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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





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





TheRamanConfocalMicroscopeSystem:Overview

Theleftrearview

Thefrontview

Synapsecamera

AndorNewtonEMCCDcamera

Halogenlampforepi‐illumination

Coarsefocusing

Finefocusing

Laserbutton

Objectiveturret

633nmlaser

830nmlaser 785 nmlaser

405 nmlaser

532 nmlaser




http://itg.beckman.illinois.edu/ ‐2‐

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


Manufacturer Laser Quantum Serial # 0420506‐82664

Model Ventus 532 50mW

Wavelength 633 nm (red) Max power 35 mW


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


Manufacturer Sacher Lasertechnik group Serial # RL‐830‐0711‐0322

Model N/A





DuringnormaloperationsoftheLaserTheRamanconfocalisaHoribaLABRAMproductandaclass3Blaserproduct,whichmeans

Laserbeamsandreflectedbeamscouldbedangerous.Diffusedreflectedbeamsinmostcasesdonotpresentanyproblem.

Lasersarehighintensitylightsourcesproducingvisibleorinvisiblelight(forexample,785nmand830nmlasersinthesystem)atspecificwavelengths.Thisconcentratedenergyinanarrowlaserbeammaycausedamagetobiologicaltissues,especiallytoeyes.Allbelowlabelsonthemicroscopeindicateahazardoussituationwherethereisariskofseriousinjurytobecausedbylaserradiation.Specialattentionmustbepaid.

Assuredoorremainsclosedandlockedfromtheoutsidewhenlaserisinuse.Theexterior“InUse”lightmustbeturnedonwheneverthelaserisinoperation

Beforeremovingorchangingobjectives,thelaserbuttononthemicroscopefrontpanelmustbein“off”position.Incaseofremovingobjectives,theemptyobjectivepositionsontheobjectiveturretmustbecoveredwithobjectivecaps.





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)





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





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





4. TurnontheLEDlampforepi‐illumination.

5. Openthecontrolsoftware–Labspecandloadsampleformeasurements.IfLabspecfreezesorgivesanerrormessage“ConnectionwithS3000fails,”followtheinstructioninthesectionofTroubleshootingtorestartthemicroscopesystem.TheLabspecsoftwaremanualcanbefoundinthefolderof“Manuals”onthewindowsdesktop.

Openthechambercarefully Carefullychangethesetwofiltersifnecessary.

Figure4Filterschamber

Figure5LEDlightforepi‐illuminationwhitelight





ShutDowntheMicroscopeSystem1. ClosetheLabspecsoftware,andlogoffthecomputer.2. DecreasetheLEDlightintensitytominimumandthenturnofftheLEDlamp(The

powerbuttonattherearofthelamp).3. Turnthe“Laser”buttononthemicroscopefrontpaneltoOFFposition.4. Turnoffthelasersthatyouhaveused.5. Cleanuptheworkingplace(Takeawayallyourstuffs;usedglassslidesorcoverslips

shouldbethrownintosharpswastebin;etc.).





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





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.





4) Yougetthreenewwindowsforthemappingacquisition.Youonlyneedtosavetheallspectradatawindow“SpIm”–clicktoactivethewindow,andsaveittoa*.ngcfile.





DefaultSystemConfigurationforGeneralUsers

PushintheDualScan/Standardplunger

Pullout–onlySynapseforgeneralusers

Pushinformicroscopemode

Usemicroscopemode

Optional

Turnitononlywhenlaserisneeded UseonlySynapse

forgeneralusers

UseonlyXYstageforgeneralusers

Optional





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”.

The left rear view - ITG : The Imaging Technology Group, Beckman

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