Olympus Fluoview FV1000 User manual

OlympusFV1000Userguide August3,2011
Olympus FV1000 MPE Microscope User Guide
A.SystemStartUp
1. Signintothelogbook.Indicatethelaserlinesyouwilluse;visiblelaserandIRlaser.
2. Turnonmercurylamppowersupplyb. 
3. Turnonmicroscopecontrollerandscanner.
Microscopecontroller‐switchONc
Scanner–switchONandturnKeytoONd.
4. Turnonlasercombinersef.
5. Turnonlaserpowersupplies
Multi‐Arg:SwitchtoONandturnKeytoON.
HeNeh:TurnKeytoON.
6. Turnoncomputeriifitisnoton.
7. Enterusername/passwordtologontoWindowsXP.
8. StarttheFV10‐ASWprogramandenterUserIDand
password.
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OlympusFV1000Userguide August3,2011
B.OutlineofAcquisitionSetting/ImageAcquisition/ImageViewerWindows

OlympusFV1000Userguide August3,2011
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C.ViewingwithTransmittedorEpifluorescenceLight
Beforescanningthesamplewithlaserlight,lookat
thesampleandfindtheregionofinterestfirstwith
eithertransmittedorfluorescentlight.
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Clickontransmittedlightbuttonj fortransmitted
lightviewingoronepifluorescencelightbutton1)for
fluorescenceviewing(Beforeclickonthelightbutton
youwillused,clickoffthepressedbuttonfirst).
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Turnthefilterwheel1!to#6(DICT)fortransmitted
lightviewing,orto#3(GreenNIBA),4(RedWIG),or
5(DAPI)forfluorescentlightviewing.Openthe
shutter1@.
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MAKESURETHESLIDEANDCOVERGLASSARECLEANANDSEALED.Place
theslideonthemicroscopestage.Focusontothesample
withthefocusknob1#(fineadjustment)orfocusbuttons1$
(fastfocusing)ontheremotecontroller(Note:thefocusknobonthemicroscopedoesn’tworkiftheremote
controllerisused).Theextent(fineorcoarse)ofmovementbyfocusknob1#canbeselectedalternativelyby
pressingF/Cbutton1%.
PressEscbutton1^tomovetheobjectivecompletelyawayfromthesampleandpressagaintobringthe
objectivetotheoriginalposition.
Adjustthetransmittedlightlevelwiththelampcontroller1& ontheImageAcquisitioncontrolwindow.
Whenyouarereadyforlaserscanning,turnthefilterwheel1! to#1position(LaserScanninglabel).
Folder/File
Explorer
Datamanager:displays
metadatacontaining
acquisitionparameters.
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OlympusFV1000Userguide August3,2011
D.ImageAcquisition
Clickoffthelightsourcebuttontoturnoff
thenon‐laserlight.
ClickonDyelistbuttonanddoubleclick
onthefluorescentdyesyouwanttouse(e.g.
FITCandTRITC)fromthelist.ClickApply
buttonb(ifAssignDyeManuallybuttoncis
checked,uncheckbeforepressingApply
button).Itwillactivatethelaserlinesandset
detectionchannelsupaccordingtothedyes
youselected.
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ChooseImagesize(default‐512x512)from
Sizepanel d.
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Pressbutton(ifnotpressed)to
automaticallyadjustHVandOffsetvalues
accordingtothescanspeedchange.Click
buttontoscanthesample.Itwill
scanfastat2us/pixelandshowlow‐quality
(pixelated)imageinaLiveViewwindow.
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Whilescanning,focusontotheregionofinterestwiththeremotefocuscontrollerorbyclickingarrowhead
buttonsinthemicroscopewindow(largearrowheadmovetheobjectivebytheextentsetbyStepSizee,
andthesmallarrowheadsmovestheobjectivebythehalfofthelargearrowheadstepsize).
ClickStopbutton tostopscanning.
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SettheLaseroutputlevelf(settheMulti‐Arlaser
to1‐5%andtheHeNelaserto1‐25%,depending
onyoursamplecondition,butnolowerthan1%).
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Setthescanspeedg to10~12.5us/pixelandclick
XYrepeatbuttontoscan(youcanscan
imagefasterorslower,dependingonyoursample).
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Detector(CHS)ChannelSetting:Adjustthe
brightnessbyHV(andGain)andbackgroundblack
level(Offset)ofindividualchannels.KeeptheHV
valuebelow700(beyond700,thebackground
noisewillshowup).
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OlympusFV1000Userguide August3,2011
YoucanoptimizetheimagequalitywithHi‐LoLUT(Look‐UpTable).ClickLUTbuttontobringtheLUT
controlwindowandselectChannelandclickHi‐Lobutton(Ctrl‐H
shortcutkeywilldothesamewithoutopeningLUTwindow).Now
theimageisdisplayedusingthisspecificlook‐uptablewherered
pixelsrepresentintensitybeyondscale(saturated)andbluepixels
represent0pixelvalue.Tomaximizethesignal‐to‐noiseratioofthe
image,adjusttheacquisitionsetting(laserpowerlevel,HV,Offset)
suchthattheimageshowsafewredandbluepixelswhileyouare
scanning.
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Oftenexcitation/emissionprofilesoffluorophoresyouareusingmay
