Olympus Fluoview FV1000 Bedienungsanleitung

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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d

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.



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).



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@.



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.

j

1) 1!

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.



ChooseImagesize(default‐512x512)from

Sizepanel d.



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.



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%).



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).



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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

c

d

g

b

f

h

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.



c

d

b

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.



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.



F.SequentialScanningMode

Usesequentialscanningtopreventpossible

cross‐talkand/orbleed‐throughbetweenthe

emissionsofthefluorophoresyouused.



ChecktheSequentialboxfanditwillbringup

thesequentialscaninformationwindow.Choose

betweenLineorFramemodeg.



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.



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.



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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e

OlympusFV1000Userguide August3,2011

G.SavingandExportingImages

1. Clickontheimagewindowtobesaved.



2. Clickon iconorselectFile/SaveorSaveasfrom

menu.



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).



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.





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.



e

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