QueenslandBrainInsJtuteMicroscopy

ZeissAxioImager/ObserverGuide
Ge#ngStarted
1. SwitchonthewhitePowersupplybox.
IfyouareusinguorescenceswitchontheHXP120andColibricontrolbox.
IfyouareusingApoTomeswitchontheApoTomebox.
2. Switchonthemicroscope(atbackleBofmicroscopestand).
3. SwitchonthecomputerandlogonwithyourUQusernameandpassword.
4. OncethemicroscopehasnishedstarJngupdoubleclicktheAxioVisionicon.
Ifapopupwindowappearsclickdonotshowagain,thenclickOK(donotrecongurethe
microscope)

Shu#ngDown
1.
2.
3.
4.

Lowerthestageandremoveyoursample.
GentlywipeanyoilobjecJvesyouhaveusedwithlens7ssue(donotusekimwipestocleanobjec;ves).
ExitthesoBwareandcopyyourlestoyourhomeorgroupnetwork/USBdrive.
TurnothemicroscopepowersupplyboxandtheHXP120/Colibri/Apotomemodules.

VisualisingaSampleThroughtheOculars
1. OnthetouchscreenaZachedtothemicroscopepressloadposi;onandposiJontheslideonstage.
Pressingthe buZonwillreturnyoutotheworkingposiJon.
2. Pressmicroscope(1)onthefarleBofthetouchscreen.
3. Youwillbeabletochangeobjec,ves,reectors(ltersets)andadjustthelightpathviathetabsatthe
top.(2)
4. YoucanuseoneofthequickbuZonsattheboZomofthescreentogetthemicroscopeready.(3)
PressFLforuorescence,BFforbrigh`ield,PHforphaseandDICforDIC/NormaskiImaging
modes.
Ifimaginguorescenceensureyouhavetheappropriateuorescentlightswitchedon
5. Checkthelightpathisallowingthesampletobeseenviatheoculars(underthelightpathtab).
AxioImager:100%tube(100%sideportisforthecolourcameraonly)andensurethepushpull
rodisallthewayin.
AxioObserver:50or100%eyepiece/vis
6. AnimagewillnowbevisibledowntheocularsadjuststageposiJonandfocusasnecessary.

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QueenslandBrainInsJtuteMicroscopy

ImagingaSampleUsingtheLiveWindow
1. Selectanimagingmodeonthetouchscreen.
FLattheboZomleBforuorescence.
BF/PH/DIC/DFfortransmiZedlightimaging.
2. Selecttheappropriatecamera(Acquisi;on>Selectcamera).
TheMRm/MR3cameraisamonochromecamerasuitedtouorescentimaging.
TheHRccameraisacolourcamerasuitedtobrigh`ieldimaging(andneedstobeswitchedonat
thewallbeforestarJngAxioVision).
3. Adjustthelightpathsolightreachesthecamera.
AxioImager:PullthepushpullrodonthesideofthemicroscopeouttothecameraposiJon.
AxioObserver:Inthelightpathmenuonthetouchscreenadjustthese#ngsto100%camera.
4. EnsuretheshuZersareopenandilluminaJngthespecimen.
FluorescenceboththereectedlightshuZer(RLshuServiathetouchscreen)andtheHXPlamp
shuZer(viatheColibricontrolpanelpresstheExt.buSon(externallightsource)thenpressthe
shuSerbuSon).
BrighUieldthetransmiZedlightshuZer(TLshuSer)(viathetouchscreen).
5. ClickontheLiveiconinthetopmenu.(1)
6. ClickExposure(belowlivewindow)toautomaJcallyadjusttheexposure.
7. Adjustexposureifnecessary.
Todothisselect"Cameras"intheworkareaontheleBofthescreenandchoosethecamerayou
areusing.Exposurese#ngsareundertheadjusttab.
8. ClickSnaptocreateanimage.Note:theimagesarecapturedbutnotsavedatthispoint.
ToautomaJcallytcapturedimagestothewindowsizegotoTools>Op;ons>DisplayandJckthe
Fitimagetowindowbox
9. ToaddascalebarclickonthescalebarbuZon(2)anddragthecursorovertheimagewhereyouwould
likethescalebartoappear.

