ICBM Spinning Disc Microscope System User Notes

Transcription

1 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly ICBM SpinningDiscMicroscopeSystemUserNotes (PerkinElmer)R2 E2 Whenyousignuponthecalendarpleaseindicate: 1)Whichobjectiveyouwilluse. 100xOilisthedefaultobjectiveonthesystem. 60xWisalsoavailable 2)Whichlaserlinesareneeded. 442,488,514,568,647nm 3)Whichdichroiccubeisneeded. CFP/YFP/CY5(labeledCFP/YFP)or FITC/Rhod/CY5(labeledrgb) 3)Ifusinglivecells,isanobjectiveheaterand/orstageheaterneeded? ICBMpersonnelwillstartthesystemupandshutitdown.Donotattemptthisyourself.Askforassistance. Facilitypersonnelwillchangeobjectivesanddichoticcubes.Donotattemptthisyourself.Youdonotwanttobeheld responsiblefordamage.askforassistance. CONTENTS GettingStarted......pg2 ObservationthroughtheOculars......pg3 RevolutionFastLZ (Fast)easier ImageWindow Rev Sutter XYZ (Slow)allowsmultifield Multifield AcquireaSubRegion DATAMANAGEMENT Thisisyourdataandyouareresponsibleforitsstorageandpreservation.TheICBMdoesnotprovidelongterm storageofuserdata. Weprovideacomputer,In Neph ToastforburningofDVDsandCDs.Pleasebringblankmediawithyou.Attheendof yourimagingsession,moveyourfilestoin Neph Toastandburn2copiesofyourdata.BlankCDsandDVDsaremuch cheaperthanthetimeandmaterialsneededtorepeatyourexperiment.afteryouhavecheckedwhetherthefileson yourcdsanddvdsarereadable,atyournextimagingsession,deletethemfromtheperkinelmerandfromin Neph Toast. 1

2 6/17/09 SC Andor iq version DRAFT fast mode only GETTING STARTED Lasers ON? (switch toward case) not on standby? (switch away from case, red arrow ) Remove stage insert. Do not bump the objective. Log in to Windows using your UserID and password. Double click the Andor iq icon to start the acquisition software. Select configuration For Basic Mode (Fast) choose (Revolution Fast LZ) For Complex Mode allows multifield, choose (Rev Sutter XYZ) If you have chosen Rev Sutter XYZ, you will sometimes get a window saying that the stage has not been registered. Check to see that the stage insert has been removed, then click START. The stage will move to its extreme positions then back to center. Replace the stage insert. You will next get a window asking you to select data path. It is best to leave this path as it is. Choose Close. Your data is saved in an internal format as you acquire images. You will save your data to another computer in a format that other programs can read at the end of your imaging session. In the Main Imaging Window Press the Select button, then choose the correct objective. (halfway down left side) Select correct optivar setting this should match the position of the optivar Unchecked = 1.0 Checked = 1.5 This must be done for the software to give you the correct pixel dimensions Optivar position shown is 1.0x. The lever to move the optivar is indicated by the large green arrowhead. To move the optivar into the 1.5 x position, gently move the lever towards the front of the microscope body as indicated by the green arrow. The large focus knob is coarse focus. The small focus knob is fine focus. The stage is moved using the joystick. Gentle motion with the joystick is preferred. Do not run the objective into the edge of the stage opening. This will damage the objective. Please be aware of the position of the objective at all times. 2

3 6/17/09 SC Andor iq version DRAFT fast mode only OBSERVATION THROUGH THE OCCULARS Transillumination Turn transillumination lamp ON and OFF with button on left side of microscope body. Control transillumination intensity with dial below the button. Turret position 5 Port position 1 Epifluorescence Turn epifluorescent illumination lamp ON and OFF using the foot pedal. If the lamp is left on, it will go off by itself after 60 seconds. Green Turret position 3 Red and Green Turret position 2 Port position 3 Set mechanical shutter to open position. Note: Mechanical shutter must be in open position for epifluorescence and for laser scanning. 3

