STANDARDOPERATINGPROCEDURE:RENISHAW INVIARAMANSPECTROMETER

PurposeofthisInstrument:TheRenishawinViaRamanSpectrometerisaninstrumentusedtoanalyzetheRaman scatteredlightfromsamplestoinferthechemistryandstructureofthematerialofinterest. Location:325HodgesHall PrimaryStaffContact: WeiqiangDing(304)6851938cell weiqiang.ding@mail.wvu.edu

SecondaryStaffContact:HarleyHart(412)4431514cellharley.hart@mail.wvu.edu The Shared Research Facilities are operated for the benefit of all researchers. If you encounter any problems with this piece of equipment, please contact the staff member listed above immediately. There is never a penalty for askingquestions.Iftheequipmentisnotbehavingexactlythewayitshould,contactastaffmember. WARNING:ThissystemusesaClassIIIblaserandemitsvisibleandinvisibleradiation. LasersafetygogglesMUSTbewornwhenoperatingthelaserasaneyehazardexists. START UP NOTE:TheRAMANspectrometer,microscope,andlasershouldbepowereddownwhennotinuse.Ifthepowerhas beenleftontoanyoftheseitems,pleaseinformaMFCFstaffmember. 1. 2. 3. 4. 5. Turnonthecomputerifitisnotalreadyon.LoginasRaman2withoutpassword. Log in your session on the FOM. Write down your name and sample information on the log book located onthecomputercart. Place the DANGER: LASER IS OPERATING sign (stored on the workbench by the door) on the outside of thedoortowarnanyindividualswhoneedtoenterthelab. TurnONtheorangemicroscopepowerswitchontheleftsideoftheopticalmicroscopebase(Figure1a). Turn ON the power toggle switch on the frontside of the PRIOR control box located to the left of the microscope(Figure1b).

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(b) Figure1.(a)Opticalmicroscopepower;and(b)motorcontrolboxpower.

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(b) Figure2.(a)LaserandPowerSupplyPositions;(b).Ramanspectrometerpowerswitch(marked). Check and remove any item placed in the path of the laser beam on the optical table (Figure 2a). Such itemwillcausedangerousreflectionsofthelaserbeamthatposesaseriouseyehazard. Toggle the power switch on the righthand side of the RAMAN spectrometer setup to ON (Figure 2b). Allow20minutesfortheCCDcameratoreachoperatingtemperature. Switch the 532 nm laser power supply toggle switch to the ON position (located on the optical table, Figure3a).Thepowerlightindicatoronthefrontofthepowersupplywilllightup. WAIT 30 SECONDS before turning on the 532nm laser key switch to the ON position to allow the power supply to stabilize. Turn the laser power supply key to the ONposition.The laser lightindicator will light up(Figure3b).Allow3minutesforthelasertostabilize.

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Figure3.532nmlaserpowersupply:(a)powerswitchintheback;(b)laserintheONposition. 2

10. OPENtheWIRE3.2softwareonthecomputerdesktop. 11. The software will give options as to which motors to reference (Figure 4). Select REFERENCE ALL MOTORSandthenchooseSTART.Thetoolwillinitializeallmotorsofthespectrometer.Ifthemotorsfail toreference,contactaMFCFstaffmemberforhelp.

Figure4:MotorReferenceOptionswindow. SYSTEM CHECK 1. 2. Turntheobjectiveturretsothatthescopeobjectivesareintherear(Figure5).Thisistopreventaccidental crashingoftheobjectivesintothesample. Lower the stage of the microscope using the Zposition adjustment knobs on the side of the microscope. Pull back the sample clip on the stage, and place the silicon reference standard (stored in the black box on thecomputercart)onthestage.Releasethestagecliptosecuretheglassslide.

Figure5.Loweredsamplestagewithobjectivesintherear. 3. Movethe5xobjectiveintopositionsothatitisabovethesiliconreferencesample.

NOTE: Whenever the sample objective is changed on the turret, the objective setting needs to be changed in theSampleReviewwindow.Thiswilladjustthescaleonthevideowindow(seeFigure6b). 4. 3 Onthefilterwheelsofthemicroscope,adjustthefilterwheelstothefollowingpositions(Figure6a):

 Upperfilterwheel=2 Lowerfilterwheel=1 Thisallowsforviewingofthesampleusingthewhitelightsourceofthemicroscopeandvideocamera(Figure6b).

