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TheMaiTaiTwoPhotonLaserSystem,ConfocalFour.OverviewandLaserSafety.NB:AllusersMUSTbetrainedintheuseoftheMaiTaiandmustreadandsigntheMaiTaispecificriskassessmentpriortouse.TheMaiTaitwophotonlaserisapowerfulinfra-red,pulsed,Class4device.Unshielded,itwouldposeahighriskofoperatoreyedamage.Beampathenclosures,interlockswitches,andshuttersbuiltintothissystem,andthematerialofthemicroscope’sincubationchambertogetherfacilitatesafeoperation.UsersMUSTNOTattempttocircumventthesesafetyprovisions,ordeviatefromtheoperationalproceduresdetailedintheMaiTaitutorial.Doingsomayresultinlaserexposure,withattendenteyedamageandlossofvision.NB:Threelowerdoorsontheconfocal’sincubationchamberdonothavesafetyinterlocks.Thesedoorsaremarked“EnsureSystemisLaserSafeBeforeOpeningThisDoor”.YoucanensurethesystemislasersafebycheckingtheZENsoftwaretoensureascanisnotinprogress.MercuryArcLampSafety.DoNotchangefiltercubeswhilstlookingthroughtheeyepieces,asyoumaybeexposedtointensewhitelight.
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Startup.
• ObtainthebluetaggedMaiTaikeyfromMicroscopyManager’soffice.
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Examinethelasersystemandmicroscope.Thesystemmustnotbeusedifanycomponentappearsdamaged,incomplete,orpartiallydisassembled.ContactIMBmicroscopystaffifindoubt.
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Ensureallthedoorsonthemicroscope’sincubationchamberareclosed,and
thattheZeissZENcapturesoftwareisNOTrunning.
• InsertthekeyintoMaiTaicontrolpanel*.Turnthekeytoon.
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Ifnecessary,startthemicroscopesystemandcomputer.LogintoWindows.Observethestartorderandnecessarydelaysfortroublefreestartup.
• StartZEN.EnsureZENstartswithouterrors.
• OntheZEN“Acquisition”screen,click“Showalltools”.
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OpentheLaserpanel,selectMaiTailaser,andopentheMaiTai’sinformation
dropdown.Youwillseeaninitialdisplayasbelow.
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ClickondropdownboxtotherightoftheMaiTainameandtoggletheselectionto“On”.
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TheMaiTaiinformationdropdownwillshowthelaser’spresentlyselected
wavelengthhighlightedinredandastatusof“CWLasing”,asthelaserbeginsitswarmupsequence.
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TheMaiTaitakesabout3minutestowarmandmode-lock.Duringthistimeit
willgraduallybuilduppowerandtransitionthrough“Busy”and“GDDPositioning”states.
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WhenreadyforuseZENwillshowtheMaiTai’sstatusas“Mode-locked”.
Usersnote:*YoumustturntheMaiTai’skeytoonbeforestartingZEN.ThoughthelasermaystilloperatewhencalledinZEN,startingtheMaiTaiafterZENwillproduceaGDDpositioningwarning.Chameleonlaserusersnote:whereasyoumustwaitfortheChameleon’sfrontpaneltodisplay“Mode-locked”beforestartingZEN,inthiscaseZENisstartedafterthelaserkeyisturnedtoon.NotethattheMaiTaiwillnotstarttowarmupuntilitiscalledfromZEN.ChangingtheMaiTai’spoweroutputandemissionwavelength.
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OutputpowercanbesetinZen’sImagingSetuppanelorintheChannelspanel,inthesamemannerastheotherlasers.
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Startwithalowpowervalue(eg.0.2%)andincreasegradually.Notethattwo
photonexcitationisnonlinear;thereisafinelinebetweenpoorexcitationandsampledestruction.
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TheMaiTaiemissiontuningrangeis690to1040nm.Typethedesiredvaluein
thedialogboxnexttothepowerslidebarandpress“enter”.ThenewlyenteredvaluewillbehighlightedinredwhilsttheMaiTaiadjusts,andbecomeplaintextoncethelaserachievesmode-lockatthenewwavelength.
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Settingupthelightpathandpinhole.Fluorophoreemissioninresponsetotwophotonexcitationmaybecapturedbyeitherthestandarddetectors,orthenon-descanneddetectors(NDDs).Thenon-descanneddetectorsareconsiderablymoresensitivethanthestandarddetectors,butarenotadjustableforcapturewavelength.Thoseusedhereareequippedwithbandpassfilters(BP500-550andBP575-610nm,detectorR1andR2,resp.)suitableforgreenandorange-redemissionfluorophoresonly.NotethatZEN’s“SmartSetup”isnottwo-photonaware;boththeexcitationandemissionlightpathsmustbeconfiguredmanually.Tocaptureviathestandarddetectors:
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OpentheZEN“ImagingSetup”panel.Settheexcitationlightpathtodirectthetwophotonexcitationtothesample,selectingeitherthe650+or760+MainBeamSplitter(MBS),asappropriatetoyourintendedexcitationandfluorophoreemissionwavelengths.
• Settheemission(orcaptureportionofthe)lightpathasnormal.
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OpentheZEN“Channels”panel.Gainandoffsetarecontrolledasnormal.Note,however,thattwophotonexcitationisconfinedtothefocalpointandthus,isinherentlyconfocal.Therefore,thedetectorpinholeshouldbesetwideopen.