MulJdimensionalAcquisiJon
MulJdimensionalacquisiJonisaccessedthroughtheMulJdimensionalAcquisiJonwindowintheworkarea.(3)
HereitispossibletocreateautomatedexperimentalprotocolsforsingleandmulJplechannelimaging,Zseries,
JmelapseandmosaicimagingoranycombinaJonofthese.
Themul;dimensionalacquisi;ontabs:

Experiment:LoadpreexisJngexperimentalprotocolsorsavenewlycreatedones.
C/colourwheel(Channel):CreatesingleormulJplechannelprotocolsforimaging(page25).
Z(Zseries):ImagemulJpleplanesthroughJssue/cells(page6).
TimeLapse:ControlsJmelapsese#ngsforliveimagingoflivingcells/Jssues.
*MosaiX:ControlsJledimagese#ngsforimagingacrossnumerouseldsofview(page910)
*MarkandFind:Controlsse#ngsforautomatedimagingaselectedlocaJonsonyourslide/dish.

(*onlyon
Green,Blue
andIndigo)

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QueenslandBrainInsJtuteMicroscopy

CreaJngaSingleChannelExperiment
1. ClickonthecolourwheeltabofthemulJdimensionalacquisiJonmenu.(1)
2. Selectthemarker/dyeyouareusingfromtherstdropdownmenu.(2)
AxioVisionwillautomaJcallyassignthisdyeacolour(youcanchangethiscolourinthesecond
dropdownlistifyouwantto).
3. UnderHardwareSe^ngs(3):
DuringAcquisi;on:selectthecorrectexcitaJonsource(HXP/Colibri)andltersetforthemarker/
dyeyouareusingseelastpageforguide.
4. UnderExposureSe^ngs:
ClickonMeasure(4)theexposurewillbeadjustedautomaJcallyandyoursampleshould
becomevisible.
AdjusttheexposurefurtherifnecessaryinthetopleBhandcornerofthewindow.
EnsuretheimagewillbecapturedwiththelinearhistogramopJon(graphiconwithadiagonal
redlinerunningthroughitattheboZomofthewindow).
WhenyouclickOKtoclosethiswindowyourexposureJmewillbesaved.
5. ClickStarttotakeanimage.(5)

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CreaJngaMulJChannelExperiment

1. Tocreateanextrachannelclickduplicate.(6)Adjusteachse#ngasdescribedaboveforyour2ndmarker.
2. Repeatthisprocessforasmanymarkersasyouhave(e.g.DAPI,Alexa488,Alexa555,Alexa647).
3. Oncenishedyoucansaveallyouchannelsundertheexperimenttab(7)clicksaveasdefaultinthe
dropdownlist.(8)
4. WhencapturingmulJchannelimages,ifyouonlyneedsomeofthechannels,youcandisablethe
channelsyoudontwantbyrightclickingthecolourtabatthetop.(9)

QueenslandBrainInsJtuteMicroscopy

Hardwarese#ngsforAxioImagers/Observers

EachmicroscopehastheopJonofusingColibriLEDsortheexternalHXPXenonlampforuorescence.
Colibri,whenusedwithlterset62,givescomparableandinsomecasesbeZerresultsthanusingtheHXPlamp,
doesntchangeintensityastheLEDsage(unliketheHXP)andturnsoninstantly(nowarmuporcooldownJme).
YouwillneedtousetheHXPifyouareusingAlexa546/555/568orCy3
Arecommendedsetupifyouwanttoobservemul;pleproteinsis:

DAPI+Alexa488/GFP+Alexa555+Alexa647

(LookoutforcrosstalkbetweenGreenandRedchannels)
Another4colourcombina;onis:

ExperimentSe^ngs:

DAPI+Alexa488/GFP+Alexa594+Alexa647
(LookoutforcrosstalkbetweenRedandFarRedchannels)
Intheexperimenttabchoose:
Beforeexperiment...
Colibri100%
ThiswillmakesureallthecolibriLEDsarebright
forbestresults.

AIerexperiment...
ColibriLEDso(Colibrionly)
orCloseRLShuSer(HXPorHXPandColibri)
StopslightfrombleachinguorescenceaBer
imageshavebeenacquired.
IfyouhaveverysensiJvemarkersitcanhelpto
havethesese#ngsselectedineachchanneltab,
especiallyifyouareimagingZstacks.

