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.