Leica SP2 Manuel utilisateur

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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LMW Microscopy Lab : Leica SP2 Page 2 `6/26/2012

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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LMW Microscopy Lab : Leica SP2 Page 3 `6/26/2012

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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LMW Microscopy Lab : Leica SP2 Page 4 `6/26/2012

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%.



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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