Leica SP2 User manual

LMW Microscopy Lab : Leica SP2 Page 1 `6/26/2012
ManualforLeicaSP2ConfocalMicroscope
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Enteryouname,thedate,thetime,andtheaccountnumberintheuserlogbook.
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Thingstocheckbeforestart‐up.
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Makesurethatyoursampleslidesarecleanandsealed.UseWindexandcottonballsorKimwipestoclean
coverslips.Fixedsamplesneedtobesealedwithnailpolish.
Checktheobjectives.The10xand20xobjectivesareDRYobjectives,andshouldNEVERhaveoilonthem!
Thereare40xand63xOILimmersionobjectives,anda63xWATERimmersionobjectivealsoonthis
microscope.Ifoilimmersionobjectiveshaveoilonthem,wipethelensgentlyonlywithlenspaper.
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SystemStart‐up.
1. Turnonthemicroscopecontrollerbandthe
ScannerLaserHeNecandPC/Standd
switches.
2. Turnonthecomputereandlogontothe
desktop.
3. TurnontheMercuryArcLamppowersupplier,
ifyoulookatfluorescentlylabeledsamples.
4. StarttheLCSprogramby
double‐clickingtheLCSiconon
thedesktop.
Onthepop‐upstartingwindowpanel,select
thePersonalthatcontainsyoursettingfor
acquisitionparametersyouhavecreatedor
Companyforadefaultsetting.
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OveviewofLeicaConfocalSoftware(LCS)
UponLCSprogramstart,thebasicimageacquisitionmenuwillappear.TheAcquirebutton
shouldappearpressed.
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ClicktheBeambutton todisplaytheBeamPathSettingas
appeared.
ClicktheZ‐Scanbutton andselectz‐Wide(thisisrequiredtofocusto
sampleontheregularslideholderbytheRemoteControlKnobs;seebelow).
WiththeObjbutton,youcanselecttheobjectiveyouwanttouse.(Amessagewindowmaypop
up,andclickOK)
WithMicCtrlbutton,youcanswitchbetweenScanmode(forlaserscanning)andVisualmode
(forviewingthroughtheeyepiece).
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BasicOperationofLeicaDMIRE2Microsocpe.
Bright‐fieldviewing:
1. Placeyoursampleslidewithcoverslip
(preferably#1.5thickness)facingdowntothe
objectiveonthestage.
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2. Forbright‐fieldviewing,turnonthemicroscope
halogenlamplightbyturningthewheel b
towardyouandturntheVIS‐SCANswitchcto
VISposition(Note:switchbacktoScanpositionforconfocalscanning,see
below)
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3. Startwithlowmagnificationobjectives(10Xor20X)firsttofocusontoyour
specimen.Youcanplacethedesiredobjectiveinpositionbyturningthe
lensturret dbyhandorbyclickingObjbutton andselectingan
objectiveyouwanttouse(recommended;seebelow).Theinformationof
theselectedobjectiveandtheirz‐positioncanbereadonthereadout
panel eatthefrontofthescope.
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4. FocusontothesamplebymovingtheobjectiveturretwiththeCoarse
FocusButtonsf(upperandlower)andtheFineFocusKnob g.Pushthe
upperCoarseButtontobringtheobjectivetotheupperlimit(“0µm”)
position–wherethemicroscopehasbeencalibratedforyoursampleto
benearinfocus.
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5. UsetheFinefocusknobtofinetunefocusing.Theextentofthefine
movementcanbesetfromS0(veryfine)toS3(coarse)bypressingthe
STEPbutton h(S2isrecommendedformostcases).
PleaseDONOTpresstheLEARN,CHANGE,orbuttons.
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6. Withmostcellsortissuesamplesstainedwithfluorescentdye,you
couldeasilyfocustoyoursamplewithafluorescencemode(see
below)orPhaseContrastmode.ForPhaseContrastmodewith10xor
20xobjectives,turntheCondenserRingi toselectPH1(for10x)or
PH2(for20x).
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7. Forusingoilorwaterimmersionlens,lowertheobjectiveturret
slightly,exposethelensbyturningtheturrethalfwaybyhand,and
placeadropofoilorwaterontheobjective(becarefulnottotouchthelensdirectlywithapplication
tools!).Placetheslideovertheobjectiveandraisetheobjectiveuntiltheoiljustspreadsoutasitcontacts
theslide.Usefindfocusknobtograduallybringyoursampleintofocus.Checkyourslideoccasionallyto
makesurethatyouarenotpushinguptheslidewiththeobjective.Thiscandamagetheobjectivelensand
yoursample!
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Fluorescentviewing:
Youcancheckyoursampletoseeifyourfluorescencelabelingworksby
usingthemercurylampilluminationandtheappropriatefiltersets.The
microscopehasfiltersetsforviewingDAPI(A),greenfluorescence(I3)
andredfluorescence(N2.1).
1. Presseithertheleftorrighthorizontalarrowbutton jonthefront
ofthescopetochoosethefilter(FLUO)set.“Scan”positionhasno
filtersetinplaceandisusedforlaserconfocalscanning.The
fluorescentlightcanbeblocked(closed)oremittedbypressingthe
Shutter 1) button(The“closed”lightshouldbeoffforfluorescent
viewing.).
