BD FACSDiVa Software - TU Dresden · The FACSDiVa software uses „Specimens“ to associate a series of tubes as a pattern or panel. Existing Specimens can be used as a template

BD LSR II and BD LSR II and FACSDiVaFACSDiVa SoftwareSoftware

Dr. Jens Fleischer, BaselDr. Jens Fleischer, BaselDr. Norbert Dr. Norbert LeclereLeclere, Berlin, Berlin

Dr. Jens FleischerBDB European Technology Center

Basel, Switzerland LSR II Basic Course Dresden January 2006

OverviewOverviewFluidics

Connectors

On/OffSample PortControlpanel

Electronics Covers

TheThe ControlControl PanelPanel

EaseEase of of UseUse

• The basic procedures to use the BD LSR II are quite similar to those forFACScan and FACSCalibur

• Controlpanelp with RUN, STDBY and PRIME.

• Flowrates LOW, MED and HI adjustable• Sample injection port with sleeve needle

and tube support arm

OpticsOptics

•• TheThe BD LSR II in BD LSR II in youryour lab has lab has threethreelaserslasers::–– 488 nm Coherent Solid488 nm Coherent Solid--State (DPSS)State (DPSS)–– 635 nm JDS 635 nm JDS UniphaseUniphase HeNeHeNe–– 407 nm Coherent 407 nm Coherent VioflameVioflame DiodelaserDiodelaser

•• Using all three excitation line allows the Using all three excitation line allows the user to select the best dyes for the user to select the best dyes for the applicationsapplications

OpticsOpticsTheThe BD BD OctagonOctagon technology technology directsdirects thethe collectedcollected light via light via

fibresfibres intointo dedicateddedicated detectiondetection blocksblocks per per laserlaser::

““Simple” schematicsSimple” schematics

WhatWhat goesgoes wherewhere

Default Filters and Default Filters and MirrorsMirrors

Changes in the configuration should be done with care. Use the chartsin the lab to check the setup before starting your measurements!

SheathSheath and and WasteWaste

•• TheThe LSR II LSR II isis equippedequipped withwith an an automatedautomated FACSFlowFACSFlow SupplySupply SystemSystem–– 20 L 20 L FACSFlowFACSFlow cubitainercubitainer as as SheathSheath–– 20 L 20 L emptyempty cubitainercubitainer forfor wastewaste–– Level Level sensorssensors withwith audibleaudible alarmalarm–– EasierEasier to to useuse thanthan refillingrefilling and and emptyingemptying

smallsmall tankstanks all 3 all 3 hourshours

StartupStartup

•• Check Check SheathSheath cubitainercubitainer, , replacereplace ifif necessarynecessary•• Check Check WasteWaste cubitainercubitainer, , replacereplace ifif necessarynecessary•• First, turn on First, turn on thethe BD BD FACSFlowFACSFlow SupplySupply SystemSystem•• Second, turn on Second, turn on thethe BD LSR IIBD LSR II•• VentVent sheathsheath tubingtubing at at thethe sideside of of thethe instrumentinstrument. Be . Be

carefulcareful notnot to to spillspill PBS PBS overover monitormonitor and and keyboardkeyboard!!•• Take off Take off thethe cleaningcleaning tubetube•• Press PRIME Press PRIME threethree timestimes•• PutPut a a tubetube withwith DI DI ontoonto thethe SIP and SIP and runrun itit forfor 10 10

minutesminutes in HI and RUNin HI and RUN•• System ready for measurementsSystem ready for measurements

ShutdownShutdown

•• Run a Run a tubetube withwith FACSCleanFACSClean forfor 10 10 minutesminutes in HIin HI•• PutPut thethe tubetube supportsupport arm to arm to eithereither sideside and and emptyempty thethe remainsremains of of

thethe FACSCleanFACSClean solutionsolution•• ThisThis will will disinfectdisinfect thethe needleneedle and and thethe outerouter sleevesleeve•• Run a Run a tubetube withwith FACSRinseFACSRinse 10 10 minutesminutes in HIin HI•• PutPut thethe tubetube supportsupport arm to arm to eithereither sideside and and emptyempty thethe remainsremains of of

thethe FACSRinseFACSRinse solutionsolution untiluntil appproxappprox. 1 ml . 1 ml isis leftleft..•• ThisThis will will washwash out out deaddead cellscells and and debrisdebris fromfrom thethe needleneedle and and thethe

outerouter sleevesleeve•• PutPut a a tubetube withwith DI DI ontoonto thethe SIP and out SIP and out thethe instrumentinstrument to to