4 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly SpinningDiscConfocalAcquisition inrevolutionfastlz (Fast) Forspinningdiscconfocalacquisition Trans illuminationlampoff,epifluorescencelampoff Turretposition5 Portposition5 InAndoriQWindow ChooseFAST Rightclicktheimagenameandchooseedit.Thiswillbetheprefix forthissessionofimages. InAcquisition/AuxiliaryDevicesWindow Undertabs Experiment Sequence LControl checkboxtoactivate EnabledandMove1st(L=wavelength) EnabledL checkboxestoactivatedesirednumberofchannels Emission check Exposure check msisgoodstartingpoint Z controlenabled check ComputedParameter Planes CurrentL chosenchannelisactivechannel channelyouwillsee whenlivescanning Setup excitation/emission Exampleshows Excitation488/Emission525/50 Excitation568/Emission600/45 Excitation647/Emission700LP Toselectexcitationwavelength,checkdesiredbox ToselectEmissionfilter,choosefromdropdownmenu Position Filter 0 OPEN 1 525/ / longpass 4 triple 5 OPEN 6 485/54, / /30 4 Use8,9for CFP/YFPFRET Whendone PressRecord.

5 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly LaserIntensitySettings inrevolutionfastlz (Fast InAcquisition/AuxiliaryDevicesWindow Undertabs Experiment Wavelength Excitation Adjustlaserlevels.(20%isgoodstartingpoint.) Laserforcurrentchannelwillbechecked.Goaheadand setaninitialintensitylevelforallofthelasersthatyouwill beusing.pressrecord. LeavetheEditLaserintensitywindowopensothatyoucan finetunethelaserintensityeasilylater. 5

6 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly CameraControlSettings inrevolutionfastlz (Fast) InAcquisition/AuxiliaryDevicesWindow Undertabs DV897_BV2302Settings Exposure. Window512:512 BinningX=Y,1x1 ShutterClose Framestobeaveraged unchecked FastLambdaZ checked DisplayControl displayeveryframe EMGainenabled Checked Valuesgenerallybetween ok Ifyouhaveasuperbrightsample,you mayneedtosetemgainlower. Pre AmpGain1.00 ExposureTime beginwith ms Timeshowninthiswindowisforactive channel. Pressrecordtokeepchanges. NoteCamerastatusframeatbottomofwindow. Tempmustbe 70forimaging.Thisnormallyjust takesafewminutes.thetemperatureisusually downto 70bythetimeyoureachthispointin setup. 6

7 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly OPTIMIZEACQUISITIONSETTINGS inrevolutionfastlz (Fast) Whatintensitylevelshouldyourimages have?itdependsonthesampleandit dependsonthetypeofexperimentthatyou aredoing.ifyouaredoingliveimagingover time,toreducephotodamageand photobleaching,youwantthelowestlaser powerandlowestexposuretimethatwill giveyouanadequateimage.youmay decidethatyoucanworkwithaslowasan intensityvalueof300.withfixedsamples thatyouwillonlyimageoncemorelaser powerandlongerexposurescanbeused. Typicallyintensityvaluesforfixedsamples varywidelybutingeneralintensityvaluesin therangeof willproduceagood image.avoidsaturation. Returntothiswindow. InAcquisition/AuxiliaryDevicesWindow Undertabs Experiment Sequence PresstheLivebuttonatthetopofthewindow.Themicroscope willbeginscanning,usingthechannelchosenincurrentl.the LivebuttonwilltogglewhenpressedtosayIdle.PressIdleto stopscanning. AtthebottomoftheImageWindow,thereisacheckboxlabeled Autoscale.Thisshouldbechecked. Withautoscalechecked,dimimagesappeargrainyandthe backgroundisgreyinsteadofblack.oversaturatedimageslose detaininbrightareasandmayevenappearsmeared(thisis calledblooming)whenapixelisoverfilledwithlight. DimImages Increaselaserintensityand/orincrease exposuretime. SaturatedImages Decreaselaserintensityand/or decreaseexposuretime.ifimageisstilltoo bright, decreaseemgain. AdjustLaserIntensityandExposureTimeforeachchannel.Press Recordtokeepsettings. 7