2. TurntheVIS‐SCANswitch 1! toScanpositiontoblockthe
transmittedbright‐fieldlight,ifnecessary.
3. Examinethesamplethroughtheeyepieces.(MakesurethatPORTSisinVISand
MAGisat1x.Ifnot,changeitselectingVisualfromMicCtrlbuttoninLCS
programwindow.)
4. Whenyouarereadytoscanyoursamplewithlaser,closetheSHUTTER1) to
preventanyunnecessarybleachingofthesamplewithmercuryarclamplight.
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SettingupforConfocalLaserScanning.
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Toturnonthelaser(s);
(1) Turnononlythelasersyouwilluse.Theydo
notneedmorethanaminuteorsotowarmup
andstabilize,soitisnotnecessarytoturnthem
onuntilyouknowyouneedthem.
(2) Argonlaser(for458,476,488,and514nm
excitationline):Turn ontheredLaserAr/ArKr
switch bandturntheLevelAr/ArKrlevelknob
ctoMIN.Then,turntheleftAr/ArKrkeydto
“ON”,andthenturnfurtherto“START”positionand
releasethekey.AdjustthelaserpowerwithLEVEL
knob c(usually9o’clockpositionissufficient).
(3) MellesGriotyellowDPSS(561nm)laser:push the
greenbutton eforafewsecondsandrelease.
(4) He/Ne(633nm)laser:turntheHe/Ne633keyfto
ON.
(5) DiodeUVlaser405nm:Turnontheredpower
button g andturnthekey h toIposition.
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LCSImagingprogramsetting
1. First,selecttheappropriatelaserlinesandsetupdetectorsforthe
fluorescentlabelsyouusedonyoursamplefromthedrop‐downLlisti
(LeicaFactorysetting). Thiswillautomaticallysetuptheemissiondetector
band‐widthj,activatetheproperdetector(PMTs)1),dichroic
beamsplitter1!,andexcitationlaserwavelengthandpower1@.Youcan
adjusttherangeandpositionofdetectorbandwidthandthelaserpower
levelasnecessary.
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2. Youcanalsochangethecolorschemeofindividualimagesintoanycoloror
greyscalebyclickingthepseudo‐colorselector 1# associatedwitheach
PMTonthewindowscreen(Note:thecolorinformationwillbesavedwithimagefileswhensaved).
3. PressModebuttontoselectScanMode(defaultisXYZ).
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4. PressFormat ,andSpeedbuttonstoselecttheseparameters(defaultsare512x512formatand
400Hzscanspeed).
5. PressZ‐scanbuttonandselectz‐WideinordertouseZPOSknob 1$ tomovetheobjectivefor
focusing(Defaultisz‐Galvo,whichisusedwithGalvostageadaptor).
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6. ClickMicCtrlbutton toselectScanmodeforlaserscanning(itwillautomaticallyshutoffvisual
outputthroughtheeyepieces).
7. PressContinuousbuttontostartscanning(itwillbecomeStopbutton).Animageorimages,
dependingonthenumberofactivedetectors,willappearontherightmonitor.
RemoteControlKnobs
8. Optimizetheimagesbyadjustingparametersincluding;
1. Thez‐position(focusing)withinthespecimen(Z‐POSknob1$).
2. Zoomfactor(ZOOMknob 1%): Defaultis1;increasingzoomwillmagnifytheimageandimprovethe
resolution,butalsowillbleachthesamplefaster!
3. PMT1,2,3,TransandSmartOffsetknobs 1^: TurnPMTknobstoincreasethedetectorsensitivitysoto
increasethesignalintensityofeachfluorescentandtransmissionimagechannel(Note:KeepthePMTlevel
under600V).Todecreasebackgroundsignal,clickthemouseoverachanneltoselectitandturnOffset
knobcounterclockwise.WithQLUTbuttonintheExperimentliveviewwindow,afulldynamicrange
ofthePMTforoptimalimagequalitycanbeobtained.Everyclickingthisbuttonchangestheviewwindow
inthreedifferentLUTmodes;pseudocolor,Hi‐Lo,andmonochrome.IntheHi‐Lomode,pixelssaturating
thePMTwillappearblueandpixelswhichareblack(0value)appeargreen.AdjustthePMTgainandPMT
offsetsuchthatthebrightestpixelsarejustunderthesaturation(afewbluepixels)andthedarkestpixels
arejustabovethezerovalue(asomegreenpixels).(AdjustLaserpowerbydraggingthelaserpowerbaron
themenuwindowifnecessary).
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Pseudocolor    Hi‐Lomonochrome
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ClicktheStopbutton againtostopscanning.(Youcansavethecurrentparametersettingwith
Savebuttonandputaspecificnameforlateruse). 
ViewingImages.
Singlebutton–
putsupfull
screenof
selected
fluorescent
channel.
Ch1,2,3,4
buttons–activate
thechannelon
thescreen.
Tiledbutton–
viewsupto3
channels
simultaneously.
Ovlbutton–
overlays(merge)
multiplechannels
intoone.
Displaybutton‐
changethe
magnificationof
thescreenimage.
Lutbutton‐open
colorlook‐up
table.
AcquiringSingleOpticalSectionImage.
MakesuretoswitchMicCtrltoScanmode.
PressSingleScanbuttontoacquireanimagebyasinglelaserscanningatascanningspeed(normally
400Hz).Theresultingimagemaydisplaybackgroundnoise.Imageaveragingisaprocesstodecreasethis
noiseandimprovethesignal‐to‐noiseratio.
Touseaveraging,clickeitherLi.A(lineaveraging,recommended)orAver (frameaveraging)
buttonandselectthenumberoflines(orframes)tobeaveraged(4lineorframeaveragingisagood
startingpoint).Then,clicktheSingleScanbutton.

