confocal 2 – zeiss 880 with fast airyscan · zen black available objective lenses: 10x air, 20x...

ACRFCancerBiologyImagingCentre TheInstituteforMolecularBioscience

Confocal2(ZeissLSM880withFastAiryscan) Page1

Confocal2–Zeiss880withFastAiryscan

Quick-startGuide

Location:Room6.029

(Updated:26/7/2019)

AcquisitionSoftware:

ZENBlack

AvailableObjectiveLenses:10xAir,20xAir,40xOiland63xOil(40xWaterisalsoavailable)AvailableLaserLines:405,458,488,514,561and633nm

BemindfulofwhatyouaredoingatALLtimes:Themicroscopeandthelensesaredelicateandveryexpensiveandshouldbetreatedwithcare.

**The40Waterand63xOilobjectiveshavebeenspecificallyselectedforhighqualityforusewiththeAiryscansuper-resolutionimagingmethodandassuchareVERYexpensive–pleasetreatthesewiththeutmostcare!

- Airyscansuper-reshasbeenoptimisedforthe40x,63xand100xlenses- FastAiryscanhasbeenoptimisedforthe20x,40xand63xlenses

SlideCleaningandPreparation:

- Makesureyourslideisscrupulouslyclean–alldust,oldoilandmountingmediashouldberemovedwithasmallamountof70%ethanol.

- IfyoucanuseahardsettingmountingmediapleasedosoandDONOTuseglitternailpolishtosealyourslidesasthiswillscatterlaserlight.Besuretogiveyourmountingmediaandsealantatleast8hourstodryproperly.



Start-upProcedure- TurntheMainswitchON(1)- Youshouldnothavetotouchthekey

sincethisshouldneverbeturnedoff- TurntheSystems/PCswitchON(2)- TurnthePCONandwaitforittobootup

completely- LogintothePCusingyourADlogindetails- TurnComponentsON(3)

o TheHXPFluorescencelampshouldautomaticallyturnONwiththecomponentsswitchabove

o IftheHXPisnotonthenswitchthisonbeforeproceeding

- Double-clicktheZENBlackicononthedesktop

- ClicktheStartSystembuttonandwaitforthesoftwaretoload

- IftheArgonlaserisrequired,itisrecommendedtogostraighttotheAcquisitiontabandswitchthelaserONtobeginwarmingup(thiscantakeupto1minuteandthelaserwillnotbefullyuseableuntilitstopsshowingRed)

SavingYourDataSaveyourDatatoC:\UserDataandcreateyourselfafolderifitdoesn’talreadyexist.DONOTsavedirectlytothenetwork,onlytransferyourfilesattheendofyoursession.

Instructionsonconnectingnetworkdrivescanbefoundonthehelpsheetateachmicroscope.

IncubationTheincubationonthe880shouldalwaysbesetat37°C.Ifyourequireadifferenttemperature,thecool-down/warm-uptimesneedtobefactoredintoYOURbookingtimeandyouareresponsibleforensuringthetemperatureisbackat37°CANDstablebeforethenextuserisbookedon.AbookingforadifferenttemperaturecanbeloggedwhilecreatingyoursessionbookinginPPMS.

ForCO2,thiscanbeswitchedONatthestartofyoursessionandsettoyourdesiredconcentrationthroughboththesoftwareandthetouch-padcontrols.PleasemakesuretoturntheCO2OFFagainonceyouhavefinishedyoursession.



