Tecnai F20 - Select Objective Aperture​; Diffraction; Check C2 Stigmation; Turn on Low Dose

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CSBCryoEMFacilityTF20OperatingInstructionsSEC
05/02/16
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22. SelectObjectiveaperture:Determinewhichobjectiveaperturetouse(asof3/28/16). 
Position1=10
Position2=20
Position3=40*mostcommonlyusedforNegativestain
Position4=100
23. Diffraction:Iftheobjectiveapertureisnotinserted,insertittotheappropriatesetting(ifthesmallpin
ispointingtotheright/towardthespecimenholderthentheobjectiveisout,ifthesmallpinpointsto
thelefttheobjectiveisin).ClickontheDiffractionbuttonontherightcontrolpanelandcondensethe
beamtoasmallpoint.Verifythattheapertureiscenteredappropriatelybyobservingthehaloaround
thesmallpoint(itshouldbeevenandcontinuous).Adjustasneeded(usuallyveryminimalchange)
andexitdiffractionmodebyclickingthediffractionbutton.
24. CheckC2stigmation:Condensethebeam,gothroughcrossoverandverifythatthebeamisround
andeven.CorrectifneededbygoingtoTuneandselectCondenser.(Toensurethatyoucanget
backtowhereyoustartedrightclickonthehighlightedsettingsandcopythemtoanotherpanelie.
“copy3to1”)Usethemultifunctionknobstoadjustthebeamtomakeitround.Clickdonewhen
finished
25. Checkobjectivestigmation(donewiththeCCD):Findfocus(canbedoneusingtheLiveViewon
DigitalMicrographorbyinsertingthesmallscreenandadjustingthefocusuntilallcontrasthas
disappeared).Oncefocushasbeenfoundselectresetdefocus(typicallyR2ontherightcontrol
board),startliveviewandgobetween0.250.5nmunderfocus(negativevalue).Observetheshapeof
theFFToftheimage.Ifitisn'tround,gotoStigmator,selectObjectiveandadjustwiththe
multifunctionknobsuntiltheFFToftheimageisanevencircle.Selectnonewhenfinished
26. TurnonLowDose(IfworkinginLowDosemode)byclickingontheLDbutton,goesfromgreyto
yellow).Thisiswhereyouwillwanttochange/determinewhichmagnificationtousewhileimaging.
Mostnegativestainprojectswilluseafocusmagnificationof100kxwithanexposuremagnification
of62kx80kx(*Thesevaluesaregoodstartingpoints).Withthelargescreendown,verifythatthe
beamiscenteredinallimagingmodes(Search,Focus,Exposure).*Centerthebeaminexposure
first!!!Verifythatthebeamiscenteredandspreadtothedesiredlevelinallmodesandcheckthatthe
spotsizeiscorrect.Oncethebeamsettingsarecorrect,clickthroughthe3modesafewtimeswhile
centeringthebeamtominimizehysteresis(beammovement),whenchangingfromonemodetothe
next.*Ifthebeamisn’tstayingcenteredaskforhelp!
27. Checktheliquidnitrogeninthedewar(fillevery23hours).
28. Whenleavingthemicroscopeforanyreasondothefollowing:
a Closecolumnvalves
b Stopthecamera
c Retractthecamera
d Putthescreendown
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