Tecnai F20 - Alignments; Open Column Valves; Eucentric Focus; Load Alignment File

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CSBCryoEMFacilityTF20OperatingInstructionsSEC
05/02/16
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III. Alignments
• Thepurposeofthealignmentofthemicroscopeistoensurethatthebeamisparalleltothecolumn
andthattheaperturesareproperlycentered.
• Alignmentsshouldbedonebeforeeveryimagingsession.
• Onlyloadthemostrecentalignmentsfile,madebytheFEIengineer.Donotsaveyourown
alignmentfile.Itwillbedeleted.TherecentalignmentmadebyFEIwillonlyneedtobetouchedup
andit'sagoodplacetostart.
1. DONOTDOALIGNMENTSINLOWDOSE
2. Makesurethat“LowDose”isturnedoff(theboxwillbegray)
3. RemovetheObjectiveAperture:MovethesmallpinontheObjectiveaperturetotherightortowhere
thepointispointingtothespecimenholder.Thisallowsyoutodoanalignmentwithouttheobjective
interfering
4. Addaspacertothesampleholderinthecompustage.*thismustbedonewiththecolumnvalves
closed!ThespaceristypicallyaBICpencap.Thisspacerallowsforthesampletoberemovedfrom
thebeampathwithoutbreakingthesealofthesamplechamber.Thespacermakesthealignment
processeasier.
5. Waitforthecolumnvacuumtorecoverafterinsertingasample.Thistakes~10minutes.Youwant
IPG1=6andIGP4=1.Thesevaluescanbefoundin“VacuumOverview.”Thisisdisplayedonthe
rightsideoftheleftcomputermonitor(Ifitisnottherethenitcanbeselectedinthebottomright
corneroftheleftcomputermonitor).
6. Opencolumnvalves.
Checkforthebeam;Ifthereisnobeamtryoneofthefollowing:
1) Movethestage(onlyifasampleisinthescope/beampath)
2) Lowerthemagnification
3) Ifyoucan’tfindthebeamcall/findme.Theremaybeabiggerproblem
7. EucentricFocus:ClickonEucentricFocusbuttonlocatedontherightpanel,thisshouldtakeyou
withinafewmicronsoftruefocus.
8. LoadAlignmentfile:GototheAlignmentstab,clickontheflapoutwindowandunderFile,findthe
mostrecentalignmentsfilefortheappropriatevoltage(120or200kV).Clickonthefile,onthe
Availableboxunderthealignmentsfile,doubleclickontheallthealignments,thenclickapply.*The
beammaygoawaybutwillcomebackwhenyouloadtheFEGregistry.
9. LoadFEGregistry:GototheSearchtab.UnderRegistries,findthemostrecentfile.Clickonthefile,
thenclickSet.Thiswillchangeyourspotsizeto3.Changethespotsizebackto5.
10. VerifySettings:Verifythatyouyoursettingsarecorrect(magnification,spotsize,beamposition).
*TypicallyIuseMag=135,000x,Spotsize=5,andcenterthebeamusingthetrackball.**The
intensityofthebeamwillneedtobeadjustedwhenchangingthemagnification
11. Makethebeamround.Checkthisathighmagnification(~135kx).Condensethebeamusingthe
intensityknobtothesizeofadimeorslightlysmaller.Athighmagnification(~135kx)thebeammay
looktriangular.GotoAlignmenttabandclickonCondenser.Rightclickonthecurrentsettingand
copythesettingtoanotherlocation(ie.copy3to1).UsetheMultifunctionXandYknobsonthe
controlpaneltomakethebeamround.Onlyadjustonemultifunctionknobatatime.Click“none”
whenfinished.
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