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.
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