ColibriSe^ngs:
DAPI,Alexa350

GFP,Alexa488,FITC

mCherry,Alexa594

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QueenslandBrainInsJtuteMicroscopy

Cy5,Alexa633/647

Hardwarese#ngsforAxioImagers/Observers
HXPSe^ngs:
DAPI,Alexa350

GFP,Alexa488,FITC

Cy3,Alexa546/555/568

DICandBrighUieldSe^ngs:

Youcancombinebrigh`ield/DICwithuorescentimaging.
KeeptheAIeracquisi7oneldempty,especiallyifyouaredoingMosaiXasclosingtheTLshuZeraBereach
acquisiJonwillslowdownimagingdramaJcally.
TheDuringacquisi7onse#ng(DICorBrighUield_Phase_DF)willensurethetransmiZedlightcomesonandthe
correctlterisinplaceBUTyouwillneedtochecklightintensity,condenserfocusandcondenserlterbefore
starJngtoimageseeGuidetoBrigh?ieldImagingformoreinformaJon.

QueenslandBrainInsJtuteMicroscopy

CreaJngaZSeriesExperiment
1. UnderthecolourwheeltabclickonthemeasurebuZon(bringingupthelivewindow)andmanually
focustothebrightestplanesettheexposureJmeforthisplanetopreventoverexposureintherestof
theZseries.
Repeatthisstepforallchannelsintheexperiment.
2. ClicktheZtabtoopentheZseriessetup(ensurethattheZstackboxisJcked).(1)
3. UnderModeselecttheStartStopopJon.(2)
4. ClickontherighthandbuZon()oppositeStartandStop(3)toopentheliveimagingwindow.
Usingthenefocusknob,focusthroughtothetop(orboZom)andclickOKforeach.
5. ForgoodZresoluJonclicktheOp;malDistancebuZon(4).However,inmanycasesyoucanusealarger
stepsize,especiallyifyouarenotusingtheZstacktocreate3Dimages.
6. ClickStarttobeginaZseriesexperimentwiththechannelsyouhaveacJvatedundertheChanneltab.
7. OncecapturedyoucanturnaZseriesintosingleaZenedimagecalledaMaximumIntensityProjec;on
TodothisclickonCutview(foundonthemenubelowthecapturedimage).ThenJcktheMIP
boxontherighthandside

QueenslandBrainInsJtuteMicroscopy

OpJcalSecJoningwithApoTome

ApoTomeusesamovinggridtochangehowtheslideisilluminated.Bytakingseveralimages,withthegridin
dierentposiJons,theApoTomesoBwareisabletogenerateasingleimagewhichexcludesalargeamountofthe
outoffocuslighttypicallypresentinuorescentimages.
TheresulJngimageslookcrisperthanthesmootherstandarduorescentimage,whichallowsnerstructuresand
detailstobeseenclearlybuttheyarealsodimmer(asthereislesslightreachingthecamera).

CalibraJngApoTome

CalibraJonmustbecarriedoutseparatelyforeachobjecJveandlterset.
1.InsertthecorrectgridintotheApoTomeslider
5x,10x,and20xobjecJve=Lgrid
40x,63xand100x=Hgrid
1. UnlockandremovetheApoTomesliderfromthesideofthemicroscope.
2. Usethetweezerstogentlyremoveandinserttheappropriategrid.
WheninserJng,matchupthewhitedotonthegridwiththeoneontheslider.
ThegridisheldinmagneJcallysoifitsnotsi#nglevelmoveitaroundslightlywiththetweezers
unJlitclicksintoplace.
OnceinplacecheckitissecurebyliBingthesliderandgentlytappingitoverthepalmofyour
hand.
3. ReturntheApoTomesliderbackintothemicroscope
2.PhaseCalibra;on
ThiscalibraJonrequiresthemirroredcalibra;onslide.
1. FocusonthecrosshairatthecentreofthesilversquareonmirroredcalibraJonslide.
EnsureyouareusingtheBFRL(brighUieldreectedlight)ltersetandtheHXPlamp.
Thelightshiningontheslideshouldappearyellowgreenanddowntheeyepiecesthecrosshair
shouldappearblack.
2. OnceyouhavefoundthecrosshairyoucanbeginthecalibraJonprocessinthedropdownmenugoto
Acquisi;on>ApoTome>PhaseCalibra;onthenfollowtheinstrucJons.
3.GridFocusCalibra;on
1. Removethemirroredcalibra;onslideandfocusonyourownsampleslide.
2. UnderthedropdownmenugotoAcquisi;on>ApoTome>GridFocusCalibra;onandfollowthe
instrucJonsprovidedtocalibrateApoTomeforeachuorescentmarkeryouareusing.
ItisnecessarytocreateanewcalibraJonforeachmarkerbutalsoforeachlightsource.

