Leica SP2 Manual de usuario

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.



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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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).



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.



Otros manuales para SP2

Otros manuales de Microscopio de Leica

Leica

Leica DVM6 Manual de usuario

Leica

Leica TCS SP8 MP Manual de usuario

Leica

Leica MATEO TL Manual de usuario

Leica

Leica M Manual de usuario

Leica

Leica DM750 M Manual de usuario

Leica

Leica DM500 Manual de usuario

Leica

Leica DM2000 Manual de usuario

Leica

Leica CME Manual de usuario

Leica

Leica ICC50 E Manual de usuario

Leica

Leica M844 Manual de usuario

Leica

Leica M165 FC Manual de usuario

Leica

Leica DM4000M Manual de usuario

Leica

Leica IC80 HD Manual de usuario

Leica

Leica DM L Manual de usuario

Leica

Leica TCS SP5 X Manual de usuario

Leica

Leica SP5 confocal Manual de usuario

Leica

Leica K3C Manual de usuario

Leica

Leica DMI6000 B Manual de usuario

Leica

Leica MC170 HD Manual de usuario

Leica

Leica F12 I Manual de usuario

Leica

Leica DMIRE2 Manual de usuario

Leica

Leica SP8-X Manual de usuario

Leica

Leica MATEO TL Manual de usuario

Leica

Leica DM300 Manual de usuario

Manuales populares de Microscopio de otras marcas

Thermo Scientific

Thermo Scientific Nicolet iN10 Guía

National

National DC3-163 Manual de usuario

KERN Optics

KERN Optics OPE-1 Manual de usuario

OPTIKA MICROSCOPES

OPTIKA MICROSCOPES B-150D Manual de usuario

Prior

Prior Lux POL Manual de usuario

Opitec

Opitec Smartscoop 115615 Manual de usuario

KERN

KERN OPO-1 Manual de usuario

Topcon

Topcon SP-1P Manual de usuario

Motic

Motic PA43 Manual de usuario

MshOt

MshOt MZ62 Manual de usuario

KERN Optics

KERN Optics OZR-5 Manual de usuario

VWR

VWR VisiScope 384 Series Manual de usuario

Nikon

Nikon ECLIPSE E200 POL Manual de usuario

ThermoFisher Scientific

ThermoFisher Scientific Continuµm Manual de usuario

ThermoFisher Scientific

ThermoFisher Scientific Continuµm Manual de usuario

Olympus

Olympus SZ61 Manual de usuario

AO Cycloptic Manual de usuario

Celestron

Celestron 44345 Manual de usuario