beclose,sotherecouldbepossiblebleed‐throughofafluorophore
emissiontoneighboringchannel.Tominimizethis,thedetecting
rangeofeachchannelcanbeadjustedbychangingSpectralSetting.
Clickon,itwillbringSpectralSettingwindow.Changetherange
ofspectrumforeachchannel(CHS1andCHS2)bysliding,widening,
ornarrowingthetabsorarrowheads.
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UseAreapanelhtorotateandzoomtheimagingarea(clickon0and1buttonstoreturntotheoriginal
viewingarea).Torotatetheviewingarea,clickonthereddotanddragclock‐orcounter‐clockwise.With
zoomedview,youcanselectthescanningareabymovingtheblue‐linedboxaround.Tozoomaspecificregion
in,clickiconanddrawaboxontheliveviewarea.Itwillzoomintheareaspecifically.
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Onceyouaresatisfiedwiththesetting,stop scanning.Setthescanspeedtoslowrates(~10‐12.5us/pixel)
(theslowerthespeedset,thebetterthesignal‐to‐noiseratio,butthemorebleaching).ClickXYbutton
toacquireanimage.Whenacquisitionisdone,a2Dviewwindowwillappear.
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E.Z‐SeriesImageAcquisition
Usethismodetoobtainopticalsectionthroughthedepth(z
dimension)ofyoursamplethatcanbeusedfor3Dvisualization.
Clickbuttontoscan.Usethearrowheadsbuttons
orthefinefocusknobontheremotecontrollertofocusinto
differentZ‐axialplanes(largearrowheadbuttonsshiftafullstep
sizeandsmallonesahalfstepsize).Whenyoufindupperlimitof
yoursample,clickEndSetbutton.Bringtheobjectivedownuntil
youfindlowerlimitandclickStartSetbutton.Determinethe
StepSizeandthenumberofSlices,whichcorrelatewitheach
other.Itisrecommendedtosetthestepsizesimilartothe
opticalsectionthicknessoftheobjectiveyouareusing,sothat
thereisnogapbetweentheopticalsectionsuponprojectioninto
3D.Thestepsizecanbefixedbycheckingtheboxb.
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OlympusFV1000Userguide August3,2011
ClickStop button.AdjusttheScanSpeedifneeded,clickDepthcbutton(“Z”willbeappearontheXY
buttontobecomeXYZ),andthenclickXYZdbutton.
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Whenacquisitionisdone,AppendNext/SeriesDone
e buttonwillappearovertheStopbutton.Click
AppendNextbuttontoaddadditionalsectionsatnext
step(enterthenumberofseriesyouwanttoadd)or
clickSeriesDonetofinishtheacquisition.Savetheimage.
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F.SequentialScanningMode
Usesequentialscanningtopreventpossible
cross‐talkand/orbleed‐throughbetweenthe
emissionsofthefluorophoresyouused.
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ChecktheSequentialboxfanditwillbringup
thesequentialscaninformationwindow.Choose
betweenLineorFramemodeg.
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Line:byline‐by‐line,itscansonechannelwith
onlyaspecificlaserlineanddetectoronandthen
sequentiallyscansthenextchannelwithonlya
anotherlaserlineanddetectoron.
Frame:itscanssequentiallyframe‐by‐frame;it
finishesscanningonechannelfirstandthenscans
thenextchannel.
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Ifthereisnobleed‐through
betweentheemissionsofsome
fluorophoresyouareusing,you
cangroupthemtogethertoscan
themsimultaneouslyasagroup.Simplyclickonthefluorophorenameonthelistanddragtotheothergroup.
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Note:itwillbegoodtosetuptheacquisitionconditionforeachchannelbeforeusingtheSequentialmode.
Todoso,firstinsimultaneousmode(uncheckedSequentialbox),settheacquisitionconditionbyturningon
onlythespecificlaserforthespecificchannel.
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ScanwithFocusx2orXYRepeatbuttontoseetocheckifthereisanybleed‐through.Ifnot,stopscanning,
andsettheScanspeedataslowerrateandclickXY(Z)button toacquiretheimage.
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OlympusFV1000Userguide August3,2011
G.SavingandExportingImages
1. Clickontheimagewindowtobesaved.
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2. Clickon iconorselectFile/SaveorSaveasfrom
menu.
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3. ASaveaswindowwillappear.Incasetheimageshould
besavedintheImagefolderofaLog‐inuser,clickon
buttonanditwilldirecttoyourimagefolder.
SelectOlympusImageBinaryFormat*.oib filetype,
typefilename,andclickonSavebutton.(Log‐inuser
anditsassociatefolderwillbecreatedasyoubecome
self‐user).
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4. Tosetupyourimagefolderwhereyoucanjumpdirectlyby
clickingMyImageFolderbutton,selectTools/Optionmenu.
ClickGeneral tabandspecifythepathofyourimagefolder
withBrowse.. button.[Uponcreatingauseraccount,each
userwillhaveitsownfolderinDdrive(Donotcreateany
folderinCdrive.).ClickOKbutton.