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(b) Figure6.(a)Filterwheelsinpositions2and1;(b)Focusedsiliconreferencesampleinvideowindow.

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Focus on the surface of the sample using the stage height adjustment knob on the side of the microscope. It may be necessary to adjust the Fstop on the bottom left of the microscope in order to focus on the surfaceofclearorhighlyreflectivesamples. Once the sample is in focus, rotate the objective turret to the 20x position and focus the sample using the stage height adjustment knob. Then rotate the turret to the 50x objective and focus the sample using the stage height adjustment knob. WARNING: Adjust the stage height SLOWLY so as NOT to crash the objectiveontothesample,whichwilldamagetheobjective. In the software program, select Measurement > NEW Measurement > 532nm1800 setup. This setup is thesavedmeasurementsetupforthesiliconreferencesample. Onthefilterwheelsofthemicroscope,adjustthefilterwheelstothefollowingpositionstoactivateRaman collectionchannel(Figure7a): Upperfilterwheel=ARROW Lowerfilterwheel=4

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The laser spot will appear in the crosshairs (Figure 7b). Using the stage height adjustment knob, focus the laserspot.

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Figure7.(a)Filterwheelspositionsformeasurement;(b)CenteredlaserspotintheVideowindow. NOTE: If the laser spot is outside of the crosshairs, DO NOT ADJUST THE CROSSHAIR POSITION. Contact a MFCFstaffforhelpinadjustingthelaserposition. 9. Select Tools > System Health > System Health Check. The tool will check the alignment position of the laser, slits, and CCD camera. If the System Health Check PASSES, then continue. If the System Health CheckFAILS, then an alignment of the system needs tobe performed and aShared Facilities staff member needstobecontacted.Recordthisinformationinthelogbook.

10. Choose the RUN icon on the toolbar to run the measurement. The acquisition can be stopped at anytimebypressingtheABORTicon.TheacquisitionscanwillappearintheSpectrumViewerwindow. 11. Select Tools > Calibration > Quick Calibration and the tool will set the offset to the silicon reference peak to521l/mm(Figure8).

Figure8.Siliconreferencesamplespectralscan. 12. Afterthecalibration,lowerthestageheightusingtheadjustmentknobsonthesideofthemicroscopeand rotatetheobjectiveturretsothatanemptyobjectivepositionisabovethesample. 13. Release sample clip and remove the silicon reference sample and place it back into the storage box. The tooliscalibratedfortheprocessingofsamples. SPECTRAL ACQUISITION Thevarioustypesofmeasurementsavailableforusersareasfollows: SpectralAcquisitionstandardmethodforspectralacquisition ImageAcquisitioncollectionoffilterspectra. Mapping Acquisition collection of spectral acquisitions over an area of the sample. If the mapping techniqueisdesiredtobeused,contactaSharedFacilitiesstaffmember.

NOTE: The following SOP is for a Spectral Acquisition. For instruction on the Mapping and Image Acquisition techniques,pleasecontactaMFCFstaffmember. 1. Placesampleontoaglassmicroscopeslidelocatedonthesamplepreptable.

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Turn the objective turret so that scope objectives are in the rear. This is to prevent accidental crashing of theobjectivesintothesample. Lower the stage of the microscope using the stage height adjustment knobs on the side of the microscope. Pull back the sample clip on the stage, and place the glass slide on the stage. Release the stage clip to securetheglassslide. Movethe5xobjectiveintopositionsothatitisabovethesample.