MicroscopeControls

- Brightfieldlightsourcecanbeadjustedmanuallybyrotatingthewheel(Int)

- OpenandclosetheshuttersusingtheTL(TransmittedLight)andRL(ReflectedFluorescenceLight)

- FocustheobjectiveusingtheCoarseorFinefocusknobs

- Controlsarerepeatedonboth

themicroscopestanditselfaswellastheremotetouch-pad

- Selectthe“Microscope”

optiononthelefthandsidetoenterhardwarecontrol



- Controlsforthehardwarecanthenbeaccessedthroughthesoftware

- Thetouchpadcontrolscanbeusedtochangebetweentheobjectivelensesautomatically

o Warningmessageswillpopupifchangingbetweenlensesofdifferingimmersionmedia(e.g.betweenoilandwaterlenses)

- ThetouchpadalsocontainsReflectorturret

optionsallowingtorotatebetweenfluorescentfiltercubessuchasDAPI,GFPorDsRed

- Incubationcontrolscanalsobeaccessed

onthelefthandcolumnofoptions- PresstheLoadPositiontolowerthe

objectivesawayfromyoursampleatanytime

CheckingThroughtheEyepieces

- MakesureyouhavetheLocatetabselected- Clickontherelevantshort-cutbuttonforthefluorescencechannelyouneed(arrow)

o SelecttheBFbuttonforbright-fieldo Thecoloursrefertothewavelengthrangeofthefluorophores

- Findyoursampleandfocususingtheeye-pieces,coarse/finefocusandjoystick

o Thetop-rightbuttonlabelledF1onthejoystickpadswitchesbetweencoarseandfinestagemovement

- OnceyouhavefinishedlookingatyoursampleALWAYSturnyourshuttersOFFtostopanysamplebleaching



ApplyingOilforaSlide- Opentheincubationchamberandtiltthemicroscopeturretback- Removetheslideandrotatetheoilobjectiveintopositionwiththesoftware- Placeasmalldropofthecorrectoilontothemiddleoftheslideorverycarefullyplacea

smalldropontothecentreoftheobjectivelens- Replacetheslide,coverslipsidedown,lowertheturretandclosethechamberbackup- DONOToveroiltheobjectiveasthiswillcauseoiltorundownthesidesandintothe

housingofboththeobjectiveANDtheobjectiveturret- Toremovetheslidesimplyopentheincubationchamberandremoveitfromthestage

inserto Itisrecommendedtowipeawayexcessoilwiththelenstissue(NOTKim-wipes)

providedandapplyfreshoilbetweeneachsample- Whenfinishedmakesuretocleantheobjectivebeforeleaving

Light-pathandIncubationControls- Despitehavingtheshortcutbuttons,

therearealsocontrolsforindividualcomponents

o Objwillallowuserstochangetoadifferentobjectivelens

o Filterwillallowuserstomanuallyselectthedesiredfluorescentfiltercube

o Shutterscanbeusedtomanuallyopenandcloseboththebrightfieldandfluorescentlightsourceshutters

- Incubationcontrolscanaltertemperature

forboththechamberaswellastheheatedsampleinsert

- ThecontrolscanalsoturntheCO2ON/OFFandalterthelevelsfortheconcentration



SettingUpKohlerIllumination- KohlerIlluminationisanimportantsetupprocedureforopticalmicroscopygivingeven

illuminationandreducedsampleheating/bleachingo Tostartwith,selecttheBFbuttonontheLocatetabtogetabrightfieldimageo FullyclosetheFieldAperture(atthetopoftheturrethead)o FocusthelightsourceusingtheCondenserFocusknobsuntilyoucanseeasharp

outsideringofthefieldaperturewhilelookingdowntheeyepieceso Ensurethelightisinthemiddleofthefieldofviewbyadjustingthetwosilver

centringscrewsatthefrontofthecondensero Oncecentred,opentheFieldApertureuntilitjustfillsthewholefieldofviewbutno

more- Thisshouldberepeatedwhenyouchangeobjectivemagnificationasitisoftendifferent

betweenlenses

AcquisitionMode- ClickontheAcquisitionTabtochangethelightpathsettingtotheconfocalscanhead- Ifnotalreadydoneso,clicktheShowAllToolstickboxtoshowtheLightpathandlaser

settings–turntheArgonlaserONifitisrequiredandallowittowarmupo TheShowAlltickboxinthetoprightofeachsettingswindowcanbeusedatany

timetodisplaymoreadvancedfeatureso YoucanalsogototheViewoptioninthetopmenuandselectShowAllGlobalto

activatethisfeatureforallwindowsatonce- TurnONanyotherlasersthatyouwillrequireforyourimagingsession