STDBYSTDBY•• Turn Turn thethe BD LSR II and BD LSR II and thethe FACSFlowFACSFlow SupplySupply System offSystem off

FACSDiVa SoftwareFACSDiVa Software

•• TheThe newnew digital digital acquisitionacquisition platformplatformintroducesintroduces a a completecomplete newnew software software conceptconcept fromfrom BDBD

•• TheThe software software runsruns on a Windows XP on a Windows XP computercomputer, Macintosh systems , Macintosh systems areare no no longerlonger availableavailable

FACSDiVa SoftwareFACSDiVa Software

•• TheThe software software usesuses a a databasedatabase serverserversoftware to manage software to manage thethe flowflow cytometry cytometry datadata..

•• All All datadata, , plotsplots, , gatesgates, , instrumentinstrument settingssettings, and , and statisticsstatistics areare savedsaved insideinside thethe databasedatabase..

•• TheThe useruser cannotcannot specifyspecify anyany pathpath forfor savingsaving, , everythingeverything isis donedone automaticallyautomatically in in thethebackgroundbackground..

TheThe mainmain layoutlayout

The BrowserManagement of your

experimental data and settings

InstrumentVoltage settings, Threshold,

Compensation

InspectorAlways shows the properties

of selected objects

WorksheetThe area for plots, histograms,Statistics and everything else

„„Floating Floating windowswindows“ (“ (alwaysalways on on toptop))

Acquisition StatusCounters for events per second,

Aborted events,Elapsed time

Acquisition ControlStarts and stops Acquisition and

data recording.

LogLog--inin

•• TheThe softwaressoftwares requiresrequires a a useruser loglog--in. Users in. Users cancan onlyonly seesee and and manipulatemanipulate theirtheir ownown datadata. .

•• OnlyOnly thethe Administrator Administrator cancan definedefine newnew usersusersand and theirtheir rightsrights..

•• TheThe Administrator Administrator cancan „Share“ „Share“ datadata withwith otherotherusersusers..

LogLog--inin

•• EachEach time time thethe software software isis startedstarted, , thethe loglog--in in menumenu appearsappears::

LogLog--inin•• DependingDepending on on thethe loglog--in, in, thethe useruser cancan

onlyonly seesee thethe appropriateappropriate datasetsdatasets::

A user can only seehis own data.

The Administrator cansee everything

HowHow to start?to start?

New New experimentsexperiments cancan bebe createdcreated via via thethe associatedassociatedbrowserbrowser iconicon oror via via thethe Menu bar.Menu bar.

New Experiment

Folders can be used to sort data sets. The foldersexist only in the database!

Global Global WorksheetsWorksheets

Be Be defaultdefault „Global „Global WorkSheetsWorkSheets“ “ offeroffer thethe possibilitypossibility to „to „rere--useuse“ “ plotsplots likelike in in CellQuestCellQuest. .

WhenWhen pressingpressing thethe „NEXT“ „NEXT“ buttonbutton, , thethe samesame plotsplots areare usedused forfordatadata displaydisplay..

Also Also savedsaved datadata cancan bebe loadedloadedintointo plotsplots..

For different For different viewsviews on on thethe samesamedatasetsdatasets youyou cancan createcreatemultiple global multiple global sheetssheets..

A A newnew experimentexperimentA A newnew experimentexperiment containscontains a „a „standardstandard“ “ instrumentinstrument

settingsetting and a and a singlesingle tube in a tube in a soso--calledcalled „„specimenspecimen“. “. WhenWhen selectingselecting "New Experiment" "New Experiment" fromfrom thethe menumenu bar bar

youyou cancan selectselect a "a "TemplateTemplate" " forfor youryour Experiment Experiment ((createdcreated fromfrom youryour olderolder experimentsexperiments).).

SpecimensSpecimensTheThe FACSDiVa software FACSDiVa software usesuses „„SpecimensSpecimens“ to “ to associateassociate

a a seriesseries of of tubestubes as a as a patternpattern oror panelpanel. . ExistingExisting SpecimensSpecimens cancan bebe usedused as a as a templatetemplate forfor thethe

nextnext, , similarsimilar tube tube patternpattern..