ImportantSe#ngsforApoTome

ClickonApoTomeintheworkareatotheleB.Underthese^ngstabselect:
1. ThecorrectgridfortheobjecJvebeingused.
2. TheGridVisibleopJonunderLiveModethiswillensurethefastestrefreshrateforlivemode
3. TheOp;calSec;oningopJonunderAcquisiJonModeifnotcheckedopJcalsecJoningwillnotoccur
4. AweakApoTomelterisrecommendedifgridlinesaresJllvisibleinthenalimage.
5. 2xAveraging(noisereducJon)isrecommendedasaniniJalse#ng.Youmayneedtoincreasethiswith
parJcularlydimsamples.
Undertheextrastabensurethedisplaynormaliza;onboxisJcked.

OnceApoTomehasbeencalibratedyoucanimageasnormal(e.g.usingLive/Snapor
Mul;dimensionalAcquisi;onexperiments).ApoTomeop;calsec;oningwilloccurwheneverthe
ApoTomesliderispushedallthewayintothemicroscope.

QueenslandBrainInsJtuteMicroscopy

MosaiX/TiledImaging(onAzure,Green,BlueandIndigo)

Beforestar;ngcheck:
1. TheorientaJonoftheliveimageisidenJcaltothatseenviatheeyepieces
Ifnecessaryrotatetheliveimagegotocameraintheworkareaandunderthegeneraltabtry
dierentcameraorientaJons
2. ScalingisONTicktheboxunderMeasure>AutoScaling
3. IfusingtheMRmcameraopentheMTB2004programonthedesktopandensurethattheInvertYAxis
boxisJckedunderMotorisedStage(InternalController)
Youwillonlyneedtodothisoncethese#ngwillberememberedonsubsequentlogins
4. IfusingMosaiXforbrigh`ieldimagingyoumayneedtoputShadingCorrec;onon(undercamera
se#ngs).EnsuretheboxisJckedbelowtheshadingcorrec;onbuSon
TosetupshadingcorrecJonsgotoablankareaoftheslideandclicktheshadingcorrec;on
buSon.

Se#ngupMosaiXCenterMode

1
ThisopJoncreatesamosaicimagearoundwhateveryouhavecenteredinthe
eldofview/livewindow.
1. OpentheMosaiXtabinMulJdimensionalAcquisiJonandJckonMosaiX.
2. Clickcenter(1).
3. Inregion,choosethenumberofcolumnsandrowsyouwantyourMosaic
imagetohave.(2)
4. Settheoverlapvalueat10%(toosmallanoverlapwillcreateproblemswith
sJtching).
5. ClickStarttocapturethemosaicimage.

MosaiX:SJtchingandConverJngTileImages

ABeracquisiJon,togetthenalimage,youneedtosJtchtheJlestogetherandconverttoasingleimage:
1. SelectS;tchingunderMosaiXintheworkarea.
2. LeavetheotherparametersastheyareandclickStarttheJleswillbereposiJonedtogivethebest
alignment.Ifthe;lesdonts;tchproperly:
UndersJtchingmodechoose:originalposi7onsandclickstartagain
Tryasearchdepthof12,andaminimumoverlapof2
CheckyouareusingthebestchannelforsJtching
3. IfyouaresJtchingmosaicimageswhicharelargerthan3x3Axiovisionwillshrinkthemwhenyourun
convert;leimagestostopthis:inthetopmenuclickonToolsthenselectOp;ons.Intheacquisi;on
tabmakethemaximumimagemuchlargerthan4096(i.e.10000000)
4. SelectConvertTileImageunderMosaiXintheworkarea.
5. Checkthatthezoomissetto1.
6. TurnontheAdjustOverlappingAreaparameter.
7. Ifyouwishtocroptheimagedrawaregionofinterestovertheregionyouwishtokeep.
8. ClickOKtocreatethesinglesJtchedimage.
QueenslandBrainInsJtuteMicroscopy

