Confocal 2 – Zeiss 880 with Fast Airyscan · ZEN Black Available Objective Lenses: 10x Air, 20x...
Transcript of Confocal 2 – Zeiss 880 with Fast Airyscan · ZEN Black Available Objective Lenses: 10x Air, 20x...
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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.
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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.
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MicroscopeControls
- Brightfieldlightsourcecanbeadjustedmanuallybyrotatingthewheel(Int)
- OpenandclosetheshuttersusingtheTL(TransmittedLight)andRL(ReflectedFluorescenceLight)
- FocustheobjectiveusingtheCoarseorFinefocusknobs
- Controlsarerepeatedonboth
themicroscopestanditselfaswellastheremotetouch-pad
- Selectthe“Microscope”
optiononthelefthandsidetoenterhardwarecontrol
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- 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
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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
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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
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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
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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
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- 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
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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
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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
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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
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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
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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
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- 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
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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!
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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)