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NOTE:Wheneverthesampleobjectiveischangedontheturret,thentheobjectivesettingneedstobechangedin theSampleReviewwindow.Thiswilladjustthescaleonthevideowindow. 5. Onthefilterwheelsofthemicroscope,adjustthefilterwheelstothefollowingpositions: Upperfilterwheel=2 Lowerfilterwheel=1

Thisallowsforviewingofthesampleusingthewhitelightsourceofthemicroscopeandvideocamera. 6. Using the stage height adjustment knob on the side of the microscope, focus on the surface of the sample. It may be necessary to adjust the Fstop on the bottom left of the microscope in order to focus on the surfaceofclearorhighlyreflectivesamples. Once the sample is in focus, rotate the objective turret to the 20x position and focus the sample using the stage height adjustment knob. Then rotate the turret to the 50x objective and focus the sample using the stageheightadjustmentknob.

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CAUTION: Adjust the stage height slowly so as to NOT crash the objective into the sample as damage to the objectivecanoccur. 8. Select Measurement > NEW > Spectral Acquisition and set the scan to the desired parameters in the SpectralAcquisitionSetupwindow:

NOTE:TheAPPLYbuttonmustbepressedtosaveanychangesmadetotheacquisitionparameters. 1).RANGETAB

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(b) Figure9.RangeTabwindowsfor(a)Staticscanand(b)Extendedscan. GratingScan:

i. Static Scan: The user sets the center point of the Spectrum Range and the system will set the scan range 500 cm1 on the center point. This scan can have an exposure time of less thanonesecondperaccumulation. ii. Extended Scan: The user sets the upper and lower limit of the Spectrum Range and the spectrometerwillscancontinuouslyovertherange.Thisscanislimitedtoexposuretimes of10secondsorgreater. Confocality:setsthesamplevolumeofthesamplecollected i. Standard:Usesalargervolumetoincreasesignalstrength. ii. High:Reducesthevolumeandsignalstrengthtoincreasedepthresolution. Configuration: i. Lasername:532nmnotch ii. GratingType:1800l/mm(vis) 2)ACQUISITIONTAB

 Figure10.AcquisitionTabwindow. Title and Description: Name the acquisition and give a description of the scan. This information willbesavedwiththeacquisition. Exposure Time: This is the amount of exposure time at the detector where longer exposure times willimprovethesignaltonoiseratio. Accumulations: This is the number of scan repetitions added together to improve signaltonoise ratios and for the removal of cosmic rays. If the cosmic ray removal box is selected, then two additional scans will be processed in addition to the number of accumulations selected Objective: This is the objective selected inthe SampleReview window and must bechanged every timetheuserchangestheobjectivemanuallyonthemicroscope. Laser Power: This is the percentage of laser power being used for the scan where higher powers translate into better signalnoise ratios. Start with lower laser powers on initial scans if thesampleissusceptibletobeingdamagedbythelaser. CosmicRayRemoval:Selectthisboxtohavethespectrometertaketwoadditionalscanstoremove randomcosmicpeaks CloseShutteronCompletion:Selectthisboxtoclosethelaserattheendoftheacquisitionandlimit laserexposureonthesample. 3)FILETAB Autosave: Select this box so that the acquisition will be saved to the folder with filename designated by the user. Select the AUTO INCREMENT box to save every scan acquisition withasequentialnumberedfilename.

 Figure11.FileTabwindow. 4)TIMINGTAB SampleBleaching:Thisisusedtodecreasefluorescencefromthesamplebyexposingthesample to the sample to the laser prior to taking a measurement. Select this box and set the timeifthisissueisoccurringonthesample. TimeSeriesMeasurements:Thisallowsfortheusertosetaspecifiedtimeinbetweenscans.

Figure12.TimingTabwindow. 5)TEMPERATURE ThisiscurrentlyNOTanoptiontousersasatemperaturestageisrequired.

6)FOCUSTRACKTAB This is used to maintain the laser focus position for time and mapping acquisitions. See Module #7 of user manual (located on the computer desktop) and/or contact a MFCF staff member for additionalinformation.

7)ADVANCEDTAB Scan Type: When an Extended Scan has been selected in the RANGE TAB, then the user has the option of using a Step Scan or a Synchroscan (or Continuous Scan). The recommended

option is the Synchroscan, but if saturation of the detector is occurring, then the Step optionshouldbeselectedsoexposuretimeunder10scanbeused. CameraGain:SelecttheHIGHsensitivityoption. Pinhole:Thesystemhasamotorizedpinholeassemblyandthisfunctionisusedtoforimproving theprofileofthelaserbeam.TheoptionINisrecommended. Laser Focus: This option controls the beam expander. A setting of 100% is a completely defocusedbeam,while0%isatightlyfocusedbeam.Ifthesamplebeingusedissubject to damage by the laser, then a highly defocused beam is recommended to reduce the powerdensityatthesample. ImageCapture:Thisfeaturecanbeusedtohavewhitelightimagesofthesampletakenbefore orafteranacquisition.