LMW Microscopy Lab : Leica SP2 Page 8 `6/26/2012
Afterscanning,theimagewillbesavedinthedirectory(temporarilyintheRAM).Savethedatatothe
harddrivewiththeSavebuttononthetopmenuiconpanel ,
andtypethefilename.(Note:Alltheimages(as*.tiff)willbesavedunderthesavedLeicafile(as*.lei)).
Computercrashorpoweroutagewillresultinlossofunsaveddata!SAVEFREQUENTLY!!
Ifyouwanttocreateanewfolderforanewsetofimages,clickNewicononthemenupanel.

LMW Microscopy Lab : Leica SP2 Page 9 `6/26/2012
SequentialScanningMode
Thisconfocalmicroscopycandetectupto
fourchannels(3fluorescenceand1
transmission)simultaneouslyaslongasall
theexcitationandemissionspectraof
fluorophoresarewellseparated.Imagingof
samplesstainedwithdifferentdyesthus
simplyrequiresmanipulationoflaserpower
andadjustmentofthedetectorbandwidth.
However,oftenexcitationofone
fluorophoremaycausetheemissionto
appearintotherangeofanother(bleed‐
through)orcanbeinducedbyneighboring
laserlines(cross‐talk),bothofwhich
producefalsesignals.Toavoidthese,the
sampleshouldbescannedsequentiallyby
collectingonefluorophoresignalatany
giventime.Therearetwowaystosetup
sequentialscanmodedependingon
fluorescenceofyoursample.
Toscansequentiallythesamplewithoutneedtouse405nmUVlaser(noDAPIstaining):
1. Choosethebeamsettingparameter
foryourfluorophores(forexample,
GFPandDsRED)byselectingtheLeica
FITC‐TRITCfromthepresetlist).Itwill
setlaserlines,dichroicmirror,and
PMTsforsimultaneousimagingofthe
twocolors.

2. Setupaconditionforone
fluorophore(i.e.FITC)byonly
activatingandadjustingthelaserlevel
andthePMT(i.e.decreasethe561
nmlaserpowerto0%anduncheck
thePMT2Activebox).

3. ClickSavebuttonbontheBeamPath
Settingpanelandtypeanameforthis
setupasyourownsetting(i.e.“GFP‐
seq”).ClickOKandthesettingwillbe
intheUsersetlist(Uinfrontofthe
settingname).

4. Repeatthesameprocessfortheotherfluorophore(i.e.TRITC)andsavetheconditionasanothersetting
(i.e.“DsRED‐seq”).MakesuretouncheckthePMT1Activecheckboxandlower488laserlevelto0%.

b

LMW Microscopy Lab : Leica SP2 Page 10 `6/26/2012
5. ClicktheSeqbuttonin the
BeamPathSettingwindow.The
Sequentialscansettingswindowc
willappear.Addthefluorophore
acquisitionsettingoneatatimeby
selectingitfromthepresetlistmenu
andthenclickAddbuttoninthe
Sequentialscansettingswindow.

