Zeiss LSM 700 User manual



InstructionLSM7002017‐12‐07.docx2017‐12‐19


ConfocalLSM700


Tableofcontents

Introduction ......................................................................................................................................2
Hardware ..........................................................................................................................................2
Objectives......................................................................................................................................2
Filterturret ....................................................................................................................................2
Laserlines......................................................................................................................................3
LightPath ......................................................................................................................................3
Startup .............................................................................................................................................4
Samplemountingandviewing .........................................................................................................5
Software ...........................................................................................................................................5
Sampleviewing ..............................................................................................................................6
Acquisition.....................................................................................................................................8
LSMChannelmodeconfocalsetting.........................................................................................8
Configuration: ........................................................................................................................8
Acquisition&parametersetting ............................................................................................10
Singleimageacquisition........................................................................................................12
Multidimensionalacquisition.................................................................................................12
Shuttingdown .................................................................................................................................17




p.2

Introduction
TheZeissLSM700isequippedwith4laserlinesandcandetectuptofourcolorsignalsatframerates
approaching5framespersecondat512x512pixels.Efficientseparationofthefluorescencesignalsby
selectivelaserexcitationcoupledtoefficientsplittingoftheemissionusingthevariablesecondary
dichroic(VSD)beamsplitterpreventscrosstalkandenablesspectralimagingaswellaslinearunmixingof
highlyoverlappingfluorophores.AmongtheadvancedfeaturesoftheVSDbeamsplitteristhatallportions
oftheemissionspectrumareutilizedfordeterminingeachspectraldatapoint.
Hardware
Themicroscope:
TheLSM700isattachedtoanuprightmicroscope(AxioImagerZ2).
Objectives

PositionObjectivelens Magnification NA coverslip Working
distanceImmersionParfocal length
1WPlan
ApochromatX401.0 02.5mmWaterdipping45mm
2ECPlan
Neofluarx10 0.3 0.17mm 5.2mmAir45mm
3Plan
Apochromatx20 0.8 0.17mm 0.55mmAir45mm
4LDLCIPlan‐
Apochromat x25 0.8 0‐0.170.57atcover
glass0.17
Water,
Glycerineand
Oil
45mm
5ECPlan‐
Neofluarx40 1.3 0.170.21mmOil45mm
6Plan
Apochromatx63 1.4 0.170.19mmOil 45mm

Filterturret

positionFiltersetExcitationfilterBeamsplitterEmissionfilter

Fluorophore
family

1FS38BP470/40FT495BP525/50Cy2,GFP
2FS43BP545/25FT570BP605/70Cy3,Al555
4

Fs49G365FT395BP445/50DAPI,BFP

5

DIC   



p.3

Laserlines

LaserLine
Solidstate5mW 639
Solidstate10mW555
Solidstate10mW488
Solidstate5mW405

LightPath


1. Thelaserscomealignedfromthemanufacturer,all4lasersareattachedtoanopticcableand
entertheconfocalscanhead.(1)
2. Thelaserlinesarereflectedbythemaindichroicmirror(beamsplitter).(4).
3. The2Galvanometricmirrorsscanthesample.(3).
4. Thereturnemissionwavelengthpassesthroughthemaindichroicmirror(4).
5. Theemissionpassesthroughthepinholeapertureandtheoutoffocuslightiseliminated.(5)
6. Thevariablesecondarydichroicbeamsplitter(VSD)iscoatedwithavariablecoatingthatbetween
420nm‐630nm,accordingtothepositionoflightincidencedeterminesthecutoffbelowwhich
wavelengththelightwillpasstoPMT1andabovethiswavelengthitwillbereflectedtowards
PMT2.(6)
7. BeforethedetectionanemissionfiltercanbeinsertedfromthefilterwheelbeforethePMT,only
longpass(LP)/shortpass(SP)filtersareavailable.(9)
8. ThelightisdetectedinoneofthetwoPMTdetectors‐PMT1forSPemissionwavelength,PMT1
forLPemissionwavelength.(7,8)

p.4

Startup
Turnonthesysteminthefollowingorder:
‐Inthepowerstripsturnonswitches12.

‐Turnonthestagecontroller(3)

‐Rotate(clockwise)thesilverkeyinthelaserselectronicunit(4)

‐Turnonthecomputerandlogin
Username:multilabs
Password:123456
‐Turnonthemetalhalidelamp(6)


6


p.5

Samplemountingandviewing
Inserttheslideordishtothestageholder.
PushintheLightswitchbetweentheocularandtheconfocal.

