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Instructions for Horiba Raman confocal imaging microscope · Raman confocal imaging microscope...
Transcript of Instructions for Horiba Raman confocal imaging microscope · Raman confocal imaging microscope...
DianwenZhang(Email:[email protected])
Raman confocal imaging microscope
Instructions for Horiba Raman confocal imaging microscope
ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnology
UniversityofIllinoisatUrbana‐Champaign
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
405NorthMathews,UrbanaIL61801Tel:217‐244‐0170;FAX:217‐244‐6219
http://itg.beckman.illinois.edu/
RevisionHistoryAuthorsandEditors RevisionDateDianwenZhang 4/20/2012DianwenZhang 12/22/2014DianwenZhang 06/28/2016DianwenZhang 05/10/2017
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
405NorthMathews,UrbanaIL61801Tel:217‐244‐0170;FAX:217‐244‐6219
http://itg.beckman.illinois.edu/
TableofContents
TheRamanConfocalMicroscopeSystem:Overview..................................................................1
StandardOperatingProcedure(SOP)forLasers.........................................................................2
Laserspecifications.............................................................................................................................................2
DuringnormaloperationsoftheLaser......................................................................................................3
Laserstart‐upprocedure..................................................................................................................................4
Lasershutdownprocedure..............................................................................................................................5
Emergencyresponse..........................................................................................................................................5
StartUpTheMicroscopeSystem.......................................................................................................6
ShutDowntheMicroscopeSystem...................................................................................................8
BasicLabspecSoftwareSettings........................................................................................................9
GeneralSpectralAcquisitionProcedure......................................................................................10
DefaultSystemConfigurationforGeneralUsers......................................................................12
Troubleshooting...................................................................................................................................13
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
405NorthMathews,UrbanaIL61801Tel:217‐244‐0170;FAX:217‐244‐6219
http://itg.beckman.illinois.edu/
TheRamanConfocalMicroscopeSystem:Overview
Theleftrearview
Thefrontview
Synapsecamera
AndorNewtonEMCCDcamera
Halogenlampforepi‐illumination
Coarsefocusing
Finefocusing
Laserbutton
Objectiveturret
633nmlaser
830nmlaser 785 nmlaser
405 nmlaser
532 nmlaser
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
405NorthMathews,UrbanaIL61801Tel:217‐244‐0170;FAX:217‐244‐6219
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StandardOperatingProcedure(SOP)forLasers
Laserspecifications
Wavelength 405 nm (blue) Max power 40 mW
Laser Class Class 3B Laser Type Continuous wave
Manufacturer ONDAX Serial # X6000
Model LM‐405‐PLR‐40‐2
Wavelength 532 nm (green) Max power 50 mW
Laser Class Class 3B Laser Type Continuous wave
Manufacturer Laser Quantum Serial # 0420506‐82664
Model Ventus 532 50mW
Wavelength 633 nm (red) Max power 35 mW
Laser Class Class 3B Laser Type Continuous wave
Manufacturer Melles Griot Serial #
Model 05‐LHP‐928
Wavelength 785 nm (invisible) Max power 575 mW (set to 330 mW)
Laser Class Class 4 Laser Type Continuous wave
Manufacturer Toptica Photonics AG Serial # XTRA_3V0_01113
Model XTRA II
Wavelength 830 nm (invisible) Max power 100 mW
Laser Class Class 3B Laser Type Continuous wave
Manufacturer Sacher Lasertechnik group Serial # RL‐830‐0711‐0322
Model N/A
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
405NorthMathews,UrbanaIL61801Tel:217‐244‐0170;FAX:217‐244‐6219
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DuringnormaloperationsoftheLaserTheRamanconfocalisaHoribaLABRAMproductandaclass3Blaserproduct,whichmeans
Laserbeamsandreflectedbeamscouldbedangerous.Diffusedreflectedbeamsinmostcasesdonotpresentanyproblem.
Lasersarehighintensitylightsourcesproducingvisibleorinvisiblelight(forexample,785nmand830nmlasersinthesystem)atspecificwavelengths.Thisconcentratedenergyinanarrowlaserbeammaycausedamagetobiologicaltissues,especiallytoeyes.Allbelowlabelsonthemicroscopeindicateahazardoussituationwherethereisariskofseriousinjurytobecausedbylaserradiation.Specialattentionmustbepaid.