CustomisingYourWorkspace

- MuchofthescreenlayoutofZENcanbecustomisedtosomeextent:o Youcanclickanddragonatoolgroup(thegreyheadingsbetweenthebluetool

menuwindows)todifferentcolumnso Individualtoolmenuwindows(i.e.Z-stackorTileScan)canberepositioned

anywhereinthescreenandsentbacktotheoriginalpositionusingthediagonalarrowinthetopright-handcornerofthetoolwindow

o ChangethedisplaysizebyslidingtheWorkplaceZoomtoolinthetoprightcorneroftheZENsoftwarewindow

- OnceyouarehappywithyourlayoutyoucansavethisusingtheWorkplaceConfigurationsavebuttoninthetoprightcorneroftheZENwindow,justbelowtheworkplaceZoomslider

- Youcanthenloadyourpre-configuredlayoutsatthebeginningofyoursession



SmartSetupforBasicAcquisition- Smartsetupallowsuserstoperformanauto-setupbysimplyselectingtheirdesired

fluorophoresandthemodeofimagingrequired- ClicktheSmartSetupbuttontoenteraguidedsetupprocedure

o Anewwindowwillopencontainingoptionforfluorophoresandtypesofsetup- Chooseyourfirstfluorophorefromthedrop-downlist(Fluorophore)- Addanyotherfluorophoresyouhave,uptoamaximumof4- Adjusttheassignedcolourofeachfluorophoreifdesired- ChoosethecorrectmodeofimagingbetweenFastest,BestSignalorSmartest(Line)

o Fastestwillimagealldyesatthesametimebuthasthehighestcross-talko BestSignalcreatesseparatetracksforeachcolour-slowerbutthebestseparationo Smartest(Line)useslineswitchingbetweentracks-gooddyeseparationwhilestill

showingallcoloursatonceBUTdoeshavesomecompromises- ClickApplywhendoneandthesoftwarewillautomaticallysetuptheimagingchannels



ManualSetupforBasicAcquisition- Toaddanewtracktoyourexperiment,click

the“+”buttononeithertheChannelsorImagingSetuptoolwindows(NewTrack)

- SelectthedetectoryouwishtousebytickingtheUseboxinthelineofCh1,ChS1orCh2intheImagingSetupwindow

o Ch1andCh2areGaAsPPMTso ChS1isthe32GaAsPSpectralarrayo ChS1canbeusedasaspectral

detectororsplitandcombinedintoseparatedetectortracks(atacompromiseofdetectorgainflexibility)

- Adjustthedetectorrange(thewhitebarabovethedetectorselection)toselectspecificemissionwavelengthranges

o IfyouneedassistanceinknowingwheretoplacethisselecttheappropriatefluorophorefromtheDyedropdownlistnexttothedetectoryouhaveticked–aspectralprofilegraphicwillthenappearshowingthepeakemissionwavelengthrangeyouneed

- YoucanalterthetracknameundertheTrackscolumnandcanchangethetrackcolourattherightsideofthetrack,bothintheChannelswindow

- TickthedesiredlaserwavelengthintheChannelswindowtoaddthislasertothetrack

o Thelaserpowersliderwillappear–setthisto2%power

- Selecttheappropriatedichroicmirrorforthelaseryouareusing(Dichroics)intheImagingSetupwindow

- Adjustthepinholetothecorrectsizefortheselectedwavelengthbyclickingthe1AUbuttonintheChannelswindow

o Youmayfindthatthesmartsetupusesalargerpinholeasastartingpoint,thisallowsforeasiersamplelocationandyoucanbedoneforyourmanualsetuptoo,thoughoptimalpinholewillalwaysbe1AUforyourconfocalimaging

