Leica SP2 Bedienungsanleitung

LMW Microscopy Lab : Leica SP2 Page 1 `6/26/2012

ManualforLeicaSP2ConfocalMicroscope



Enteryouname,thedate,thetime,andtheaccountnumberintheuserlogbook.



Thingstocheckbeforestart‐up.



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.



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.



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.



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).



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.



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)



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.



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.



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.



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).



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!



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.



SettingupforConfocalLaserScanning.



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.

1$ 1%

LMW Microscopy Lab : Leica SP2 Page 5 `6/26/2012

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.



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).



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

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).



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).



Pseudocolor    Hi‐Lomonochrome

1$ 1%

LMW Microscopy Lab : Leica SP2 Page 7 `6/26/2012

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.



Andere Handbücher für SP2

Andere Leica Mikroskop Handbücher

Leica

Leica Sp5 WLL STED Betriebsanleitung

Leica

Leica StereoZoom Bedienungsanleitung

Leica

Leica ES2 Bedienungsanleitung

Leica

Leica VOH IV Bedienungsanleitung

Leica

Leica F12 I Bedienungsanleitung

Leica

Leica S4 E StereoZoom Bedienungsanleitung

Leica

Leica DMS300 Bedienungsanleitung

Leica

Leica M Bedienungsanleitung

Leica

Leica TCS SP8 MP Bedienungsanleitung

Leica

Leica MC120 HD Bedienungsanleitung

Leica

Leica DM LSP Bedienungsanleitung

Leica

Leica BM E Bedienungsanleitung

Leica

Leica TCS SP5 X Bedienungsanleitung

Leica

Leica S Series Bedienungsanleitung

Leica

Leica DM EP Bedienungsanleitung

Leica

Leica BM E Bedienungsanleitung

Leica

Leica DMLS Bedienungsanleitung

Leica

Leica SP5 II Bedienungsanleitung

Leica

Leica DM LM Referenzhandbuch

Leica

Leica S Series Bedienungsanleitung

Leica

Leica DMi8 Bedienungsanleitung

Leica

Leica ICC50 W Bedienungsanleitung

Leica

Leica MC170 HD Bedienungsanleitung

Leica

Leica Z6 APO Bedienungsanleitung

Beliebte Mikroskop Handbücher anderer Marken

Thermo Scientific

Thermo Scientific Nicolet iN10 Leitfaden

National

National DC3-163 Bedienungsanleitung

KERN Optics

KERN Optics OPE-1 Bedienungsanleitung

OPTIKA MICROSCOPES

OPTIKA MICROSCOPES B-150D Bedienungsanleitung

Prior

Prior Lux POL Bedienungsanleitung

Opitec

Opitec Smartscoop 115615 Bedienungsanleitung

KERN

KERN OPO-1 Bedienungsanleitung

Topcon

Topcon SP-1P Bedienungsanleitung

Motic

Motic PA43 Bedienungsanleitung

MshOt

MshOt MZ62 Bedienungsanleitung

KERN Optics

KERN Optics OZR-5 Bedienungsanleitung

VWR

VWR VisiScope 384 Series Bedienungsanleitung

Nikon

Nikon ECLIPSE E200 POL Bedienungsanleitung

ThermoFisher Scientific

ThermoFisher Scientific Continuµm Bedienungsanleitung

ThermoFisher Scientific

ThermoFisher Scientific Continuµm Bedienungsanleitung

Olympus

Olympus SZ61 Bedienungsanleitung

AO Cycloptic Bedienungsanleitung

Celestron

Celestron 44345 Bedienungsanleitung