LMIC Leica SP5 BASIC use guide (Version 2)
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10/2010 LMIC Leica SP5 BASIC use guide (Version 2) Start-up ‐ Uncover microscope, tilt illumination arm back, move objectives all the way down (objectives will be down unless you moved them up). ‐ Turn on the Hg‐lamp if you will need it. ‐ Turn on the laser power switch (switch on the right) and turn on the laser key. ‐ Turn on Scan box (middle switch). ‐ Turn on computer/microscope. ‐ Log‐on, form should come up automatically. If computer is already on click on the icon for “shortcut to iCenter application” to bring up the sign‐in sheet. o add your IU username o the account number to be charged o and the last name of the lab PI. ‐ Start software (LAS AF icon on desktop). o Be sure configuration is on “machine”. ‐ After stage initialization, move illumination arm back to upright position. ‐ You are now ready to look at something.
Transcript of LMIC Leica SP5 BASIC use guide (Version 2)
10/2010
LMICLeicaSP5BASICuseguide(Version2)
Startup
‐ Uncovermicroscope,tiltilluminationarmback,moveobjectivesallthewaydown(objectiveswillbedownunlessyoumovedthemup).
‐ TurnontheHg‐lampifyouwillneedit.‐ Turnonthelaserpowerswitch(switchontheright)andturnonthelaserkey.‐ TurnonScanbox(middleswitch).‐ Turnoncomputer/microscope.‐ Log‐on,formshouldcomeupautomatically.Ifcomputerisalreadyonclickontheiconfor
“shortcuttoiCenterapplication”tobringupthesign‐insheet.o addyourIUusernameo theaccountnumber
tobechargedo andthelastnameof
thelabPI.
‐ Startsoftware(LASAFiconondesktop).o Besureconfigurationison“machine”.
‐ Afterstageinitialization,moveilluminationarmbacktouprightposition.‐ Youarenowreadytolookatsomething.
Microscope
‐ Itonlyworkswellwhenitisclean!Pleasedoyourparttokeepitclean.‐ Insertslideinslotinz‐galvostageinsert.(Samplepointsdowntowardobjective).Ifyouwillneed
oil,putasmalldroponthecoverslipbeforeyouloadyourslide.o Avoidgettingoilonthenon‐oilobjectives–ifyoudo–askforhelp,wewillcleanthe
objective.‐ Findyoursample–Istartwiththe10Xobjective(ithasalongworkingdistanceandwon’ttouch
theoilwhenitisintherightfocalplane).
ScopeOperation–lotsoflittlebuttons.Softwarecontrolsmostthings,manyareautomatic.
LeftSide:
1. ChangeTL–changestransmittedlightfrombrightfieldtoDIC.
2. ChangestoFluorescencelightpath–Canalsodothisbyjustselectingafiltercubeonfrontpanel.
3. Changesfiltercubes4. ChangeCS–changestoconfocalmode5. Changesaperturediaphragm
(automaticallyadjusted)6. Lightintensityadjustment7. Fielddiaphragm–howmuchoffieldof
viewyousee.8. Brightfieldlightswitch
Center:
1. Alllighttoeyepiece.2. Cameraportselection.Willhappen
automatically.3. Magnificationselection–wedon’thave
thisfeature–leavebuttonalone.4. Ifyoupress#3,pressthisbuttontoget
back.5. Fluorescenceshutter(makesnonoise).6. GreenFluorescencefiltercube7. RedFluorescencefiltercube
8. Analyzercube9. Emptyslotforimaging10. DAPIFluorescencefiltercube11. Emptyslot
RightSide:
1. Pressandholdtomoveobjectiveuptosetposition.
2. Setbutton.Tosettopposition,pressandholdsetbuttonwhilepressingtopbuttononcetoclear,againtoset.
3. Pressandholdtomoveobjectivedown.Pleasedon’tsetbottomposition.
4. Changeobjective(toleft)5. Changeobjective(toright)6. Switchfromdrytoimmersion
objective.