- SettheGain(Master)toaround500asastartingpoint



- Toaddfurthertracks,repeattheprocessaboveforeachadditionalfluorophoreyouwishtouseasmanytimesasyouneedforthefluorophoresyouhaveonyoursample

o HighlighttheindividualtrackintheChannelswindowtoeditsettingsforthisspecifictrack

o Itispossibletohavemultiplechannelsonasingletrackusingmorethanonedetectoratthesametime

o Whenusingasinglecolour,itisrecommendedtousegreyscaleduetothehighersensitivityofviewingthiscombination,thoughformultipletracksassignthedesiredcolourtoeachchannelindividuallyusingtheLUTdropdownlistattherightsideoftheChannelslist

- Whensettingupmultipletrackskeepcross-talk/bleedthroughinmindo Separateoutchannelsthatmayhavecrosstalkintoseparatetracks(forexample,

DAPIwilltendtoshowupinaGFPchannelsifbothlasersareonsoshouldbeplacedinseparatetracks)

o Coloursthathavecloselyoverlappingspectramaybedifficulttoseparateoutwithoutmorecomplexspectralun-mixingtools

ImageAcquisition- Onceyouhavesetupyourchannelsgoto

theAcquisitionModewindowandsetuptheimagesettings

- SetthePixelresolutionofyourimageo 512x512and1024x1024arethe

mostcommonoptionsforroutineimaging

o Beawarethatthemorelines=slowerscanning

o Optimaluses“Nyquist”sampling- ClickMaxforscanspeed(ifyouhaveanoisy

imageyoucandropthespeedoneortwolevelstohelpimprovesignal:noise

- Setthebit-depthofyourfinalimageo 8-bit=256colours;12-bit=4096

colourso 16-bithas65,535colourvaluesbut

isNOTrecommended

- Adjusttheamountofaveragingyouwishtouseforyourimages



o Averaging(beitframeorline)helpstoimproveyoursignal:noisebyimagingyoursamplemultipletimesandtakingtheaverageintensityofeachpixelacrossallthoseimages

o YoucanswapbetweenLineandFrameandevenchangetoAccumulation(addingthevaluesratherthanaveragingthem)byclickingtheShowAllbuttoninthetoprightoftheAcquisitionModewindow,revealingfurtheroptions

- Bi-directionalScanmodecanberevealedwiththeShowAllbutton,thisallowsscanninginbothforwardANDbackwardscandirections,increasingscanspeed,becarefultonoteanymisalignmentinthescandirectionswhentheyareinterlacedtogetherandadjustthecalibrationdirectionsifrequired

- ByclickingtheShowAllbutton,youwillalsogainaccesstoanumberoftranslationalandrotationoptionsintheScanAreasection,allowingyoutomovetheareatobeimagedandrotateit

- Zoomwillzoomintosmallerareas,effectivelychangingtheresolutionofyourimagewithoutincreasingyourpixelnumberatthecostofasmallerfieldofview

o A2xzoomisequivalenttochangingfrom512pixelsto1024pixels- TheResetAllbuttoncanbeusedtoresetallthetranslationalandrotationchanges,aswell

asthezoomfactor,backtotheirdefaultvalues

Capturingyourfirstimage

- ClicktheLivebuttontobeginascanofyoursampleo Thiswillgiveyouafast,repeatingframeoftheimageasperthechannelsetupo Inlineswitchingmodeallchannelswillbeincludedinthescanimmediatelyo Ifyouhavesetupmulti-trackbutareusingframeswitching,thiswillcaptureasingle

trackbeforeswitchingthesettingsandcapturingthenexttrack,thiscanbecluckyanditisrecommendedtountickallbutonetrackandadjusttracksettingsindividually

- Adjustyourfocussothatthebrightestpartofyoursampleisinfocus,remembertheconfocalwillblockoutoffocuslightsothesampleneedstobeinthefocalplanecorrectlybeforecontinuing