8 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly Z SCAN AcquiringanImageVolume inrevolutionfastlz (Fast) InAcquisition/AuxiliaryDevicesWindow Undertabs Experiment Sequence Z controlenabled checked Z controlmovefirst unchecked ComputedParameter Planes FastScan checked Stepsize typeinastepsize.agoodplacetostartis0.5µm. CurrentZ PressGoCentre Thiscentersthepiezoinitstravel 100µm. Q:HowdoIchooseastepsize? A:Itdependsonthesample. Somestartingpointsare: 1/2theopticalresolutioninz(~0.5µm) or z=thex,ypixelsize But,dependingonwhatyouaretryingto visualize,youmaybeabletotakez steps2xthisormore. Commonlyusedstepsizesonthissystemrange from µm PressLive.Findandfocusoncellofinterestusingfocusknobonmicroscopebody(Donotuseknobonscopeafterthis step) UsingslidernexttoCurrentZorarrowsunderStepthroughplanes,movetothestartpositionforthe3Dvolume,then pressstart.movetotheendposition,thenpressend. InAndoriQWindow PressRun. ThefirsttimethatRunispressed,adialogueboxopensandsaysthattheshutter isnotopen,doyouwanttoopentheshutter.clickyes. Thisdialoguewillnotappearagaininthecurrentsession. ClickOKtostartprotocol. TheimagesareautomaticallysavedintheImageList.Youwillexportallofyourimagesattheendofyoursessionina formatreadablebyothersoftware.ifyouhaveseveraldifferentsamples,youshouldrenameyourimagesasyou acquirethem.seeimagelist,page10. 8

9 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly TIMELAPSE inrevolutionfastlz (Fast) SetupparametersformultichannelandZ scan. IntheAndorIQwindow,rightclickonRepeat,then chooseedit.typeinnumberofrepeats. IntheAndorIQwindow,rightclickonInterval.Default Intervalisnopausebetweenscans.Typeindesired interval. 9

10 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly IMAGELIST ChoosingImagelistonthemenubar ofanyofthewindowswillopenthe ImageManger,whichdisplaysallof yourimagesalongwiththeir acquisitioninformation. RenameImage AndorIQsoftwareautomatically namesandsavesimages.stacksand timeseriesarenamedbeginningwith theprefixthatyouassignedatthe beginningofyourimagingsession (usuallythedateandanexperiment identifier)andhaveanumber appended.snapsarenamedwiththe activechannelnamewithanumber appended. Ifyouhavemorethanonesample group,eitherchangetheprefixfor eachsamplegroup,orrenameyour filesasyouacquirethemtoavoid confusionlater. WhentheImageListisopened,the mostrecentimageacquiredappears atthetopofthelist.ifadditional imagesareacquiredwiththelist open,theydonotautomaticallyappearonthelist.clickrefreshandthemostrecentimageacquiredwillappear atthetopofthelist. TheRenameImagebuttonallowsyoutospecifyadifferentnameforthecurrentlychosenimage.Selectanimageby clickingonitintheimagemanagerwindow.clickrenameimage.typethenewnameintothetextboxandclick OK.ThenewnamewillnowappearintheImageManagerwindow. DeleteImage TheDeleteImagebuttonwilldeletecurrentlyselectedimagesfromtheImageManager. SaveImage Ifyouareanewuser,logontotheCD/DVDburningmaching,In Neph Toast.Loggingonwillcreateyouruserfolderon In Neph Toast.Nowlogoff.ItisnotnecessarytostayloggedonwhilesavingyourimagestoIn Neph Toast. 10

