TIMELAPSEDATAANALYSISTOOLBOXV2

FabriceDUPRAT,july2014,duprat@ipmc.cnrs.fr,

Thisisasetofseventoolsdesignedforanalysisoftimelapsedataexportedfromimagingsoftware(ex:calcium
imagingfromMetafluor).Tool2isspecializedforfluorescencedatabutthe6othertoolscanworkonmanykind
ofdata.MadewithOriginProversion8.5andshouldbecompatiblewithversion9.

Inthetoolbarthe7toolsaredisplayedinaworkfloworder:

1Changetheallworksheetlongnames(and/orshortnames)withexperimentalconditions,
2Transformallfluorescenceintensitiesintoratioand/orcalciumconcentration,
3Graphicallyremoveoutliers(deleteplot+correspondingdatacolumn),
4Graphicallynormalizeallplotsto0orto1,
5Measurebaseline,peak,andplateauofallplots,
6Fitmultipleplotstogether,
7Collectanalyseddatainthesameworksheet.

Ifyouareusingmytoolspleaseusethefollowingreference:

DupratF.2014,http://www.ipmc.cnrs.fr/~duprat/scripts/

AlsoavailableonOriginLabfileexchangesite:http://www.originlab.com/fileexchange/

Helpfile
Displayinformationssimilartothepresentdocument.

Tool1:Changenames
Allowtoeasilychangeshortandlongnamesofallworksheets.Westronglyrecommendtheuseoflongnamesto
describethedataandletOrigindealwithshortnames(Screenshot#1).Shortnamesmuststartwithaletter,donot
usespecialcharacters(.,;*).

Screenshot#1
Tool2:Fluorescencedataanalysis
Thisisthemaintool,itperformsanalysisofdatafromimageanalysisofratiometricornonratiometricfluorescent
probes.Thereare5partsintheparameterswindow(seeScreenshot#2).

Screenshot#2

Before starting the tool, it is recommended to spacely arrange the window on the desktop, because windows
createdbythetool(calculatedvaluesandgraphs)willusethesamewindowsizeandposition,seeScreenshot#2a.

Maketheworksheettobeanalysedactive(simplyclickonit)beforestartingthetool.

Screenshot#2a

Part1:Structureofthedata

Screenshot#2b
EntertheTimeunitforgraphlegend.Togetridofheadersandlabelrow(ifpresent),entertheFirstcolumnwordto
detectlabelrow(Xcolumn),thisrowandallrowsabovewillbeignored.Otherwise,themacrowillproposetostart
analysisfromthefirstrow.InScreenshots#2band#2c,thewordis"Time",alllinesbetween1and12areignored.

Screenshot#2c

EntertheNNumberofcolumnsforeachseries.TheactiveworksheetmuststartwithoneXcolumn(time)andfor
each region of interest (R1, R2, R3, ) there must be the repetition of the same serie of Y data (recorded
wavelengthsWave1,Wave2,Wave3,,oralreadycalculatedratio,orothercolumnsofdata)(seeScreenshot#2c).

Foraratiometricsignalanalysis(ex:Fura2)entertheRankofthecolumnstouseforlowwavelength(ex:340nm)
andhighwavelength(ex:380nm).Fornonratiometricsignal(s)enterthesinglewavelength(s)(ex:Fluo4,488nm).
You can analyse at the same time a ratiometric signal (2 columns of data) and up to 3 non ratiometric signals (3
columns).Forexample,inscreenshot#2c,theyare2recordedwavelengthsand1ratio,theworksheetcolumnsare:
X, R1Wave1, R1Wave2, R1Ratio, R2Wave1, R2Wave2, R2Ratio, R3Wave1, R3Wave2, R3Ratio, .... To select the
wantedYcolumns,indicatetherankofeachYcolumntobeanalysedwithineachserie.Exampleinscreenshot#2b
and#2c:Ranks1and2forlowandhighwavelengthsofaratiometricsignalareentered,Ratio(rank3)isnotentered
andwillbeignored.Nosinglewavelengthwillbeanalysed(enteredvaluesare0).

