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 MC120 HD Manual de usuario

Leica

Leica S Series Manual de usuario

Leica

Leica DVM5000 Guía del usuario

Leica

Leica StereoZoom Manual de usuario

Leica

Leica M205A Manual

Leica

Leica M500 OH3 Manual de usuario

Leica

Leica ES2 Manual de usuario

Leica

Leica DM3 XL Manual de usuario

Leica

Leica M60 B Manual de usuario

Leica

Leica SP8 AT CIAN Manual de lista de piezas

Leica

Leica ARveo Manual de usuario

Leica

Leica DM300 Manual de usuario

Leica

Leica MicroStar IV Manual de usuario

Leica

Leica DM1000 LED Manual de usuario

Leica

Leica DM500 Manual de usuario

Leica

Leica OH4 Manual de usuario

Leica

Leica DVM6 Manual de usuario

Leica

Leica VOH IV Manual de usuario

Leica

Leica MATEO TL Manual de usuario

Leica

Leica DM E Manual de usuario

Leica

Leica M620 F20 Manual de usuario

Leica

Leica EZ5 Manual de usuario

Leica

Leica DM750 M Manual de usuario

Leica

Leica Stellaris 5 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