It is possible to “pre-define” a tube list, but you can always duplicate a specimen after measurement of all associated tubes.

Pre-definition is not required.

TheThe firstfirst plotsplotsAt At thethe toptop of of thethe WorksheetWorksheet WindowWindow youyou will find will find thethe "Tool "Tool

Palette"Palette"

Clicking on the “Clicking on the “DotPlotDotPlot” icon activates the creation of ” icon activates the creation of DotPlotsDotPlots. Just click once on the worksheet to create a . Just click once on the worksheet to create a dotplotdotplot of “standardof “standard--size”. Dragging and holding the size”. Dragging and holding the mouse creates a mouse creates a dotplotdotplot with the size of with the size of courcour choice. choice.

In addition do In addition do dotplotsdotplots, the software offers contour/density , the software offers contour/density plots and histograms.plots and histograms.

DotPlotsDotPlotsThe DotPlot can be activated byclicking onto the white border area.

The Inspector window shows informations about the activated plot(s):

Acquiring DataAcquiring DataDataData acquisitionacquisition isis controlledcontrolled via via thethe „„AcquisitionAcquisition

ControlControl“ “ windowwindow

Data acquisition is started and stopped with the „Acquire“ Button

During acquisition, data saving is initiated by pressing the„Record“ button.

The „Restart“ button is available during acquisition to restartthe acquisition and/or data recording.

AcquiringAcquiring datadata

The acquisition control window shows also the tube name, an event counterand the elapsed time since the acquisition has started.

The user also controls which events are counted and stored:

Storage Gate: Which events are recorded and saved

Stopping Gate: Which cells are counted

Events To Record: How many cells should be recorded (in the Stopping Gate)

Events to Display: How many events are shown “on-line” in the acquisition plots

TheThe instrumentinstrument controlcontrolTheThe „Instrument“ „Instrument“ windowwindow controls all controls all parametersparameters of of

thethe instrumentinstrument. . ItIt consistsconsists of different of different tabstabs, , whichwhich all all relaterelate to to specificspecific instrumentinstrument functionsfunctions..

Instrument Instrument -- ParametersParameters

TheThe Parameter Parameter tabtab showsshowsthethe instrumentinstrumentparametersparameters and and theirtheirsettingssettings. . TheThe useruser cancanchangechange thethe PMT PMT voltagevoltage, , thethelinear/linear/logarithmiclogarithmicsettingssettings, and , and cancanselectselect whetherwhether AreaArea, , HeightHeight and and WidthWidth forforthethe parameterparameter shouldshouldbebe acquiredacquired. . TheThedigital digital electronicselectronics has has no no parameterparameterlimitationlimitation!!

Instrument ParametersInstrument Parameters

The „Add“ and „Delete“ buttons allow the user to delete unwantedparameters, thus savingspace for the recordeddata sets.

Note that the parameterscorrespond to fluorochrome names, notchannel numbers. Theassociation betweenchannels, lasers and fluorochromes isexplained later.

Instrument Instrument -- ThresholdThreshold

The digital electronicsallows the user to definemultiple thresholds.

Thresholds can be set on any parameter from anylaser.

Threshold can beconnected with logicalOR and logical AND, so that either one of thethreshold conditions isfulfilled or all conditionsmust be met by an event.

Instrument Instrument -- CompensationCompensation

The digital electronicsallows the compensation of all parameters againsteach other. Thus, thecompensation starts to getincreasingly complex in multicolor applications.

The software allows„Autocompensation“ ifsingle color stains areavailable, reducing theworkload for instrumentsetup during multicolorexperiments.

Instrument Instrument -- RatioRatio

The instrument canautomatically calculate theratio between any twoparameters, even fromdifferent lasers.

Calcium flux:Ratio Fluoro3/FuraRed

Ratio Indo(bound)/Indo(Free)

Absorption ratio for dyesexcitable by different wavelengths

Instrument Instrument -- LasersLasers

Users should in mostcases not change thesesettings. All values aresetup during installation of your machine.

Please use this tab only if aBD representative asksyou to do so.

Instrument Instrument -- StatusStatus

The Status tab shows anyerrors that the instrumentencounters.