5. Oibfiletypecontainsallthemetadataincludingallthe
acquisiotnparametersanditcanbeopenedinFV10‐ASW
program.Thelightversionofthisprogramisavailablefor
installinginuser’sowncomputer.Otherwise,theimage
filecanbeexportedasotherfileformatsothatitcanbeopenedandprocessedinotherimagingsoftware.

Toexportimages:
1. SelectFile/ExportorFile/ExportMulti‐Tiffmenu.ClickMy
ImageFolderbuttonifthecurrentfolderisnotyours.

2. WithExportMulti‐Tiffoption,itsavestheimageasa
singletifffilethatcancontainmultipleframes,likez‐or
timelapse‐series.ThisfiletypecanbeopenedinImageJ
software.

ChecktheSavePropertiesAsASCIITextboxtosavethe
metadatathatcontaininformationabouttheimage
acquisitionsetting.Thisdatawillbeusefultofindouttheimagingconditionlaterwithotherprograms.


OlympusFV1000Userguide August3,2011
3. File/Exportcommandbringsawindowwith
differentoptions.
Thiscommandwillexportimagesintoa
folderthatcontainindividualimagesofeach
channel,z‐steps,ortime‐points.

SelectFiletypebforexport.
ROIOverlaycsectionallowssavingthefile
withROIinformation.
OutputFormatd:
RGBColor:thered,green,andbluechannel
willbesavedincolorasindividual*.tiffilesin
afolder.
RGBColor+GrayScaleboxchecked:the
colorchannelswillbesavedingrayscaleas
*.tiffilesinafolder.
MergeChannel:asinglemergedcolorimage
willbesavedasa*.tiffile.

FrameRatee determinetheplayspeedof
themoviewhenthefileisexportedasAVI.
ChecktheAVIboxtocompressthemoviefile.

4. ChecktheSavePropertiesAsASCIITextbuttonifyouexportthemetatdata.ClickSavebutton.

5. TransferyourimagedatatoCDorUSBflashmemory.




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b
c
d e

OlympusFV1000Userguide August3,2011
G.SystemShut‐DownProcedure
Wipeofftheoilfromanyoilobjectivesyouhaveused
duringyoursession.

Loggingoffandtransferringdata.
1. ExittheFV10‐ASWprogram.
2. Makesurethatyoutransferyourimagefileto
CDorUSBflashmemory.Allimagefilesmore
thanamontholdwillberemovedduring
monthlyclean‐up.
3. LogoffWindowsXP.Ifsomeonesignsupforuse
withinanext1hourorso,leavethesystemON.
Ifnot,shutdownthesystemasfollows.

ShuttingOffthesystem.
1. ShutdownthecomputerB.
2. TurnthemercurylampoffC.
3. TurnthemicroscopecontrollerkeytoOFFandturn
offbothscannerandcontrollerI/Oswitchesd.
4. Turnoffthelasercombiners e,FV10‐MCPSUand
FV10‐MP‐LCU.
5. TurningofftheMulti‐Arlaser fbyturningthekey
switchtoOFFfirstandthenturntheI/Opower
switchtoOFF.
6. TurnthekeyofHeNelasergtoOFF.

c
b
d
f
g
e