Assuredoorremainsclosedandlockedfromtheoutsidewhenlaserisinuse.Theexterior“InUse”lightmustbeturnedonwheneverthelaserisinoperation
Beforeremovingorchangingobjectives,thelaserbuttononthemicroscopefrontpanelmustbein“off”position.Incaseofremovingobjectives,theemptyobjectivepositionsontheobjectiveturretmustbecoveredwithobjectivecaps.
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
405NorthMathews,UrbanaIL61801Tel:217‐244‐0170;FAX:217‐244‐6219
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Whenusing785nmand830nmlasers(invisible),allusersarerequiredtowearlasergogglesthroughoutexperiments.
Usershouldavoidwearingreflectiveaccessories/jewelry(e.g.watches).
Laserstart‐upprocedureThelasercontrollersareshowninFigure1.Onlyonelaserissupposedtobeusedatatime;
DoNOTadjustthesettingsonthelasercontrollers.
Forthe405nmlaser(Figure1(a)),plugthepowersupplytothenearbypoweroutlet.Forthe532nmlaser(Figure1(b)),turnonthekeyswitch,andpressthe“Enable”buttonwhenitstartsflashing.Forthe633nmlaser(Figure1(b)),turnonthekeyswitchonthelasercontroller.Forthe785nmlaser(Figure1(c)),turnonthekeyswitchlocatedontheopticaltableinfrontofthe830nmlasercontroller.Forthe830nmlaser(Figure1(b)),pressonthepowerbutton,waitfor30secondsandpresstheshutter“on/off”buttonon.
Figure1 (a)405nmlaserpowersupply;(b)532,633and830nmlasercontrollers;(c)785laser
Shutter
633nmlasercontroller
532nmlasercontroller
830nmlasercontrollerPower
(b)(a) 405nmlaserpowersupply
785nmlaser“on/off”switch
(c)
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
405NorthMathews,UrbanaIL61801Tel:217‐244‐0170;FAX:217‐244‐6219
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LasershutdownprocedureForthe405nmlaser(Figure1(a)),unplugthepowersupply.Forthe532nmlaser(Figure1(b)),turnoffthekeyswitchonthelasercontroller.Forthe633nmlaser(Figure1(b)),turnoffthekeyswitchonthelasercontroller.Forthe785nmlaser(Figure1(c)),turnoffthekeyswitchlocatedontheopticaltableinfrontofthe830nmlasercontroller.Forthe830nmlaser(Figure1(b)),pressoffthepowerbuttononthelasercontroller.
EmergencyresponseINEVENTOFEMERGENCY:
‐ Turnoffalllasersimmediately.‐ Dial911.‐ Ifalasereyeinjuryissuspected,havetheinjuredpersonkeeptheirheaduprightand
motionlesstorestrictbleedingintheeye.Aphysicianshouldevaluatelasereyeinjuriesassoonaspossible[1].
‐ Ifthereisafire,leavethearea,pullthefirealarm,andcontactthefiredepartmentbycalling911.Donotfightthefireunlessitisverysmallandyouhavebeentrainedinfire‐fightingtechniques[1].
‐ InformthePI,thegroupsafetyofficer,andthebuildingmanagerassoonaspossible.Ifthereisaninjury,thePIneedstosubmitareportofinjurytoRiskManagement.Provideasmanydetailsaspossible[1].
‐ InformtheDRSat217‐333‐2755assoonaspractical.Provideasmanydetailsaspossible[1].
References
[1]UIUCDRSLaserSafetyManual.http://www.drs.illinois.edu/site‐documents/LaserSafetyManual2014.pdf
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
405NorthMathews,UrbanaIL61801Tel:217‐244‐0170;FAX:217‐244‐6219
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StartUpTheMicroscopeSystem
1. Turnonthelaser.
2. SetupthelaserpathtomicroscopeFollowingthenoteontheyellowlabelbesideeachplunger(#1‐4inFigure2),setupthefourfrontplungerstoguidethelasertothelaserentranceportofthemicroscope.