If an error occurs, the color ofthe tab changes to red, signalling that something iswrong.

The Clear button at the lowerright corner deletes thewarning messages.

AcquisitionAcquisition StatusStatus

The Acquisition Status window included a counter for events over the threshold(Events/Second) and a sum counter (all eventssince Acquire has been pressed).

The Abort Rate and Abort Count show howmany cells are „lost“ from the analysis becausethey were too close together to be separated as single events.

Processed Events shows how many cells havebeen analysed and is always close to theThreshold Count.

Elapsed Time shows how long you have beenacquiring the data.

WorkingWorking WithoutWithout Global Global WorksheetsWorksheets

In order to In order to switchswitch to to thethe „Standard „Standard WorkSheetsWorkSheets“, “, clickclick on on thethe smallsmall greengreentriangletriangle in in thethe upperupper leftleft cornercorner of of thethe tooltool palettepalette..

NowNow youyou cancan createcreate different different plotsplots forfor eacheach tube. In tube. In additionaddition, , eacheach tube tube nownow has has itsits ownown setset of of populationpopulation..

„Standard „Standard WorksheetsWorksheets“ “ areare good good forfor a a longlong list of list of tubestubes withwith different different analysisanalysis requirementsrequirements, , oror ifif youyou wantwant to to putput plotsplots of different of different tubestubesonztoonzto thethe samesame printprint pagepage

HoweverHowever, , bebe carefulcareful whichwhich mode mode isis currentlycurrently activeactive: A : A greengreen tabtab meansmeans"Global "Global WorksheetWorksheet", a ", a greygrey tabtab „Normal „Normal WorksheetWorksheet".".

User User PreferencesPreferences• Tube specific Worksheet: Each Tube gets itsown Worksheet

• Start Acquisition on pointer change: Savesone mouseclick on Acquisition

• Show GUID: Unique ID for each tube (useful?)

• Remove Tube-Specific Instrument Settingson Duplicate: Always use the sameInstrument Setting in an Experiment, evenwhen one tube is changed.

•Save Analysis after Recording: Copies all Plots from the Template onto the normal Worksheet after Recording

• Automatically Update Instrument Setting: Use always the latest setup even in "old" experiments

User User PreferencesPreferences

• Interval Gate Default Color: HistogramGates per Default have no color to reduce clutter

• Quadrant Gate color: Quadrants arealso not colored to reduce datacluttering

•• Default Background Color Default Background Color sincesince versionversion 4.1 4.1 isis „White“ „White“ ((equalequal to to printoutprintout))

•• CanCan bebe setset to to anyany colorcolor bybythethe useruser

•• In In somesome instancesinstances, , especiallyespecially whenwhen updatingupdatingexistingexisting databasesdatabases, , itit cancanoccuroccur thatthat youyou getget blackblackdotsdots on on blackblack backgroundbackground, , alwaysalways openopen thethe Population Population HierarchyHierarchy to check to check thisthis!!

•• EachEach useruser cancan definedefine his his ownowndefaultdefault templatestemplates forfor newnewexperimentsexperiments..

•• Default Global Default Global WorksheetWorksheet: BD : BD recommendsrecommends to to useuse thethe global global worksheetworksheet as as standardstandard methodmethod to to reducereduce screenscreen clutterclutter..

ExportingExporting TemplatesTemplates

After choosing "Export" the user can select the folder in a drop-down box. If you type in a new name, the folder isgenerated.

New New InspectorInspector viewview forfor recordedrecordedtubestubes

•• TheThe newnew inspectorinspector viewview forforrecordedrecorded tubestubes showsshows thetheinstrumentinstrument settingssettings in a different in a different way way comparedcompared to to thethe „live „live settingssettings“ “ shownshown beforebefore

•• ItIt isis notnot possiblepossible to to changechange thethevolatevolate settingssettings oror to to deletedelete / / addaddparametersparameters AFTER AFTER acquisitionacquisition

•• ItIt isis possiblepossible to to chnagechnagecompensationcompensation offlineoffline

New New scalingscalingIn In thethe digital digital electronicselectronics therethere areare no no errorserrors in in thethe determinationdetermination of of

thethe logarithmiclogarithmic valuevalue forfor lowlow intensitiesintensities. . IndeedIndeed, , itit alwaysalways lookslooksup up thethe mathematicallymathematically correctcorrect valuevalue..