Viathetouchpad:
1. Choosethepreferredobjective


2. ChoosetheproperfiltersetandopentheTL\RLshutter:

Software
‐ LogintoBookitLabandactivateyourreservationtostartBlackZen2.3 
‐ ToacquireimagesclickStartSystem 

Filterset

p.6

Sampleviewing
IntheLocatetabchoosethepreferredobjectivefiltersetandopentheilluminationshutter
RL\TL


SettheTransmittedlight(IncasetheTLisneeded)
Clickthetransmittedlightlampicon,clickONandsetdesiredbrightness(orviathe
microscope).
SetthefilterturretpositiontoDICandthecondenserbelowthestagetoI,IIorIIIaccording
totheobjectivelens(seeobjectivelensinfoontouchpad).
IfyouareacquiringtransmittedchannelsetupKohlerillumination:
1. Bringthesampleintofocus. 
2. Closethefieldstopusingthemotorizedaperturebutton(F)locatedattherightofthe
microscopeuntilyoucanseeatleastoneedge

3. Adjustthecondenserheightuntiltheedgesofthediaphragmimagearecrisp. 
4. Centerthediaphragmimageusingthetwocenteringscrews. 
5.
Openthefielddiaphragm,justuntiltheimagefillsthefieldofview. 

p.7




2,5
3
4

p.8

Acquisition
LSMChannelmodeconfocalsetting
InthesoftwaregototheAcquisitionmaintooltab
PullouttheLightswitchbetweentheocularandtheconfocal.

Configuration:
ItishighlyrecommendedtoseekBCFstaff’sadvicefornewfluorophores(exactname)ornew
combinations.

Chooseone:
LoadasavedconfigurationfromtheExperimentManager



Reusesettingsofanimage:openanimageandbelowtheimageclickReuse
Newsetup:click,choosethedesireddyesfromthelist,choosethescanning
methodandclickApply





















p.9

SelectshowalltoolandopentheImagingsetuptab
Note:makesurethatthechosenconfigurationiscorrectandsuitsthefluorophores;make
changesifneededitishighlyrecommendedtocheckalldyesspectrumsinaspectra
viewer
ThermoFisher
Chroma

1. Laserlines
2. Maindichroicmirror
3. PMTs:PMT1andPMT2
4. Variablesecondarydichroicbeamsplitter(VSD).
5. EmissionFilterselection
6. EmissionspectrumwithGraphicrepresentationofthelaserline,thepositionofthe
VSDandemissionfilter.
7. TransmittedlightPMT‐atransmittedlightimage(nonconfocal)canbeaddedtothe
confocalimage(itrecommendedtoaddtothelongestwavelengthtrack).

1
2
3.2
4
5
6
7
3.1

p.10

Acquisition&parametersetting
Pulloutthelightpathswitchbetweentheocularandtheconfocal

Parametersetting
OpentheAcquisitionmodetab

Scanmode:frame(inmostcases);
  linescanmodecanbeusefulforphysiologyexperiments.
Framesize: 512X512
Speed: 8
Averaging:
Method:mean(inmostcases):
inlowsignalwithlownoiseSummethodmaybeuseful
Number:1
Mode:frameforfixativesample,forlivecellsandincaseofunstabledyeline
modewillbemoresuitable
BitDepth:definedthedynamicrangeoftheimage,choose8bit\12bitaccordingto
thebiologicalquestion.
Direction: ‐‐‐‐>(moreprecise)
Scanarea:slightnudge,zoomandrotation


p.11

OpentheChannelstab

Checkandselectonlytheshortestwavelengthtrackandclickthe1AUbutton
Startscanincontinuousmode
ChecktheRangeindicator(belowtheimagecontainerintheDimensions
tab).
Setyourimageparameterstoachieveimagewithoutsaturation(saturation=redpixelsin
theimage)andthecorrectblacklevel(black=bluepixelsintheimage)
oSetthePMTgainto550‐600.
oSettheLaser%‐itisrecommendedtousethelowest%aspossibleandto
increasegain(master).
oIncaseoflowsignalwithhighlaser%andhighgainyoucanadddigitalgain
(upto2).
oSettheoffsetleveluntilyoureachthePMTthreshold(bluepixelwillappearin
theimagewhilescanninginrangeindicatormode).
Stopthescan
Selectanothertrack;setitspinholetothesameμmsectionasthefirsttrackandsetits
parametersandsoon
Stopthescan


p.12

Singleimageacquisition
IntheAcquisitionModetabselectthepreferredzoom(alternativelyyoucaneasily
positionthecroppingframetozoominviathecroptoolbelowtheimageinthe
Dimensionstab)

Scanoneframetocheckposition.
CheckalltracksintheChanneltab,IntheAcquisitionModetabclicktosetthe
framesize.
Setscanningspeedto6
Setaveragingnumberto2‐4(accordingtotheSNR)
PressSnapinthestartbuttons
Savetheimage:pressintheImagesandDocumentstab(righthandsideofthe
window)andsaveinthedirectory:
D:\Usersdata\PIname\MM‐YYYY\Username\YYYY‐MM‐DD

Multidimensionalacquisition

Zstack
IntheMultidimensionalAcquisitionoptionsbelowthestartbuttonscheckZstack;anew
tab(Z‐stack)willappearundertheMultidimensionalAcquisitionSection.
Settheinterval:pressSmallestforthesystemrecommendation(Nyquistcriterion)
WithonechannelcheckedandIntheAcquisitionmodetab:
Framesize 512X512
Scanningspeed8
Averagingnumber1

p.13

Startscanincontinuousmode
DefinethesignalboundariesbymovingthefocuswheelsandpressingSetFirstinone
boundaryandSetLastinthesecondboundary.