Part2:Globalanalysis

Screenshot#2d

SelectifyouwanttoDisplaytagsongraphs.Tagsareassumedtobetextwithexperimentalconditionslocatedin
firstYcolumn(secondcolumnoftheworksheet)betweenthedata(seeScreenshot#2g,row283).

SelectifyouwanttoAnalyseallworksheetsoftheproject,makesuretheyallcontaindataattheappropriateand
identicalformat(firstcolumnX,andseriesofYdataasdescribedabove).Ifneededselectlinesineachworksheet
fornormalization(R/R0,Rmin/Rmax,orSinglewavelength)beforestartingthetool.

Part3:Probesparameters

Screenshot#2e
EntertheNamesoftheprobes(1ratiometricandupto3nonratiometric),andthekD(integervalue)andUnitofkD
forthecalciumconcentrationcalculation(Screenshot#2e).

Part4:Ratiometricsignalanalysis

Screenshot#2f

SelectifyouwanttoSubtractbackgroundfromfirstserieofdata(ROIR1)toalltheotherdata.

Selectwhattograph:

Simpleratiooflow/highwavelengthonly(ex:340/380forFura2)

Rationormalizedtoselectedrows(R/R0).ForeachROI,aftercalculatingtheratioforallrows,theratio
from selected rows are averaged and divided to all the ratios. Before starting the tool, you must select at
least 3 rows in the active worksheet or in all the worksheets (if analysing all worksheets in one go). In
Screenshot#2gallvaluesbetweenlines280and298willbeaveragedforeachcolumn.

Calciumconcentration.CalculationwillbeperformedaccordingtoGrynkiewiczetal.1985(J.Biol.Chem.
260(6):34403450),youmustselectanormalizationmethod(seebelow).

Simpleratio+Calciumconcentration.Bothratioandcalciumconcentrationwillbedisplayed.

Selectnormalizationforcalciumconcentrationcalculation:

Rmin/Rmaxfromselectedrows.Youmustselectatleast6rowsbeforestartingthetool,thefirst3rowsare
usedforRminandlast3forRmax(inScreenshot#2g,foreachROI,valuesbetweenlines280and282willbe
averagedforRmin,andvaluesbetweenlines296and298willbeaveragedforRmax).
Rmin/Rmax(autodetect).Enterauniqueword(tag)presentinfirstcolumnofYdataindicatingtheendof
theRmincondition(ex:0CaEGTA+ionomycin)andthebeginningofRmaxcondition(ex:saturatingcalcium
+ionomycin),weassumethatyouhaveperformedRminbeforeRmaxandnottheopposite(inScreenshot
#2gthewordis"Ev8_20Caiono").Theaveragedvaluesofthe3rowsbeforethedetectedwordareusedfor
Rmin(inScreenshot#2g,lines280to282),thelast3rowsofthecolumnforRmax(inScreenshot#2g,lines
296to298).Notethatselectedrowsareignoredwhenautodetectisused.

Nota:Onlyonerangeofrowscanbeselected,soyoucannotperformnormalizationtoselectedrowsforratiometric
andnonratiometricsignalsatthesametime,otherwisetheywillusethesameselectionofrows.

Screenshot#2g
Part5:Singlewavesignalsanalysis(nonratiometricsignal)

Screenshot#2h

Ifarank(not0)isenteredinPart1(Screenshot#2b)agraphofthecorrespondingcolumnswillbegenerated.Thus,
foreachwavelength,selectifyouwanttoSubtractbackgroundfromfirstserieornot,andNormalizefromselected
rowsornot.Ifnormalization,beforestartingthetool,youmustselectatleast3rowsintheactiveworksheetorinall
theworksheets(ifanalysingallworksheetsinonego).