HoweverHowever, , thethe logarithmiclogarithmic scalescale cancan onlyonly displaydisplay numbersnumbers fromfrom 26 26 to 260.000 in to 260.000 in thethe 44--decade decade displaydisplay ((whichwhich isis recommendedrecommended).).

In In thethe 55--decade decade displaydisplay itit isis 2.6 to 260.000 2.6 to 260.000 channelschannels. .

TheThe electronicselectronics actuallyactually calculatescalculates intensitiesintensities fromfrom ––2600 to 2600 to 260.000. 260.000. EverthingEverthing belowbelow 26 in 26 in thethe 44--decade decade scalingscaling thereforetherefore isisan "an "onon--axisaxis" " eventevent, as , as therethere isis no no logarithmiclogarithmic valuevalue to to plotplot..

TheThe "way out": "way out": UseUse a different a different axisaxis scalingscaling. . TheThe "bi"bi--exponential" exponential" sclaingsclaing isis linear at linear at thethe beginningbeginning and and convertsconverts to to logarithmiclogarithmic at a at a certaincertain point.point.

ExampleExample 1 1 –– Normal Normal loglog--scalescale

ExampleExample 1 1 –– BiBi--exponentialexponential

BiBi--ExponentialExponential

BiBi--exponential exponential scalingscalingshowsshows also also nicelynicelythethe effecteffect of of compensationcompensation..

TheThe upperupper panelpanel isisoverover--compensatedcompensated, , thethe effecteffect isis clearlyclearlyvisiblevisible in in thethe bibi--expoentialexpoential displaydisplay..

CorrectCorrect CompensationCompensationcancan bebe nicelynicely seenseenin in thethe lowerlower panelpanel..

BiBi--Exponential Exponential –– WhereWhere to to activateactivate??

The bi-exponential scaling is activated for each plot in theInspector. Thus, it is possible to have bi-exponential and normal log scale on the same page.

Population borders drawn in bi-exponential displays cannot be displayed in "normal" plots, but are always calculated on bi-exponential scaling.

The same holds true for the borders of population from "normal" log scales. They are not drawn in bi-exponential plots, but calculated based on log scale.

DensityDensity PlotsPlots

Densities and Contour Plots canbe generated with the same icon

Final formatting is done via the„Inspector“ Window.

Note that density plots can also be calculated on bi-exponential scaling, showing the populationdistribution.

CopyCopy and Paste...and Paste...

TheThe FACSDiVa Software FACSDiVa Software nownow supportssupports CopyCopy and Paste and Paste to to generategenerate PowerPoint PowerPoint PresentationsPresentations oror WordWord--DocumentsDocuments ((oror anyany otherother formatformat) ) withwith flowflow cytometry cytometry datadata..

Alternatively, Plots can be exported as graphics files. Alternatively, Plots can be exported as graphics files. However, only JPGHowever, only JPG--format is supported, leading to format is supported, leading to loss of quality if resized after import to PowerPoint.loss of quality if resized after import to PowerPoint.

Hinged QuadrantsHinged Quadrants

In many cases rectangular quadrantsare not the correct solution.

The FACSDiVa Software now offers "hinged" quadrants, which can be moved individually to gate the cells of interest without the need to create four different polygon regions.

Hinged QuadrantsHinged Quadrants

In many cases rectangular quadrantsare not the correct solution.

The FACSDiVa Software now offers "hinged" quadrants, which can be moved individually to gate the cells of interest without the neeed to create four different polygon regions.

OffsetOffset QuadrantsQuadrants

•• SometimesSometimes itit maymay bebeusefuluseful to to offsetoffset oneone sidesideof of thethe quadrantsquadrants to to adjustadjust clearlyclearlyoverlappingoverlapping populationspopulations..

•• YouYou cancan eithereither offesetoffesetquadrantsquadrants oror useuse thethehingedhinged versionversion. . ItIt isis notnotpossiblepossible to to useuse bothbothversionsversions simultaneouslysimultaneously!!

Final Final recommendationrecommendation::

Just Do It!

BD FACSDiVa Software - TU Dresden · The FACSDiVa software uses „Specimens“ to associate a series of tubes as a pattern or panel. Existing Specimens can be used as a template

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