Figure13.AdvancedTabwindow. 9. Onthefilterwheelsofthemicroscope,adjustthefilterwheelstothefollowingpositions: Upperfilterwheel=ARROW Lowerfilterwheel=4

Thelaserspotwillappearinthecrosshairs(Figure14).Usingthestageheightadjustmentknob,focusthe laserspot.

Figure14.CenteredlaserspotinVideowindow. 10

NOTE:Ifthelaserspotisoutsideofthecrosshairs,DONOTADJUSTTHECROSSHAIRPOSITION.Thelaserposition needsadjustedandaSharedFacilitiesstaffmemberneedstobecontacted. 10. Turnofftheroomlight. 11. ChoosetheRUNicon onthetoolbartorunthemeasurement.Theacquisitioncanbestoppedatany timebypressingtheABORTicon. 12. If another acquisition is required and spectral acquisition settings need to be changed, select Measurement > Setup Measurement option. The scan parameters from the previous acquisition will still bestoredintheSpectralAcquisitionSetupwindow.Ifthescanparametersneedtobesavedforlateruse, selectMeasurement>SaveMeasurement. 13. To remove the sample from the microscope stage, lower the stage height using the adjustment knobs on thesideofthemicroscopeandrotatetheobjectivesturretsothatanemptyobjectivepositionisabovethe sample. 14. Releasethesampleclipandremovethemicroscopeslidefromthestage. 15. See Module #3 of user manual for data manipulation techniques under the Analyze tab of the Wire 3.2 software. 16. If another sample is to be processed, then proceed back to step #1 of this section. If all necessary acquisitions,havebeenacquired,thenproceedtotheSYSTEMSHUTDOWNsectionoftheSOP. NOTE: Users should make sure that they copy their data off the computer in a timely manner. The Shared ResearchFacilitiesisamultiuserfacilityandthereforecannotguaranteethatthesavedfilewillnotbemodifiedor deleted. DATA OPTIMIZATION 1.Toimprovesignaltonoiseratio a.Increaseexposuretime b.Increasenumberofaccumulations 2.Toeliminatestrongbackgroundduetofluorescence a.Decreaselaserpower b.Quenchfluorescencebyexposingsampletoincidentlaserlightforaperiodoftime c.Changeexcitationwavelength 3.Toavoidsaturatedsignal a.Reducelaserpower b.Reduceexposuretime 4.Toavoidlaserablationonthesample a.Usealowermagnificationobjectivelens b.Defocusthelaserspot

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c.Reducelaserpower DATA ANALYSIS NOTE: Below lists two basic data processing procedures. Please refer to M3 Data manipulation module (on computerdesktop)foradditionalinformationorcontactaMFCFstaffmember. 1. Baselinesubtraction 1) OpenthespectrumintheViewer. 2) Select Processing > Subtract Baseline. A new Viewer opens with the spectrum in the top half and the resultsofanybaselinesubtractioninthelower(Figure15). 3) The default baseline is fitted between the two end points of the spectrum. Use left mouse click to add morepointstothebaseline. 4) Use right mouse click to get the context menu. Select Properties > Cubic Spline Interpolation from the menu. 5) SelectAcceptfromthecontextmenu. 6) Youwillbeaskedtokeepthecorrection.NOTE:IfyouselectYes,youwilloverwritetheoriginalfile.You mayneedtokeepabackuporrenamethefile.

Figure15.Baselinesubtractionexample. 2. Smoothing 1) SelectProcessing>Smooth. 2) A new window will open with the sample spectrum at the top and the result spectrum below (Figure 16). 3) To modify the degree of smoothing, select Properties from the context menu (right mouse click) to see theSmoothPropertieswindow. 4) You can save the resulting smoothed spectrum by select Processing > Smooth > Accept > Current dataset.