- AdjusttheGain(Master)andLaserpowertoincreasethebrightnessofyoursignalo Findabalancebetweenthetwo;toomuchlaserpowerincreasesbleachingwhile

toohighagainincreasesbackgroundnoise(tryandstaybelow850gain)o Avoidgoingtoohigh–overexposedpixelsarelostinformation!UsetheRange

Indicatortooltocheckforoverexposedpixelsthatwillshowupinred- AdjusttheOffsettosetthebackgroundcut-off–againbecarefulnottogotoofaroryou

couldremoverealsignalifitisweaker,(usetherangeindicatorhereaswelltoshow‘Zero’pixelsinblue)

- Ifsettingupformulti-track,repeattheprocessforeachchannelinturn- Settheimageacquisitionsettingssuchaspixelcount,averaging,speedetc.- StoptheLivescanandthenclickSnaptoacquireyourimage- SavetheimageoutusingthesaveiconorthroughtheFile>Saveoptionsinthetopmenu

o Topreservehardwareinformationandscalingsaveasthe*.CZIfileformat



AdvancedImagingSetupYoucancombineanyoftheadvancedsetupoptionstogether,suchasZ-Stacks,Time-SeriesorTileScantocreatemorecomplexexperiments.ToactivateanyoftheseoptionssimplytickthesettingyouwishtohaveaddedundertheAcquisitionTabattheleft-handside.

Z-Stacks

- ClicktheLivebuttontogetanupdatingviewtoassistinfocussing- Selecttherangeofyour3dZ-Stack

o ForFirst/Lastmethod,startbyfocussingtothetopofyourobjectandclicktheSetFirstbutton,nextfocustothebottomoftheobjectandclickSetLast

o ForCentermethod,focustothemiddleofyourobjectandclicktheCenterbutton§ NotethattheCentermethodisonlyavailableoncetheShowAllboxisticked§ Centermethodisidealformulti-positionacquisitionwheretheCenterofyourZ-

StackwillbetakenastheZco-ordinateofyourposition§ RangeSelectwillperformanX-Zscan,showingthesamplefromaside-viewwith

threelines;tworedlinesrepresentthetopandbottomofthestackwhileagreenlinerepresentsthecentreofthescan–theselinescanthenbemovedtoadjusttheZ-Stacksettings

- ClickStoptohalttheLiveView- SettheZslicethicknessbyeithermanuallyenteringtheIntervalsizeinmicronsorbyclickingon

theOptimalButtono Intheexampleshown,theoptimalsizeissetto7.13um–thisisapproximatelyNyquist

sampling,butshouldyouwishhighaccuracyitisrecommendedtousetheNyquistcalculatortofindtheexactdistancerequired

o YoumaychoosetousetheSlicenumberratherthanIntervalsize- ClickStartExperimenttobeginacquisition(NOTSnapasbefore),theexperimentwillcaptureall

channelssetwithall3dslicesNOTE:YoucanalsoselecttocaptureallchannelsforeachsliceoramoreefficientwayistocaptureafullZ-Stackforasinglecolourbeforeproceedingtothenexttrack



Time-Series

- ActivatingtheTimeSeriesoptionallowsuserstosetupfortime-lapseimagingtocapturemoviesofliveevents

- SettheCyclenumber(thisisthenumberoftimestheacquisitionsetupwillrepeat)- SettheInterval(thisisthetimethesystemwillwaitbetweencapturingoneimageand

startingthenext)o Use0.0asyourvalueifyouwishtoimageasfastaspossible

- ClickStartExperimenttobeginthetime-lapsecapture

Positions

Addingmulti-pointcoordinatesallowsformoreefficienttime-lapseimaging,grantingmultipledatapointsforthesametimespentonthemicroscope