Fortheuserswhouse633nmlaseranddon’tuseSuperhead,pleasealsocheckthefifthplunger(Figure3)inthesystem:
3. Checkthetwoopticalfiltersinsidetheslidingchamber(Figure4)ontopofthemicroscope.Ifthelabelsoftheinstalledfiltersdoesn’tmatchthelaserwavelength,changethem.
Plunger1
Laserentrance(ahole)tothemicroscope
Plunger3
Plunger2
Plunger4
Plunger5needstobepulledupfortheuseof633nmlaserwhenusingthemicroscope.
Figure2 Thefrontplungers
Figure3 Therearplunger
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
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4. TurnontheLEDlampforepi‐illumination.
5. Openthecontrolsoftware–Labspecandloadsampleformeasurements.IfLabspecfreezesorgivesanerrormessage“ConnectionwithS3000fails,”followtheinstructioninthesectionofTroubleshootingtorestartthemicroscopesystem.TheLabspecsoftwaremanualcanbefoundinthefolderof“Manuals”onthewindowsdesktop.
Openthechambercarefully Carefullychangethesetwofiltersifnecessary.
Figure4Filterschamber
Figure5LEDlightforepi‐illuminationwhitelight
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
405NorthMathews,UrbanaIL61801Tel:217‐244‐0170;FAX:217‐244‐6219
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ShutDowntheMicroscopeSystem1. ClosetheLabspecsoftware,andlogoffthecomputer.2. DecreasetheLEDlightintensitytominimumandthenturnofftheLEDlamp(The
powerbuttonattherearofthelamp).3. Turnthe“Laser”buttononthemicroscopefrontpaneltoOFFposition.4. Turnoffthelasersthatyouhaveused.5. Cleanuptheworkingplace(Takeawayallyourstuffs;usedglassslidesorcoverslips
shouldbethrownintosharpswastebin;etc.).
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
405NorthMathews,UrbanaIL61801Tel:217‐244‐0170;FAX:217‐244‐6219
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BasicLabspecSoftwareSettings
1. Selecttherightlaserline.2. Selecttherightdensityfilter.ThenumbersareopticaldensityOD=Log(Power
transmissionfactor).Thedashlinesgivenodensityfilterinuse.3. Theconfocalpinhole(Hole)controlsmicroscopespatialresolution.Forbestoptical
resolution,setitto200‐300μm,orotherwiseusethemaximumvalue950μm.Fillinadesiredvalue,click“enter”onkeyboard.
4. Clicktheredthunderbuttontoinitializethe“Hole”.5. Theslitsize(Slit)determinesspectralresolution.Fillinadesiredvalue,click“enter”on
keyboard.6. Clicktheredthunderbuttontoinitializethe“Slit”.7. Selectthedesiredgrating.Bydefault,only300g/mmand1800g/mmareavailable.300
g/mmforbroadspectralrange,butpoorspectralresolution,while1800fornarrowspectralrange,butgoodspectralresolution.
8. Payattentiontotheunit.Followthebelowpicturetochangeitifnecessary.
9. Setthecentralvalueofthex‐scaleofthespectrum.Changeitforvaryingspectralrange
under“singlewindow”modespectralacquisition.Fillinadesiredvalue,click“enter”onkeyboard.
10. Clicktheredthunderbuttontoinitializethe“Spectrometer”.11. Selecttheobjectivelenstobeused.Itmustbesetrightbeforeyourmappingacquisition.
Alldefaultobjectivesarelongworkingdistance(LWD)lens.12. Theprefixofyourdatafilename(optional).13. Makesurethisis“XYZMotorized”.14. Thesethreeredthunderbuttonsallowyoutodefinethecurrentsamplepositiontobe
theoriginofcoordinatesinthesystem.Usuallyitisneededtoclickthebuttonforz‐axisbeforeyoudoz‐axisscanningmapping.
15. Theexposuretimeforrealtimespectralacquisition.Theunitisseconds.
1 2
3
74 6 10 1315
11 14
5 89171216
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OperationmanualofRamanconfocal
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16. Theexposuretimeforsinglespectrumacquisitionormappingacquisition.Theunitisseconds.