Se#ngupMosaiXRectangleMode

ThisopJonallowsyoutoshapeamosaicimagearoundyourregionofinterest,oruseanoverviewimagetotrace
regionsyouwantimaged.
1. OpentheMosaiXtabinMulJdimensionalAcquisiJonandJckonMosaiX.
2. ClickRectangle.TheSetup...buZonbelowwillbecomeacJveclickSetup...
3. Underthestagetabmovethedesiredobjectintothecentreoftheeldofview(1)thenpressthecentre
buSon(2)thismarkstheposiJonofthecentreofthemosaic.
4. Youcanmovetotheedgesoftheobjectyouwishtoimage,andexpandthemosaictotbyusingthe
expandbuZon.(3)
5. Ifyouneedtotrimthemosaic,movetheviewtowhereyouwanttheedgeofthemosaictobeandpress
theappropriatetrimbuZon.(4)
6. IfyouwantanoverviewoftheareayouareimagingclicktheoverviewimagebuSon.(5)
HereyoucanchoosealowerpoweredobjecJvethenclickAcquiretocaptureanoverview.
Youcantracetheregionyouwanttoimageonthisoverviewthenswitchbacktoahigher
poweredobjecJvetoacquireyournalimage.
7. Whenyouhavenishedse#ngupyourmosaicclickOKtoleavetheMosaiXsetup.

QueenslandBrainInsJtuteMicroscopy

SavingImages

TosaveanImageyoucanclickthesavebuZoninthetopmenu.
Thiswillsavetheimageasa*.ZVIle,whichcanbeonlyopenedbyAxiovisionandFIJI(free
downloadablesoBware)
ItisbesttokeeptheZVIleasyouroriginaldatabutifyouneedtheimageforapowerpoint
presentaJonorwanttoopenitinphotoshopyoucanexportyourZVIlesasTIFlesseebelow.
Youcansaveyourimagestothedesktopbutattheendofeachsession,either:
Moveyourlestoyourgroupshareorpersonalnetworkshare.
MoveyourlestodriveD:forstoragethere.Or,
MoveyourlestoaUSBsJck/portableharddrive
YoucansetupautomaJcprexesandsuxesforyournamingyourimagesunderTools>Op;ons>Naming
Chosethecategoryyouwishtochangethese#ngsforasthereareseparatespecicaJonsfor
eachcategory(forexamplesingleacquisiJonversusmulJdimensionalacquisiJon).
YoucanalsosetthesoBwaretoautomaJcallysaveeveryimageyoucaptureinTools>Op;ons>Storage

ExporJng*.ZVIlesasTIFs

IfyousavelesdirectlyasTIFsyoumayloosedataandhavenowayofge#ngitback,alsoanychangesyousave
toTIFsarepermanent.ItisbesttosaveaZVIlethenconverttoTIFformat.
TomakethingseasieryoucansaveZVIlesasyougoandthenconvertalloftheselestoTIFsinabatchatthe
endofyoursession.
Todothis:
1. InthetopmenuclickFileandthenselectExport
2. IntheexportwindowthatopenschoosewhereyouwouldlikeyourTIFlestosave(1)(e.g.aTIFexport
folderonyourdesktop).
3. UnchecktheCreateprojectfolder(2)(unlessyouareexporJngZStacks/mosaiXimages).
4. ClickthebatchbuZon(3),thenchoosetheZVIlesyouwanttoconvertbyclickingAddles...(4)(you
canselectmulJplelesusingtheshiBkey,orwholefolders).
5. Tickonall4topopJons(5)
Ifyouareusingmorethan3colours(i.e.notRGB)uncheckmergedimagesonly.
6. ChooseTIFintheletypeselecJon(JPEGformatcompressesyouimagesandreducesimagequality)
7. Tickon:(6)
Applydisplaymappingskeepsany
1
adjustmentsyouhavemadetothe
histogram/brightness/contrast
2
3
Burninannota;onskeepsscale
barsvisibleinimage
8. ClickRunBatch(7)(notStart)

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QBIFluorescentMarkerGuide

QueenslandBrainInsJtuteMicroscopy

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