- ActivatethePositionsoption- Movetothecellorsample

areaofinterestandfocususingtheLivebutton

- ClicktheAddbuttontoaddtheX,YandZcoordinatesofthisaretothePositionList

- Repeattheprocessforanysubsequentareasyouwishtoimage

- Shouldyouwishtochangeanyposition,clickonthepointinthelist,clicktheMoveTobuttonandreadjusttheposition,clickUpdatetooverwritethispositionwiththenewcoordinates

- Toremoveasingleposition,clickonthepositioninthelistandthenclicktheRemovebuttontodeleteitfromthelist

- RemoveAllwilldeletetheentirelistofpositions- UsetheSavebuttontosavethecoordinatelistandLoadbuttontoimportandreusea

previouslysavedpositonlist- ScanOverviewImageallowstheusertoscanalargeareaatlowresolutionandutilisethis

asaquicktemplatescanthatcanbeusedtomoreeasilyidentifypositionsofinterest- TheSampleCarriersectionallowstemplatestobeusedtoassistnavigation



TileScans

Thisfeatureallowsmultiplesmallerfieldsofviewtobestitchedtogethertoformonelargeimage,givinguserstheabilitytohaveamuchbroaderoverviewoftheirsamplewhilemaintainingtheresolutionofthecurrentobjective.

- SelecttheTileScanoption- UsetheCenteredGridtabtocreatesetsizemosaics

o Setthenumberofhorizontalandverticaltilesrequired–thesedonothavetobesymmetrical

o Thesoftwarewillusethecurrentstagepositionasthecentreofthetile- BoundingGridallowstheusertosetspecificX-Ycoordinatestoincludeinthefinaltilescan

o MovetothefirstpointofinteresttoincludeinthetileandclicktheAddbuttono RepeatthismoveandAddprocedureforallremainingpointsofinteresttoincludeo Thesoftwareautomaticallycreatesalargertiletoencompassallofthese

coordinates- ConvexHullworksinthesamewayasBoundingGridbutwillexcludeanytilesthesoftware

deemsnotrequired,savingtimebyavoidingtheneedtoscanblankareasorareasofnointerest

- SetyourOverlapsizeto10%minimum,thisallowsthestitchingsoftwaretomatchtherepeatingpatternstoprovidea“pixelperfect”finalimage

- AvoidusingOnlinestitching,thissometimesfailssoitisbettertomanuallystitchpostacquisition

- ClickStartExperimenttobeginthetilescanacquisition- OncecompletedgototheProcessingtab,clickontheStitchingmethod,selectthecurrent

imageandclickApplytocreateastitchedversiono YoumaywishtoalsoselecttheparametertocreateaNewOutputforthestitched

imageratherthanreplacetherawdata



CombiningSettingsforMoreComplexExperiments

- Anyofthesettingsabovecanbecombinedtocreatemorecomplexexperiments,forexample:AZ-stackcanbecombinedwithatime-seriesandmulti-positions

o Clickthetick-boxesofmultiplemodulestoactivatethemforthesameexperiment- UsetheExperimentDesignermoduletocreateevenmorecomplexexperimentsetups

o Thisallowsyoutocreate“multi-blockexperiments”o Eachblockiseffectivelyauniqueexperiment,allowingforcomplexdesignasthey

actindependentlyofeachothero Theblocksexecuteafteroneanother,canhavepausesplacedinbetweenblocks

andthenloopthewholeexperimenttocreatecomplextime-lapses§ Beawarethatthetimingwithwaitsignalsmaynotbe100%precisesothere

maybesomesmallmicroseconderrorsintimeintervals

Bleaching(FRAPandFRET)

- ActivatingtheBleachingmodulewillalsoactivateRegionsandTimeSeriesaswellsincethesewillberequiredalso

o TodeactivateBleachingyouwillstillneedtountickalloftheothermodulesindividually

- Usetheregionstooltoselectaspecificareaoftheimage,youmayselectanysizeorshapeyourequireandevenaddmultipleregions

o Theregioncanbeusedforbleachingaspecificareabutcanalsobeusedforanalysisofthatareatoo

