Zeiss LSM 510 Inverted User manual

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UseoftheZeissLSM510Inverted

Firstlypleasebeawarethatthismicroscopeshouldbetreatedwithrespectandcareatalltimes.

Rulesofuse:

ThisMicroscopecanonlybeusedbyMastersbyResearchorPhDstudents,Postdocsandmembersofstaff.

YoumustreceivetrainingfromAnnWheelerbeforeyouusethismicroscope..

Themicroscopelensesmustbecleanedaftereveryusageandtheequipmenttreatedcarefullyatalltimes.

Ifyouhaveanyproblemsatallwiththemicroscope,nomatterhowtrivialtheymayseempleaseseeAnn

Wheeler,AmandaWilsonimmediately.

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REMEMBER:Youhave5GBofdiskspaceonthismicroscope.Checkbeforeyoustartifyouhaveroomforyour

experiment.Ifnotdeleteyourolddata.

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Toswitchonthemicroscope.

SwitchontheMercuryarcbulb

Switchontheremotecontrolbutton

Switchonthecomputer

Cleantheobjectivelensyouwanttousebeforeyoustartwork

1.Startthesoftware

ClicktheZENshortcutbutton

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Clickstart

system

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Thescreenlookslikethisonceyouhaveswitchedeverythingon.Thecontrolmenuisontheleftandyouworkfromtop

tobottom.Yourimagesarestoredontheright.

Lightpath(eyes

andlaseris

controlledhere)

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2.TurnontheLasers

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ToturnonlasersselectLASERmenu,RHSofworkspace.Dropitdownusingthesmallarrowintheleftcorner.

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Selectthelasersyouneedforyourexperimentand

turnthemon

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FortheArgonandEnterpriselasersdropdownthe

LaserPropertiesarrow

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FortheArgonlaser:switchitontostandby,waituntilithas

warmedupandthenswitchon,andsetoutputto6.1A.



NBisthisusesmorethan50%ofthelaseroutputsee

Ann/Amanda

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FortheEnterpriselaser:switchitontostandby,waituntilithas

warmedupthenswitchon,andsettheoutputto100%

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3.SelecttheConfigurationyouneed

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PickImagingConfigurationfromthedropdownmenu

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Selecttheconfigurationyouneed

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 Clicktheicontoactivateyourchosenconfiguration

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4.SettheMicroscopeupandfindyourspecimen

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ClicktheOcularbuttontosendthelighttoyoureyes(toprightareaofthe

workspace)

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Thiswillallowyoutocheckyoursamplesdownthemicroscopebeforeyoustart

laserscanning

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IntheLightPathcontrolmenu:



Thisopensthetransmitted(Brightfield)light

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Selecttheobjectivelensthatyouwanttouse

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Selecttheappropriatefilter(DAPI/FITC/TRITC)

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ClickthefluorescenceshutterOpen

toseefluorescentlight

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5.Startconfocalscanning

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ClickLSMbutton(toprightoftheworkspace)

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ClicktheFastbutton(foundontopleftoftheworkspace)ClickSplitonthelefthandsideof

your  image(thissplitstheimage

accordingtochannelsused)



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



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6.Setthelevelsforeachchannel

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Clickthecolouredregionundereachchannel(inthepanelunderneaththe

imagewindow)toactivatethe‘rangeindicator’palette;

theimageswillnowgogreyorRedandBlue

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

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Makesurethechannelsmenuisinyourworkspace,ifnotclicktherighthandsidearrowto'float'themenuout

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Foreachchannelandchangethefollowingineachtoobtaingoodimage:



1)Pinhole:clickon1tomakepinhole1airyunit(Pleasenote:ifyou’re

doingcolocalisation,refertheworksheetontheBALMwebsite)

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2)Mastergain:increaseleveluntilyouseered,thenreduceagainuntil

redjustdisappears

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3)Offset:decreaseleveluntilyouseeblue,thenincreaseagainuntilblue

justdisappears

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7.Resizeyourimagesusingthecroptool.

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TostopscanningatanytimeclickStop

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StopscanningandpressCrop(bottomofscreen)

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Simplyputthecroppingwindow(Redboxwithcrosshair)overtheregionyouwantto

crop

Changetheposition,sizeorrotationofthecroppedareabydraggingtheedgesofthe

cropboxwiththecursor

Startscanningagainandyourcroppedimagewillappear

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8.Setupthescanningparametersandselectingtheareayouwanttoimage

Onceyouarehappywithyourimagestopscanning

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GototheAcquisitionModetabontheLHSofthe

workspaceandfloatoutifnotinworkspacealready.

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Generallyforanicedetailedpicture:

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Framesizeshouldbe1024x1024

Scanspeed:7

Datadepthshouldbe16bit

Scanaverageshouldbe4

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Optionalifyoudon'twanttousecrop

Toalterthescanareadropdownthearrow

Whiletheimageisscanning:



Youcannowexpandorreducethescanarea,

Zoomin



orturnitaroundasrequired



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9.Takethepicture

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ClickSingleinthescanmenu

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10.SavingyourImage(s)



GototheOpenImagessectiononthetoprightofthe

workspace



Selecttheonethatyouwanttosave



Clickthefloppydiskicontosavetheimage

(pleasesavetoyourfolderinUsersonE‐drive!!)

Toconverttotifgotothefilemenu(topright)

andclickExport.



Toexportjusttherawdatathatyouhavepicktheformat(.tif)clickexportRaw

dataSingleplane(normalscan)orSeries(Zstackortimelapse)

Toexporttheimagedisplayincludinganyoverlaysoranalysisthathaschoose

exportContentsofImagewindoworFullresolutionimagewindowSingleplane

(normalscan)orSeries(Zstackortimelapse).

Choose'selectfilenameandsave'tosave

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12.TotakeaZstack

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GototheZ‐Stacktoolbar(foundontheleftsideoftheworkspace)anddropitdown

ClickontheticktoactivatetheZstackmenu(youcan't

collectazstackwithoutthis)Itswhitewhenactivated



Chooseifyouwanttoselectfirstandlastplanesorto

collectdatafromacentralpoint(BALMstaff

recommendselectfirstandlastingeneral)



ForstackscollectedinselectFirstandLastmode

Enteryourrequiredintervalforzstack

SelectKeepinterval

Note:useOptimalIntervalifyouwantthebest

resolvedstack(for3Dimagegeneration)



ClickFasttostartscanning

Movetothebottomyoursampleandhitsetlast

Movetothetopofyoursampleandclicksetfirst



ClickSTARTtocollecttheZstack



Youcanviewthezseries(andtimeseries)asitis

beingcollectedusingtheGalleryoption



13.Collectingatimeseries/timelapse