaptureDepthto8‐bitmonointhepulldownmenu.

‐Setthepreview(pvw)andacquisition(acq)areas(Fullis1376x1040).



4.OpenaliveimagePreviewbutton.

‐Adjustfocusandlighting(3)onthemicroscope.

‐Adjustcameraexposure.

‐SethowtheimagefileistobesavedusingtheImageTab.

 ‐Formultipleimages/movies,checkthe‘EnableMultipleImagesCapture’box,andsetcontrols.



5.Capturetheimage.

‐StopthelivePreviewusingthe‘Stop’button.

‐Clickthe‘Snap’button,andanimagefilewillappear.

‐Savetheimage.Ifitwastakenin12‐bitmono,thecomputercan’topenit.Youcouldconvertitusing

File>BatchConversionandselectthesourcefileandthedestinationfolderandfiletype(TIFFisbest).



6.Takemeasurementsinthesavedimage.



‐Selecttheobjective(spatialcalibration).Clickthecaliperbuttononthemenubar,andselectthe

objectivethatisinuse.

‐ClickthemeasureDistancebuttononthemenubartoopena‘MeasureDistance’popupwindow.

‐Clickanddragtomeasurethefeature(inmicrons).

‐Tosavetheannotation,clickthecamerabuttononthemenubar.Anewfileiscreated.SaveitasaTIFF.



7.Toinsertascalebar,clickthecaliperbuttononthemenubar,anddouble‐checkthatthecorrectobjectiveisselected.

‐Selectthe‘Marker’buttoninthepop‐upwindow.

‐A‘SpatialcalibrationMarker’pop‐upwindowappears.

‐Choosecolor,markerlength,andnon‐destructivemode.

‐Changethepositionsbydouble‐clickingonthebarorthelabelBEFOREclosingthemessagewindow.

‐Tosavethescalebar,clickthecamerabuttononthemenubar.Anewfileiscreated.Saveit.



8.TaketheimagesonaflashdriveortransferthemtotheLab_xfericon.

Filesareautomaticallydeletedin7days.



9.ClosetheImageProPlussoftware.



10.Leavethecomputeron.

Troubleshooting

1.White(brightfield)lightisn’tmakingittothesample.

CAUSE&REMEDY

Lampnoton.

Turnonthelamp(1)andlooktoseeifbothbulbscomeon.Onelampisunderthe

stage(13)andtheotherisintherearlamphousing(33).

Turnonthemercurylamp(18)(ifithasbeenoffforatleast30minutes).Wait5‐10

minutesforthebulbtostabilize.



Wronglightsourceorincorrectlightpath.

Selecttheocularpath(2)toallowthesampletobeseenintheoculars.

Selectreflected(top‐side)ortransmitted(underneath)whitelightwithswitch(4).

Selectreflectedwhitelightvs.fluorescentlightwithslideknob(30).

Opentheshutter(20)forreflectedlight.



Intensityislow.

Turn(3)uptheintensityoflightsource.

DisengagetheNDfilters(32).

Openthefieldirisdiaphragm(13).

Opentheapertureirisdiaphragm(22)andfieldirisdiaphragm(21).

SelectBF,DForDICmirrors(17)whenusingwhitelight.



2.PROBLEM:Whitelight(brightfield)istoointense.

CAUSE&REMEDY

“Preset’(4)ispushedinwhichautomaticallyturnsonthemaximumlightvoltage.

Deselectthebutton(greenlightwillgooff).



Intensityisturneduptoohigh.

Dial(3)itdown.



3.PROBLEM:Fluorescentlightisnothittingsamplecorrectly.

CAUSE&REMEDY

Lampnoton.

Turnonthemercurylamp(18)(ifithasbeenoffforatleast30minutes).

Wait5‐10minutesforthebulbtostabilize.



Wronglightsourceorincorrectlightpath.

Selecttheeyepiecepath(2)toallowthesampletobeseenintheoculars.

Selectreflectedlight(top‐side)switch(4).

Selectfluorescentlightwithslideknob(30).

Opentheshutter(20)forreflectedlight.



4.PROBLEM:Can’tcaptureadigitalimage.

CAUSE&REMEDY

Cameraisnotturnedonorstabilizedyet

Turnonthecamera(34)attheblackpowerswitchandwait2minutes.



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