- UsetheStartBleachingafter#scanstocaptureasetnumberofimagesbeforethebleachstep(a“before”snapshot)

- Youmayalsorepeatthebleachafteracertainnumberofimages

- Setthenumberofiterationsforthebleachstep

o Moreiterationsmeanalongerbleachstepwhichmayberequiredforsamplesthatyouarestrugglingtobleacheffectively

- UsetheDifferentScanSpeedoptiontoslowdownthebleachstep

o Slowerspeedmeanslongerbleachstepandlongerpixeldwelltime,resultinginamoreeffectivebleachstep

- SafebleachforGaAsPallowsforextraprotectionforthemoresensitivedetectors



- YoumayalsochoosetobleachinadifferentZposition- UseZoombleachifyouwantafasterbleach

o Notethisisaroughscanandthereforewon’tbeasaccuratetoyourROIofchoice- TickthelaseryouwishyouusetoONandsetthepower,typicallyuse100%forthebleach

lasero YoucanalsochoosetoassigndifferentlaserstodifferentROI’s

- SettheTimeSeriesuptoanumberofrepetitionstosuityourexperiment,thisneedstobesetto3ormoreforasuccessful“before”and“after”imageexperiment

o Failuretosetthisproperlywillresultinasinglebleachstepbutnootherimaging- ClickStartExperimenttobeginthebleachexperiment- UsetheMeanROItoolintheImageWindowtodisplayananalysisoftheROIoverthetime

courseoftheexperimento FortypicalFRAPstudiesyoushouldseeaninitiallevelofsignalforthe“before”

imagesfollowedbythebleachstepcausingadecreaseinsignal,thenarecoveryofthefluorescenceovertime

o Notetherecoverywillneverreach100%andinsteadplateausafterawhileatalowerintensitythantheoriginal

- YoumayalsohaveaFRETanalysistoolavailablethatwillanalysethe“before”and“after”intensitiesandperformaratio-metricstudyofAcceptorBleachingFRET

SavingandTransferringFiles- Anyfilesthatareunsavedwillshowontheright-handsideimagelistwithasmallyellow

exclamationmarksignnexttoito IfyoucloseallwindowsorcloseZEN,thesoftwarewillwarnyouofanyunsavedfiles

andgiveyouonelastchancetosavethem(ensuretheyaretickedandyouwillbepresentedwithasavewindow)

- Tomanuallysaveafile:o Ensurethefileistheactivewindow(doubleclickthefileontheright-handsidelist

orclickthetabofthatfileintheimagewindowtabso Clickthesaveiconontheright-handsideoralternativelyusethemenuoptionFile>

SaveorFile>SaveAso EnterasuitablenameforthefileandselectthelocationtosaveittoandclickOK

§ DONOTusespacesornon-alphanumericcharactersinyourfilenames§ NEVERsavetotheC:drive–alwayssavelocallytotheDatadrive§ Makesuretogiveyourfileameaningfulname§ WritedatesinfilenamesasYYMMDD(i.e.190129)toassistinchronological

orderingunderWindowso Alwayssaveasa*.CZIfilewherepossible;NEVERsaveasaJPEG

- Onceyouhavefinishedyourimagingsession,transferallyourrecentfilestothenetworkdriveofchoice

o Remember,theacquisitioncomputerisNOTbackedupandareregularlywipedofoldfilessomakesureyoucopythefilesoffIMMEDIATELY



o IMBSharenetworkdrives,(bothyourownpersonaldiveandyourgroupdrive),arefullybackedupandyoushouldalwayscopyfileshereforarchiving

o Ifyoudonotseethenetworkdrive/sintheMyComputerareaofWindowsExplorer(pressWindows-Etoopen)youmayadditbyclickingMapNetworkDriveandaddingthenetworkaddressandclickOK