6. Keepthescanmodedat“between
lines”.DonotclosetheSequential
scansettingswindow.
Youcansavethissequential
acquisitionsettingsbySavebutton
e intheSequentialscansettings
window.

7. Starttheacquisitionbyclicking
SingleScanorSeriesbutton
dependingonyourimaging
condition.

























c
d
e

LMW Microscopy Lab : Leica SP2 Page 11 `6/26/2012
Toscansequentiallythesamplewithneedtouse405nmUVlaser(DAPIstaining):
DuetothehardwareforUVlaser,
sequentialscanningofaspecimen
containingUVdye(i.e.DAPIorHoechst)
needsadifferentconfiguration.Hereis
anexampleofspecimenstainedwith
DAPIandFITC.
1. ForsettingupDAPI,doubleclickL‐
405onlyfromthepresetlist.Itwill
makeUVlaserandPMT1active.
StartscanningbyclickingContinous
button.
2. Whilescanning,adjusttheimage
withPMT1knobandOffsetknob
(andlaserpowerifnecessary).
3. Stopscanningandselectthenumber
oflineaveragingwiththeLi.Abutton.
Savethesettingasdescribedabove
(forexample“trainingblue”).
4. Next,doubleclickonL‐488DDfrom
thepresetlistanditwillmake488nm
laserandPMT2activeandturnoffUV
laserandPMT1.Startscanningand
adjusttheimagequalitywithPMT2
knobandOffsetknob(and488nm
laserpowerifnecessary).
5. Stopscanningandselectthenumber
oflineaveragingwithLi.A.buttonas
above.Savethesettingas“training
green”.
6. ClickSeqbutton andadd
“trainingblue”and“traininggreen”
settingfromtheUser(U)list.
7. Selectbetweenframesfromthe
Modedrop‐downlistandcheck
theLine‐Averageboxfromthe
Parameterlist.
8. ClickSingleScanorSeriesbutton
toacquiresequentialimage
capturing.
9. Tofindanewregionforanew
image,closetheSequentialScan
Settingswindow.Double‐clickon
oneofthesettingyoucreated.ClickContinuousbuttonandfindanewregionofinterest.
10. Onceyoufindaregion,stopscanning.ClickSeqbuttontobringupthesequentialsettingandacquire
sequentialimagingwithSingleScanorSeriesbutton.

LMW Microscopy Lab : Leica SP2 Page 12 `6/26/2012
AcquiringZ‐seriesimages.
Z‐seriesallowsacquisitionofopticalsectionsthroughthecertainvolumeofyoursamplethatcanbeusedfor
making3Dimages.
1. Tosetthezrange,pressContinuousbutton,turnZ‐POSknobincounterclockwisetofocusontoaspecificz
positionofyoursampleandclickEndbutton(itwillappearpressed).
2. TurnZ‐POSinoppositedirection(clockwise)tofocusonadeeperregionofthespecimenand
clickBeginbutton(pressed).
3. ClickStopbuttontostopscanning.
4. (optional)ClickSeriesbuttontocheckthethicknessofthez‐seriesanddeterminethe
numberofopticalsections.
5. ClickSectbuttontoselectthepredeterminednumberofopticalsection,ortoselectOthers…
6. SelectingOthersbringsupaz‐configurationwindowtodeterminethenumberofsections
andspacing(stepsize)between
sections.Enteradesiredstepsize
(~1µmfor63xobjective)andclick
Calculatebuttonnexttothe#
Section.Itwillautomatically
calculatethe#ofsections
accordingtheStepsize.PressOK.
7. Choosethenumberofaveraging
usingLi.A.button andclick
“Series”button tobegin
Z‐seriesstackimaging.
8. Withseriesbuttonsintheviewer
window,youcanviewthe
individualsectionsorprojectall
thesectionsintoone2Dimage.
ChooseMax.(maximumpixel
projection)orAvg.(averagepixel
projection).
WithSect.Button,youcan
visualizecross‐sectionofthe
specimen.