Tool3:Deletecolumnfromgraph
Deleteacolumnofdatafromitsplot.Thegraphlayermustbeactivebeforestartingthetool.Selectifyouwantto
Rescale after deleting or not and to Update the legend after deleting or not (Screenshot #3). Click OK and then
select the plot to be deleted by double clicking (or simple click + arrow + enter) on the plot, the corresponding
columnofdatawillalsobedeletedfromworksheet.Thetoolautomaticallyrestartstoletyoudeleteanotherplot.
ClickCanceltostopthetool.

BEWAREUNDOISNOTPOSSIBLE.

Screenshot#3
Tool4:Normalizationofmultiplecurves
Allowtonormalizeto0and/orto1allplotsfromagraph.The graphlayertobeanalysedmustbeactive.Choose
normalizationtoapply(Screenshot#4a)andthendoubleclick(orsingleclick+valid)on1pointtosetthetimepoint
(X)fornormalizationto0(subtracteachcorrespondingYvalue)orto1(divideeachcorrespondingYvalue).Forboth
normalizations,doubleclickon2points(orsingleclick+validtwice),firstlyfornormalizationto0andsecondlyfor
normalization to 1 (Screenshot #4b). The values around the selected time point are averaged according to the
choosenNumberofpointstoaverageandthenusedfornormalization.BEWAREORIGINALDATAAREMODIFIED,it
isindicatedinthelongnameofcolumns(Screenshot#4c),UNDOISNOTPOSSIBLE.

Screenshot#4a Screenshot#4b

Screenshot#4c
Tool5:Baseline,peak,andplateauanalysis
Perform baseline, peak, and plateau measurement of large number of curves in one go. The graphlayer to be
analysed must be active. Select a Range on the graph, enter Name of experimental condition and N Number of
pointstoaverage,Directionofpeaktobefound,ifyouwanttoShowstatisticsonthemademeasurements(mean,
sem, sd, n number). The N first points are averaged for baseline, the N last for plateau. The peak is searched in
between. Delta amplitude between baseline and plateau, and between baseline and peak are calculated with
statistics (Screenshot #5b) or without statistics (Screenshot #5c). Select if you want to Keep the same results
worksheetwindow(called"AnalysePeak"),itwilladdeachanalysisoneafteranother,butthestatisticsaredisabled
(Screenshot#5a).Togetnextresultsinanewworksheet,simplyrename"AnalysePeak"tosomethingelse.

Screenshot#5a

Screenshot#5b

Screenshot#5c
Tool6:Multiplefitanalysis
Perform fitting of large number of curves in one go. The graphlayer to be analysed must be active. Enter your
Experimentalconditions,Choosefittingfunction,youcanKeepthesameworksheetforoutputofresults(called
"AnalyseFit",youcannotchoosethisoptionandplottheanalysis),itwilladdeachanalysisdataoneafteranother.
SelectifyouwanttoShowtheplotsoftheanalysisandtheNumberofcolumnsandNumberofrowsonthefinal
graph(Screenshot#6a),youcanasktographhundredsofplots(newgraphpageswillbecreated)butthespeedand
crashpossibilitydependsfromyourcomputerperformance.ClickOKandthendoubleclickon2points,orsingleclick
+valid(confirmedbyaredcircle)tosetthelimitoftheanalysis(rangeofXvalues)(Screenshot#6b).

Screenshot#6a Screenshot#6b

Theoutput(Screenshot#6c)isaworksheetwithplotindex,thenameofsourcegraphpage,experimentalcondition,
fittingresult,Rvalue,numberofpointsintherange,startandendXvaluesoftherange,andfittingparameters(for
BoltzmanitisA1,A2,x0,anddx,seeOriginfittingfunction).Ifplottingtheresults,experimentalpointsareinblack
andfittingcurveinred,plotindexisindicated,legendsareidenticaltooriginalgraph(Screenshot#6d).

Screenshot#6c

Screenshot#6d
Tool7:Copyworksheets
Give group names to define 2 to 5 groups of data, and then attribute the groups to the needed worksheets
(Screenshot#7a).Itwillcopyallthedatafromallselectedworksheetsinto2to5worksheetsinanewworkbook,
correspondingtodefinedgroups(Screenshot#7b).

Screenshot#7a

Screenshot#7b