Zeiss LSM 700 Manual de usuario

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InstructionLSM7002017‐12‐07.docx2017‐12‐19

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ConfocalLSM700

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Tableofcontents

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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

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

p.2

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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

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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

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Filterturret

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positionFiltersetExcitationfilterBeamsplitterEmissionfilter

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Fluorophore

family

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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

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Fs49G365FT395BP445/50DAPI,BFP

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5

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DIC   

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

p.3

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Laserlines

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LaserLine

Solidstate5mW 639

Solidstate10mW555

Solidstate10mW488

Solidstate5mW405

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LightPath

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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

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Startup

Turnonthesysteminthefollowingorder:

‐Inthepowerstripsturnonswitches12.

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‐Turnonthestagecontroller(3)

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‐Rotate(clockwise)thesilverkeyinthelaserselectronicunit(4)

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‐Turnonthecomputerandlogin

Username:multilabs

Password:123456

‐Turnonthemetalhalidelamp(6)

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

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p.5

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Samplemountingandviewing

Inserttheslideordishtothestageholder.

PushintheLightswitchbetweentheocularandtheconfocal.

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Viathetouchpad:

1. Choosethepreferredobjective

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2. ChoosetheproperfiltersetandopentheTL\RLshutter:

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Software

‐

LogintoBookitLabandactivateyourreservationtostartBlackZen2.3

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‐

ToacquireimagesclickStartSystem

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

Filterset

p.6

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Sampleviewing

IntheLocatetabchoosethepreferredobjectivefiltersetandopentheilluminationshutter

RL\TL

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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:

Bringthesampleintofocus.

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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

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Adjustthecondenserheightuntiltheedgesofthediaphragmimagearecrisp.

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Centerthediaphragmimageusingthetwocenteringscrews.

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Openthefielddiaphragm,justuntiltheimagefillsthefieldofview.

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p.7

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

2,5

3

p.8

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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

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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

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

p.9

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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).



3.2

6

3.1

p.10

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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



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