Stage/Focuscontroller(salt&peppershaker):
• Top–stagemovementinX• Bottom–stagemovementinY• Back–Focus• Buttonsonleft–togglebetweenfastand
precisestagemovement.• Buttonsonright–togglebetweenfineand
coursefocus.
LASAFSoftware
‐ Configurationo Turnonlasersyouneed.o SetlaserpowerforArgonlaser(20%isagoodplacetostart).o Lookatobjectivelist–listedforeachisthemax.resolution.Keepthisnumberinmind
whenzooming,etc.(ItisOKtooversampleabout40%).‐ Acquisition
‐ o Establishlightpath.
TheeasiestwayistothisistoselectpreloadedLeicasettings(redarrowabove)thataresimilartoyourneeds.Thiswilldomanythings.Itturnsonlasercontroller,laseroutput.PMTs,andsetswindows.
ThenclinkonfluorophoreselectionbarforeachPMTandpickdyeyouactuallyused.
Adjustcollectionwindowsasnecessary.Makesureyouarenotcollectinglightoverlaseroutput!Windowshouldbeatleast10nmawayfromlaserwavelength.
o Selectobjective(redcircleabove).Payattentiontowhatimmersionmediaeachobjectiverequires.
o PickAcquisitionMode(xyz=“normal”).Theothersdosomenicethings.Askifyouneedhelp.
o Format–pickimagesize.Keepinmindmax.resolutionofyourobjective–andremember,smallerisfaster.
o Speed–defaultis400Hzandisagoodplacetostart.600Hzisasfastasitcanscanwithoutzoomingin.Slowerspeedsequalsbetterqualityandmorebleaching.
o Seewhatyouhave–hitlivebutton–youshouldgetanimage.Taketimetooptimizeimage–maybeonaspotyoudon’treallycareabout.
Focus–Usethez‐controlknoboncontrolpanel–thisistheonlyfocusthecomputerunderstands.
Ingeneral,highgainandlowlaserpowerisgood.Iftoograiny,turnlaserup.Startwithgainaround1000.
Checkforbleed‐through(turneachlaserdown). Turnonquick‐lookuptable(rainbowicononrightscreen).Bluepixelsaremaxed
out(white),greenpixelsareblack.Forbestimagestryafewbluepixelsandafewmoregreen(=mostdynamicrange).
ClickZoom,drawboxonimagewhereyouwanttozoom(remembermax.resolution).
Pinholeisautomaticallysetatoptimum–canopenitifnecessary(morelight,lessconfocaleffect).
Ifdesired,setZ‐series,findtopandbottom. HitCaptureforsingleimage,orStartforseries.
‐ ClickExperimenttab.HitSaveAll(Untilyouhitsaveall–yourimageswillbelostifthecomputercrashes)
ShutDown
‐ Firstturnoffkeyswitchforlasers.Leavelaserpowerswitchon!Thisturnsthelasersoffbutleavesthecoolingfansontoavoiddamaginglasers.
‐ Cleanobjectives!‐ Transferdatatoserverandsign‐out.
o Savealldatatotransfertoonefolder(youcanonlytransferonefoldertoserverperlog‐in)
o click“selectdatadirectory”buttonandnavigatetoyourfolder.
o Fillinatleast“ProjectTitle”soyoucanfindyourdownloadontheserver.
o Hit“Sign‐Out”
Pleasedeleteimageswhenyouaresuretheyaresafelytransferredtotheserver.Youshouldneverhaveimagesonthescopecomputerformorethan12sessionsofimaging
‐ Shutdowncomputer(ifyouneedtoworkonthecomputer,youmayleaveiton).
‐ Turnoffscanhead.‐ TurnoffHglamp.‐ Coverscope(don’tcoverHglamp).‐ Cleanuptables,etc.‐ Whencomputerscreensaredarkandthelaserfancable/ventstopshaking(lasercoolingfanhas
stopped)itissafetoturnoffpowerswitchestolasersandcomputer.