17. Setthenumberofaccumulationsperspectralacquisition.
GeneralSpectralAcquisitionProcedure
1. Checktheabovementionedsettingsatthebottompanelofthesoftwareandmakesuretheyareallcorrect.
2. Checkthe“Laser”buttononthefrontpanelofthemicroscopeandmakesureitisonthe"OFF"position.
3. Startvideobyclicking .4. YoushouldbeabletoseetheLEDwhitelightshiningonyoursamplethroughthe
objecivelens.Adjustthemicroscopefocustogetyourinterestedsampleareaintothe
objectivefocalplaneandthenstopthevideobyclicking .Youcansavetheimageinthevideowindowtoa*.ngvorotherimageformatfile.
5. Turnoffthefluorescenceroomlight,butyoumaykeeptheHalogenceilinglighton.Nowyouarereadyforspectralacquisition.
a. Ifyouaregoingtodoreal‐timespectralacquisition ,turnonthe“Laser”buttononthemicroscopefrontpanel,click tostartitandclick tostopit.Youcansavetheacquiredspectrumtoa*.ngsfileandconvertittoa*.txtfile.
b. Ifyouaregoingtodosinglespectrumacquisition ,followthebelowsteps:1) Ifyouhavecheckedthe“extendedrangeacquisition ”andknownthere
isnothingneededtochange,skipthisstep.Orotherwiseclick ,selecteither“Singlewindow”or“Multiplewindows”mode.Ifyouaregoingtousethe“Multiplewindows”mode,youalsoneedtosetforaspecificspectralrange.
2) Turnonthe“Laser”buttononthemicroscopefrontpanel,click togetasinglespectrum.Everytimeyouclick ,yougetonemorespectrum.
3) Usingthemenu“File→SaveAll”,youcansaveallacquiredspectratoasingle*.ngs ile,oryoucanalsousethemenu“File→SaveAs”tosaveasingleselectedspectrumtoa*.ngsfileorconvertittoa*.txtfile.
c. Ifyouaregoingtodomappingacquisition ,followthebelowsteps:1) Settouse“SingleWindow”modeinthe“extendedrangeacquisition ”.2) Setthemappingproperty .3) Turnonthe“Laser”buttononthemicroscopefrontpanel,click tostart
mappingacquisition.
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
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4) Yougetthreenewwindowsforthemappingacquisition.Youonlyneedtosavetheallspectradatawindow“SpIm”–clicktoactivethewindow,andsaveittoa*.ngcfile.
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DefaultSystemConfigurationforGeneralUsers
PushintheDualScan/Standardplunger
Pullout–onlySynapseforgeneralusers
Pushinformicroscopemode
Usemicroscopemode
Optional
Turnitononlywhenlaserisneeded UseonlySynapse
forgeneralusers
UseonlyXYstageforgeneralusers
Optional
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OperationmanualofRamanconfocal
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Troubleshooting1. Problem:WhenstartingLabspec,itbecomesfrozenorshowsthebelowerrormessage
beforethefinalmainwindowcanappear.
Solution:Followthebelowstepstorestartandresetthemicroscope.
1) CloseLabspec.SometimesyoualsoneedchecktheWindowsTaskManagertomakesurethatthereisnoaprocesscalled“NGSLabspec.exe”runninginbackground.
2) Restartthemicroscope.Findthebelowsocketandthemicroscopepowerboxbesidethelasercontrollersontheopticaltable.Turnoffthesocketswitchpointedbytheyellowarrow.Waitmorethan1minuteandthenturnitbackon.Nowthemicroscopeisrestarted.
3) StartLabspec.Labspecshouldrunnormally,andafterafewcommondevice
initialization,itwillshowthebelowstagecalibrationmessage.Beforeyouclick“OK”,makesurethatnosampleisloadedortheloadedsamplewon’thittheobjectiveswhenthestagemovinginitsfullrange.Click“OK”tostartthestagecalibration.
4) Initializethethreekeymicroscopecomponents‐“Hole,”“Slit”and
“Spectrometer”.Seethebelowimage.Clicktheredthunderbuttonatthetop‐rightonthe“Hole”controlpanel,waituntiltheinitializationprocessiscomplete,andthenseparatelyrepeatthisprocedurefor“Slit”and“Spectrometer”.
MicroscopySuite,ImagingTechnologyGroupBeckmanInstituteforAdvancedScienceandTechnologyUniversityofIllinoisatUrbana‐Champaign
OperationmanualofRamanconfocal
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