Withallchannelschecked,intheAcquisitionmodetab:
Framesize Pressoptimal
Scanningspeed7
Averagingnumber2‐4
Press
SavetheImage
Note:therearemoreadvancedZstackoptionsinthistabsuchasbrightnesscorrection
overz.
Timeseries
IntheMultidimensionalAcquisitionoptionscheckTimeseries;anewtab(TimeSeries)will
appearundertheMultidimensionalAcquisitionSection.
Definenumberofcyclesandtheinterval
Note:incaseoflivecellimagingavoidhighlaserintensity

IntheAcquisitionModetab:
Framesize Pressoptimal
Incaseofbigframesizewithlongframescantimeitrecommendedtoincreasethe
zoomandsetanewframesizewithlesspixelsbypressingoptimalagain.
Scanningspeed: 7‐8
Averagingnumber1‐2
Press
SavetheImage

p.14


Tile
IntheMultidimensionalAcquisitionoptionscheckTilescan;anewtab(TileScan)will
appearundertheMultidimensionalAcquisitionSection.
Definethenumberoftiles
Definetheoverlap%‐recommendedis8%‐15%
Calculatesliderotation:InthemainmenuchooseMacroTilescanrotation
InthepopupwindowpressCalibrateandwaituntiltheprocessisfinished(therotation
anglewillbeexportedtothetilescantab)thenpressclose.


Note:Onlinestitchingisnotarecommendedoption,theprocesscanbeconfiguredinthe
processingmaintooltab.
IntheAcquisitionmodetab:
Framesize: Pressoptimal
Zoom: Itisrecommendedtosetthezoomabove1.2
Scanningspeed:7
Averagingnumber:2
Press
SavetheImage
Stitching:IntheProcessingmaintooltabStitchselectyourinputimage
pressApply.
Incaseofunevenilluminationthestitchingprocesscanbedonewithmoreoptionsin
theZENbluesoftware.

RegionsandBleaching
IntheMultidimensionalAcquisitionoptionscheckBleaching;theBleaching,Timeseries
andRegionstabswillappearintheMultidimensionalAcquisitionSection.
IntheBleachingtabdefine:
Startbleachingafternumberofscans

p.15

Repeatbleachafternumberofscans
Theamountofiterations
Thelaseranditsintensity
Moresettingcanbedefinedinthebleachingdialog.

DefineoneormoreROIs:
AfterasinglescanintheRegionstabchooseoneoftheROItoolsanddrawonthe
acquiredimage;thedimensionscanalsobesetmanuallyintheWidth/Heighttextboxes.
DefinewhichprocesswillbeperformedintheROI–Acquisition/Bleach/Analysis.

DefineTimeseries:
IntheAcquisitionmodetab:
Framesize Pressoptimal
Scanningspeed7ormore
Averagingnumber1‐2
Press

p.16

Savetheimage
TheintensitydatafromtheROIcanbeviewedintheROImeantabintheimagewindow
Note:Theregioncanbedefinedinothertypesofexperimentswithorwithouttimelapse
andcanalsobeusedasatoolofzoominginbycheckingFitFramesizetobounding
rectangleofregions.
Positions
Apositionslistcanbeaddedtoamultidimensionalexperimentinwhichallofthesettings
willbeacquired(channel,acquisitionmode,Zstack,tile,timeseries).
ThePositionstabcanbeausefultoolinmanagingalargetileandforquickslidescanning
(viatheocular)withlowmagnificationbeforemovingtohighmagnificationwithsmallfield
ofview.

AddapositionbypressingPositionsbelowtheimagecontainerandclickingontheimage
containerthatwasjustacquiredatthecurrentstageposition
CheckPositionsattheMultidimensionalAcquisitionoptions.
AtthePositionstabmovetoapositioninthelistbyselectingandpressingMoveto
ApositioncanalsobeaddedbypressingAddinthePositionstab

Note:IfyouusethepositionslistonlytolocatesavedpositionsuncheckPositionsfromthe
MultidimensionalAcquisitionoptionsbeforepressingStartExperiment,otherwiseyour
settingswillbeacquiredateverypositioninthelist.

NoteyoucanusemultipleoptionsinthemultidimensionalscanningforexampleZstack&tile
Timeseries,bleaching,ROI&Zstackandpositions.


p.17

Shuttingdown
‐Makesureallthedataissaved.Thedatawillbeautomaticallysavedtotheserverduringthe
night.PLEASEDONOTUSEANYUSBFLASHDRIVEonthiscomputer.
‐Cleananyoilimmersionobjectivesyouusedwithlenspaperandpetroleumether,twice.
‐ClosetheZENsoftware
‐LogofffromyouraccountintheBookItlabwindow
‐Checkifanotheruserhasareservation;ifyesverifyhis/herarrival.Ifyouarethelastuserforthe
dayorthenextreservationisintwohoursorlater,continuewithsystemshutdown.
‐Turnoffthemetalhalidelamp(6)
‐Turnoffthecomputer(5)onlyonweekends
‐Turnoffthelaserelectronicunit(4)
‐Turnoffthestagecontroller(3)
‐Turnoffswitches21Intheswitchbox
‐Coverthemicroscope