§ Yourpersonaldriveandgroupdrivewillbelocatedat\\IMBShare.imb.uq.edu.au\<username>(replace<username>witheitheryourusernameoryourgroupfoldername

ReusingPreviousSettings- Itisvitallyimportantwhenwantingtohaveanyquantitativeanalysisperformedonyour

datasetsthatyoumaintainthesamesettingsconsistentacrossimages- Savingoutasa*.CZIfileformatcreatesafilecontainingalotofmetadataassociatedwith

thehardwareofthesystem- Toreusesettingsusingthismetadata,loadinyoursavedimageandclicktheReusebuttonin

theZENsoftwareo Thiswillwriteallthesettingstothesoftwareandyoucanusethesystemasifyou

werestillinthesameimagingsessiono Beawarethatthesesettingsarenottransferablebetweenmicroscopesastheymay

havevariationsonhardwareconfigurations- Youcanalsosaveoutyoursettingstothedrop-downexperimentsettingsfolderatthetop

oftheZENsettingswindowtoavoidneedingaccesstosavedimageso Thiswillbesavedforyourprofilesonotavailabletootherusers

ShutDownProcedure- TurnthelasersOFFinthesoftware

o WaituntilthecoolingfansstopfortheArgonlaser(upto5min)beforeturningtherestofthehardwareoff

o Saveyourfilesandtransferthemtothenetworkdrivewhileyouarewaiting- Exitthesoftwareandwaitfor1minforallbackgroundprocessestofinishclosing- Removethesampleandcarefullycleantheobjectivelensofanyoilusingthelenstissue

provided(pleaseDONOTuseKim-Wipesforthis)- ShutdownthePConcethelasershavecooled

o YoushouldhearanaudibledropinthenoiseoftheLasosArgonlaserfan- TurntheComponentsswitchOFF- TurntheSystems/PCswitchOFF- TurntheMainSwitchOFF- DONOTturnofftheLasosArgonlaser(boththekeyandthepowerswitchshouldbelefton)- DONOTturnoffthelaserinterlockkeyPleaserememberthattheopticsonthesemicroscopesareverydelicate,pleasetreatthemicroscopesgentlyandwithrespect–theyareVERYexpensivetoreplaceandmayrequiresignificantdown-timeofsystemsaswellashighcosttothefacilitysopleaseBECAREFUL!



SystemHardwareSummaryMicroscopeStand:ZeissAxiovert200MInvertedMicroscope

ObjectiveLensList

- PlnApo10x/0.45DICII(WD=2.0mm)- PlnApo20x/0.8DICII(WD=0.55mm)- PlnApo40x/0.95KorrDICIII(WD=

0.25mm)- PlnApo40x/1.3OilDICIIIUV-IR(hand-

pickedforAirySR)(WD=0.21mm)- PlnApo63x/1.4OilDICIII(hand-pickedfor

AirySR)(WD=0.14mm)

LaserLinesAvailable

- 405nm- 458nm- 488nm- 514nm- 561nm- 633nm

MainBeamSplitterDichroics- 458- 458/514- 458/561- 488- 488/561- 488/561/633- 80/20

DichroicsBeamSplitterforSinglevMulti-channel:

- BP420-460+LP500- LP525- LP570- LP660- LP460- SP615- Mirror(100%internaldetectors)- Plate(100%Airydetector)

AiryscanEmissionFilters:

- Plate- Blank/None- BP420-480+BP495-550- BP420-480+BP495-620- BP420-480+LP605- BP465-505+LP525- BP495-550+LP570- BP570-620+LP645

LambdaDetectorWindowSizes:

- 3.0nm(requires96passes)- 4.5nm(requires64passes)- 8.9nm(1passonly)- 17.8nm(1passonly)- 26.7nm(1passonly)- 35.6nm(1passonly)

confocal 2 – zeiss 880 with fast airyscan · zen black available objective lenses: 10x air, 20x...

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