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 Figure16.Curvesmoothingexample 3.Zap UseZapfunctiontoremovestraybandssuchascosmicraysfromthespectrum. 1) Openthespectrumofinterest. 2) Select Processing > Zap from the menu. A new Viewer will open with the sample spectrum at the top andtheresultspectrumbelow(Figure17). 3) Theupperspectrumhasazapregionbetweentwoverticalblacklines.Grabeachlineinturnandadjust the position of the zap region to just enclose the band to be removed. You may need to use the zoom functiontoisolatetheband.

Figure17.Zapfunctionexample. 13

4.Curvefitting 1) SelectAnalysis>CurveFittoopenthecurvefitwindow. 2) Zoomintoaregionthatcontainsthebandandsomebaselinedataateitherside. 3) Usethemousetopositiontheapproximatecentreoftheband. 4) Clicktoaddthebandandrepeatforthecentersofotherbandsiftherearemultiplebands. 5) You can adjust the center or width markers for each band using the mouse and cursor to get a better roughfit. 6) UsemouserightclicktogetthemenuandselectStartFittofitthepeak(Figure18). 7) Youcansavethecurvefitfilefromthecontextmenu.

Figure18.Curvefittingexample. 5. Peakpick 1) SelectAnalysis>Peakpicktoopenthecurvefitwindow(Figure19). 2) YoumayadjustthethresholdsbyselectingAnalysis>Autosetthresholds.

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 Figure19.Peakpickexample. SYSTEM SHUT DOWN 1. 2. 3. 4. 5. 6. 7. 8.

TurnthelaserpowersupplykeytotheOFFposition.Thelaserlightindicatorwillturnoff. SwitchthelaserpowersupplytoggleswitchtotheOFFposition.Thepowerlightindicatoronthefrontof thepowersupplywillturnoff. TogglethepowerswitchontherightsideoftheRAMANspectrometersetuptoOFF. TurnOFFthepowerswitchonthefrontsideofthePRIORcontrolbox. TurnOFFthepowerswitchontheleftsideofthewhitelightsourceforthemicroscope. CLOSEtheWIRE3.2softwareonthecomputerdesktop. Checkthatallrequiredparametersarerecordedinthelogbook.Signoutonthelogbook. Cleanupworkareabydoingthefollowing: a. b. c. Safetyglassesarereturnedtothepropercaseontopofthecomputercart. The silicon reference sample is placed back in the storage case located on top of the computer cart. RemovetheWARNING:LASERINUSEsignfromthefrontdoorofthelab.

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Properlydisposeofanyusedmaterialsi.e.alphawipes,glassslides,etc.

10. SignoutoftheFOM.

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EMERGENCY PROCEDURES Ifnooneisavailableandthemachineisnotactingasexpected,theusershoulddothefollowing: PressABORTontheWIRE3.2software. TurnthekeytothelaserpowersupplytotheOFFposition. TurnoffthexraysbypressingtheredOFFbuttononthepowercontroller. Do not leave the machine running in an abnormal state. If the machine cannot be placed in the default state, immediatelycontact: PrimaryStaffContact: SecondaryStaffContact: WeiqiangDing(304)6851938weding@mail.wvu.edu HarleyHart (412)4431514 harley.hart@mail.wvu.edu

Then, if possible, the user should stay by the tool while trying to contact a Shared Facilities staff member. If it becomesnecessarytoleavethetoolthentheusershouldleavealarge,legiblenoteattheRAMANstatingthetool isDOWN. The user should then complete a Tool Issue Report form and submit this form to ESB G75D. The user should also addcommentusingtheFOMsoftwareindicatingthetoolstatus. If a dangerous situation is evident (smoke, fire, sparks, etc), ONLY if it is safe to do so, the user should turn off power to the system by switching the toggle switch on the right side of the tool and the laser power supply key and toggle switch to the OFF position. The user should then notify all other persons within the Hodges Hall 325 labtoevacuateandleavethelabimmediately.Theusershouldthencontactproperemergencypersonnelfroma safeplace.

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