11 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly ImagesaresavedwithintheAndorsoftwareduringacquisition,buttheyareinaformthatcanonlybeaccessedfrom withintheandorsoftware.attheendofyourimagingsessionyoumustsaveyourimagestoanothermachinein TIFFformat.TIFFformatisreadablebyalargenumberofotherimagevisualizationandprocessingprograms. Filescanbesavedoneatatimeorasagroup.AndorIQsoftwarehasdifferentdialogueboxesforindividualand groupsaves. SaveImage MultipleImages Highlightthefilesthatyouwishtosavebyclickingonthenamesin theimagemanagewindow.tohighlightmorethanonefile,hold downctrlandcontinueselectingfiles. ClickSaveImage.TheMutiSaveOptionsandSaveMultiplefiles dialoqueboxeswillopen. ChooseTIFF(singleimagefiles)*.tif Uncheckthedeletetheimagesbox. (Deletethissessionsimagesthenexttimethatyouusethespinningdisc microscope,afteryouhavemadeacopyoncd/dvdoronyourownlab machine.) ClickOK TheBrowseforFolderdialogueboxwillopen.BrowsetoyourfolderonIn Neph ToastandclickOK. GotoIn Neph ToastandburntwocopiesofyourdataontoCDorDVD. Spotcheckthatyoucanopenyourimageswhenyougetbacktoyourown lab.deleteyourdatafromtheimagemanagerandfromin Neph Toast duringyournextimagingsession. SaveImage singleimage Highlightthesinglefilethatyouwishtosavebyclickingonthename intheimagemanagerwindow. ClickSaveImage. Browsetowhereyouwanttosavethefiles In Neph Toast ChooseTIFF(singleimagefiles)*.tif Givethefileaname(filename.tif) ClickSave 11

12 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly IMAGEWINDOW(windowislabeled AndorCameraWithFastZandWavelength) IntensityScaling AtthebottomoftheImageWindow,thereisacheckboxlabeledAutoscale.Thisshouldbechecked.TheAndorcamera acquires14bitimages(2 14 levelsofgrey).mostmonitorscanonlydisplay8bitimages(2 8 levelsofgrey).whenyouclick autoscale,thesoftwarewillscalewhatevernumberofgreylevelsofsignalthatyouhave,upordown,tofitwithinthe8 bitdisplay.thisdoesnotchangeyourdata,itjustmakesvisualizationeasier.movethemouseovertheimagetoseethe intensityvalueforthatareaoftheimage.thex,ycoordinatesandtheintensityvaluewillbedisplayed atthebottomoftheimagewindow.todisplayahistogramoftheimageintensityvalues,press: toopentheimagecontrastcontroldialoguebox. 12

13 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly ColorandColoroverlay Whenyouhaveacquiredamultichannelimage,thesecontrolswillbeavailableinthelowerleft oftheimagewindow.whenmultiisunchecked,onechannelatatimeisdisplayedandyoucan togglebetweenchannelsbypressingthebuttons.youcanchangethedisplaycolorforthe channelsbyrightclickingonthebutton,thenchoosingacolorfromthelutmenu.tooverlay twochannels,checkthemultiboxandpressbothbuttons.keepinmindthatthisisdisplay coloronlyanddoesnotaffectthedata.dataisacquiredasgreyscale,intensityvalues. ScrollingthroughZ,TimeandFields InthelowerleftoftheImagewindow,therearetabsthatcorrespondtothedimensions acquiredinanimage. ToscrollthroughZ,clickonthetablabeledZandmovetheslider. Toscrollthroughtime,clickonthetablabeledTimeandmovetheslider. Tomovebetweenfieldsinamultifielddataset,clickonthetablabeledXYandmovethe slider. 13

14 6/17/09SCAndoriQversion1.9.1DRAFT fastmodeonly AcquireaSubRegion Snapimageofspecimenwithchannelthatletsyoudefinedesiredarea. IntheImageWindowToolbar(AndorCameraWithFastZandWavelength) UsingImage ToolDefineRectangle drawarectangularroi(regionofinterest)aroundthesubregionthatyou wanttocapture PressDeviceSetupButtonandopen: Acquisition/AuxiliaryDevicesWindow DV897_BV2302Settings Exposuretabs. ClickontheWindowbutton(ItwillchangetosayFullChip) Snapimagetoconfirmthatcorrectregionissetup Acquire3D,multichannel,timelapseasusual.SubRegionwillbeappliedtofutureimages.Thisisusefulwhenyouwant tospeedupacquisition. Toreturntothenormal512x512,clickontheFullChipbutton.ItwillchangebacktoWindowandthesubregionisno longerselected.toclearthedefinedregion,rightclickontheimageandchoosedeleteall. 14