Tecnai F20 - Select C2 Aperture; Center the Condenser Aperture (C2); Gun Tilt; Gun Shift

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CSBCryoEMFacilityTF20OperatingInstructionsSEC
05/02/16
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12. SelectC2aperture:DeterminewhichC2aperturetouse(asof3/28/16). 
Position1=50
Position2=70
Position3=100*mostcommonlyusedfornegativestain
Position4=150
13. CentertheCondenseraperture(C2):Usingtheintensityknob,condensethebeamandgothrough
crossoverandmonitorthatthebeamspreadsevenlyaboutthecenter.Ifadjustmentsareneeded,
centerthecondenseraperture(C2manually)byveryslightlyturningthecenterandleftsideknobsof
theC2Apertureuntilthebeamiscenteredonbothsidesofcrossover.
14. Checkthatthebeamisstillround.Ifnotfollowtheprocedureoutlinedinstep#11
15. GunTilt:Lowerthemagnificationto~10,000x.Toadjusttheguntilt(minimizeexposuretime)goto
theTunetabandselect“GunTilt”underDirectAlignments.Bringthelargescreendown,movethe
smallscreenintothebeampath(physicaltabtotheleftofthemicroscope).Usingthemultifunction
knobs(settoguntilt)adjustthebrightness.Keepaneyeontheexposuretime(“Meas.Exp.”onthe
monitor)tomakeitaslowaspossible.Clickdonewhenfinishedwithalignment.Movethesmall
screenbacktoitsoriginalposition(outofthebeampath).
16. GunShift:PerformthegunshiftinAlignments.ClickonGunShiftandfollowthedirections.The
magnificationandspotsizewillbesetautomatically.Theideabehindthisalignmentistocondense
thebeamandusingthemultifunctionXandYknobstocenterthebeam.Followtheinstructioninthe
lefthandpanelintheFEIuserinterface.Inordertogetamoreaccuratealignmentbesuretoclick
“NormalizeAll”(typicallysettoL2ontheleftcontrolpad)beforeandaftereachstep.Repeatthe
entireprocedure23timesuntilthebeamremainsinthecenter.Click“done”whenfinishedwith
alignment.
*Thegunshiftneedstobedonewithasampleinthemicroscope(orasampleinthescopewitha
spacer/pencapinplace).IFnotthenthethemicroscopewillbeepandyouwillgetanerrormessage
whichsays“xraysafety:Spotsizeclippedto5.”
17. BeamTiltXandY:Adjustbeamtiltpivotpoints.Changethemagnificationto10,000x.Condensethe
beamtoadot.ClickonBeamTiltppXunderDirectAlignments.Adjustthebeamsothatthebrightest
spotisaroundthecenter,nottheedges(withmultifunctionknobs).Click“done”whenfinished.Do
thesameforppYandclick“done”whenfinished.Changethemagnificationto135,000xandredo
bothpivotpointsXandY.
18. BeamShift:AdjustbeamcenterbyclickingonBeamShiftunderDirectAlignments,thencenterwith
multifunctionknobs.Click“done”whenfinished.
19. Removespacer:Closethecolumnvalvesandthenremovethespacer/pencapfromthecompustage
20. Wobbler:GototheSearchtab,flapoutmenuandclickonWobbler.Thisistosetuptheproper
Zheight(eucentricheight).Thegoalistominimizethemovement(wobbling)andonlyhavethe
samplemoveevenlyabouttheZaxis.AdjustZbyclickingonthe+/buttonsontherightcontrolpad
untilthemovementisminimized(itissuggestedthatyoufindafeatureonthegridoragridbarto
keeptrackofthemovement).Oncedone,clickonWobbler(thebuttonwillgofromyellowtogrey,it
takesamomenttocometoafullstop).
21. Rotationcenter:Withthescreendownandthesmallscreeninserted,clickonRotationCenterunder
DirectAlignmentsandminimizethebeammovement,makingitwobbleevenlyaboutthecenter.Set
themultifunctionknobstothelowestmovementpossibleandcarefullyminimizethewobbling.
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