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Tocaptureviathenon-descanneddetectors:
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OpentheZEN“ImagingSetup”panel.Ensureyouselect“ShowAll’inthebar’sheadertodisplayallavailableoptions.
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Selectthe“NonDescanned”button.Thenon-descannedlightpathwillbe
displayed.
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SelecttheappropriateMainBeamSplitter(MBS)foryourtwophotonwavelengthandfluorophore(asabove).
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SelecttheappropriateSecondaryBeamSplitter(BS-MP355/690+®orBS-MP-
760)todirectyourfluorescenceemissiontothenondescanneddetectors.
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SelectoneorbothoftheNDDchannels.Thesehavefixedbandpassfilterscoveringtheemissionranges500-550and575-610nm,respectively.
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OpentheZEN“Channels”panel.Ensure“Integration”isselected.Thealternative,“PhotonCounting”doesnotformanimage.
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ControlofNDDgainandblacklevelisasnormal.Note:thereisnopinholeintheNDDlightpath.
Warning:Thenon-descanneddetectorsareextremelysensitiveandeasilydamagedbyexcesslight.StartwithLOWlaserpowersanddetectorgainsettings.Collimation.Theinfra-redemissionofatwo-photonlaserfocusestoaslightlydifferentzplanethanthelightfromvisiblelasers.Themagnitudeofthisdifferenceiswavelengthdependent.Laserfocuscanbeadjustedtocoincidenceviathecollimator.
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Selectawelldefinedobjectorpointinyoursampletowardsthecentreoftheimagefield.Captureareferenceimageusingtheappropriatevisiblelaser.
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OpenZEN’s“Maintain”screenandselectthe“AdjustPinholeandCollimator”
panel.
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UnderCollimators,selectthe“LSMCOLLI_NLO”optionfromthedropdownbox;thedescriptionLSM710CollimatorNLOwillbedisplayedtotherightoftheselection.YoumustensurethatyouselectthiscollimatorandNOTthe405/UVdevice.
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Switchbacktothe“Acquisition”screenandclick“New”tocreateanewimagecontainer.Setthisupfortwophotonexcitation.Initiatea“Live”or“Continuous”scan.Then,whilstscanning,switchbacktothe“Maintain”screen.
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Thecollimatorisadjustedtobringthelive,twophotonproducedimageofyour
sampleobjectorpointtothesamefocusobtainedinthevisiblelaserimage.Collimatorpositionisbestadjustedwithslow,smoothmotionofthescrollbar.
• Click“StoreCurrentPos”.
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Switchbacktothe“Acquisition”screentostopthelive\continuousscan.
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Comparethevisiblereferenceimagetothetwophotonimagetoverifyyour
setting.Note:thisproceduremaywellbleachyoursample.Collimationisbestperformedonanexpendablesample.Changingthe“GDD”Setting.Imagesharpness,confocalityandexcitationefficiencyareinfluencedbythegroupvelocitydispersionofthelaserpulse.Thesystemstoresasetofdefault“GDD”compensationpositionsforeachobjectiveandemissionwavelength.Thesecan,however,bealteredtoattemptoptimisationforyourparticularsampleandwavelengthneeds.
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GDDpositionsareshownintheMaiTaiinformationdropdownontheZENLaserpanel.
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Thedefaultvaluecanbealteredbymovingtheslidingbar,usingthearrows,ortypingavalueintothevaluebox.TheGDDcontrolwillfreezeandthevaluewillbehighlightedinredastheMaiTaiadjusts.Bepatient.
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DONOTClickthe“Store”button.Doingsoaltersthedefaultvalueinthe
databaseandthusaffectsallotherusers.Beawarethat,ifyouchoosetoaltertheGDDposition,youareexpectedtofirstnotetheoriginalvalue,andresettothatpositionpriortoshuttingdowntheMaiTaiandZEN.Wesuggestalsothatyoutakenoteofyoursample’sGDDvalue,sothatyoumayeasilyre-enteritinfuturesessions.
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Note:1)Adjustinsmallincrementsandtestbyimaging.2)Remember,theGDDsettingisglobal;youralterationwillaffectallotherusers.Ensureitisresettotheoriginal,defaultvalueatthecloseofyoursession.Notethatifyouhaveaccidentallyclicked“store”foryouraltered,sampleGDDvalue,youMUSTre-entertheoriginalvalueandclick“store”againbeforeturningofftheMaiTaiandclosingZEN.Shutdown.
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OpentheZENLaserpanel,selectMaiTailaser,andopentheMaiTai’sinformationdropdown.
• IfyouhavealteredtheGDDsetting,resetittoitsinitialvalue.
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ClickondropdownboxtotherightoftheMaiTainameandtoggletheselectionto“Off”.
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Wipeupanyoilormediaspiltonthemicroscope,themicroscopestage,and
tables,cleanthemicroscopeobjectivesofoil,andsettheincubationchamberbacktothedefaulttemperature,asappropriate.
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ShutdownZENandlogoutoforshutdownthethecomputer,asappropriate.
• TurntheMaiTaicontrolkeytoOff,extractthekeyandreturnittothe
MicroscopyFacilityManager’soffice.Note:YoumustturntheMaiTaioffinZENbeforeturningtheMaiTai’skeytooff.TurningthekeyoffwhileZENshowsthelaseronwillresultinlossofcommunicationwiththelasermodule.Zenmustberestarted,withouterror,beforecommunicationcanre-establish.DarrenJ.Paul.Revision3.2016-05-20.