LMW Microscopy Lab : Leica SP2 Page 13 `6/26/2012
AcquiringTimeSeriesimages.
Timeseriesallowstime‐lapseimagingoflivesamplestostudythechangesanddynamicsofyourobjectof
interest.Thetime‐lapseimagescanbesavedasmultipleTifffilesandAVIfilesforplayinginmovieprograms.
1. Tosetobtaintime‐
seriesimages,
selectscanModeas
xyt.Itwillactivate
Timebuttons
.
2. ClickTimebutton
toopenthe
time‐lapsesetting
windowandsetthe
parameterssuchas
thetimeintervalbetweenframes
andthenumberofframes.
3. Checkoneofthreeparametersand
enterthedesirednumbersintothe
othertwoparametersHitEnterkey
onkeyboard,whichwillcalculate
thevalueforthechecked
parameter.(forexampleshownin
right,check“CompleteTime”and
enter5secforTand100for
Frames.Enterkeywillcalculatethe
CompleteTimeastotaltimeof8min15s.)
4. ClickApplybutton.
5. ClickSeriesbuttontostartthetime‐lapseseriesacquisition.
(Option)ClickonAverorLi.A.buttontosetthenumberofaveragingperframe.Note:Ifyourobjectof
interestchangesormovefasterthanscanningspeed,averagingwillgeneratedistortedorghostimages.
Toincreasethesignal,useslowerscanspeedbyclickingSpeedbutton.
Set time
interval
Set the number
of frames

LMW Microscopy Lab : Leica SP2 Page 14 `6/26/2012
Processingandexportingimages:Theoriginalimagesaresavedassingletifffiles;forexampleatwo‐color
channelimageissavedintwotifffiles‐‐oneforeachchannel.Youcansaveacurrentscreenimageafter
imageprocessingsuchasbrightness‐contrastenhancementandmergingchannels(usefulforpowerpoint
presentation).

1. Adjustimagesandputannotationsyouwant,suchasscalebar.
2. ClicktheDisplaybutton andselect1:1todisplayimageinexactpixeldimension.
3. Clickontheimageyouwanttoexportandright‐clickonthemousetobringthecontextmenu;Send
To>Experiment>Selection(raw)/Selection(snapshot)/All(snaphot).Selectoneoftheseoptionsandit
willexporttoanewimagefile.
Selection(raw):exportthecurrentselectedimageonly(noannotationsincluded).
Selection(snapshot):exportthecurrentselectedscreenimagewithallannotations.
All(snapshot):exportallthescreenimageswithannotations.

4. Fortimeseries,youcangothroughorplaythetimelapsemoviewithsequencebuttons(First,Next,Play,
Prev,Last).ForZ‐series,MaxorAvgbuttonsprojectsthedifferentsectionsintoaprojected2Dimage.You
canalsovisualizethecross‐sectionofthespecimenwithSect.button.
5. Savethe*.leifolderfilewheneveryouaddnewimages.

LMW Microscopy Lab : Leica SP2 Page 15 `6/26/2012
SavingImages.
Thisisaveryimportantforyoureffortanddata.Computercrashorpoweroutagewillresultinlossof
unsaveddata!SAVEFREQUENTLY!!Tosavetheimagingdatafiles,click“Save”buttonatthetopofthe
window.SelectDdriveandusersfolder.Createyourfolder,renametheExperiment#andsave.

Sincethestoragememoryinthecomputerwillbefilledupquicklyandthislackofharddrivespaceprevents
imageacquisition,alluserfileswillbedeletedaftertwoweeks.Besuretocopyyourfilestoaflashmemoryor
CD/DVDdisks.

Shut‐downProcedure
Pleaseenteryourtimeofuseinthelogbookandnoteanyproblemsandsuggestionsduringyourtime.
1. Cleantheoilfromtheobjectivesonlywithlenspaper(notKimwipe).Cleanthemicroscopeandstage.
2. ClosetheLCSprogram.
3. CopyyourdatafilestoaflashdriveorCD.Usersareresponsiblefortheirownfiles,asthereisno
automaticbackupatpresent.
4. Logoffthecomputer(notturningoffthecomputer)andturnoffthemicroscopecontrolbox.
5. Ifthereissomeonewhowantstouse,leavetheinstrumentON.
6. Ifyouarethelastuseroftheday:
1)Turnoffthemercurylampb.

2)MinimizethepoweroftheAr/ArKrlevelc for>3minbeforeturningitoff.

3)Around3minafterminimizingtheAr/ArKr
level,turnthekey dto“off”andwaitfor10
mintocooldownthelaser.Itisreadytoturn
offtheredbuttone switch.

4)FortheHe/Ne
633laser,turnthe
key ftooff
position.

5)TurntheMelles
GriotDPSSlaseroff
bypushingtheOFF
(red) gbutton.

6)For405nmlaser,
turnthekey hto
offpositionand
thenturnoffthe
redbutton i.

7)ExitwindowsandshutdownthepowerforPCstand j,theScanner/LaserHeNe1),andtheLaser
Ar/Krewiththeredbuttons.Finally,turnoffthemicroscopecontroller1!.
b
c
d
e
f
g
h
i
j
1)
1!