AutomatedObjectDetec/onandTracking

CharlesT.AndersonAssistantProfessorofBiology,PennState

data:bit.ly/2eOl5cu

Somervilleetal.,Science2004

plantcellwallsarebuiltofpolysaccharidenetworks

-cellulose

-hemicelluloses(e.g.,xyloglucan)-pec/ns

-glycoproteins

celluloseismadeattheplasmamembranebyCelluloseSynthesisComplexes(CSCs)

data:bit.ly/2eOl5cu

CSCscontainCelluloseSynthase(CESA)proteinsandmovethroughtheplasmamembrane,guidedbycor/calmicrotubules,astheextrudecelluloseintothecellwall

plasmamembranecellwall

cytoplasm

microtubule

CSC

cellulose

wecantrackCSCsinvivotodeterminetheratesandpaOernsofcellulosesynthesisinplantcells

data:bit.ly/2eOl5cu

datacollec/onandprocessing

5µm

-usestackregplugintocorrectforsampledriQ

-applyBackgroundSubtract(slidingparabola,10pixelradius)

-imageGFPCESA3every10secfor~12.5minusingspinningdiskconfocal

GFPCESA3,180Xspeed

data:bit.ly/2eOl5cu

UsingthePar/cleTrackerPluginforImageJ1)downloadImageJandGFPCESA3/melapsedataat:bit.ly/2eOl5cu

2)openGFPCESA3par/clesdatasetinImageJ3)openthePar/cleTrackerPlugin(Plugins>Par/cleDetector&Tracker>Par/cleTracker)

UsingthePar/cleTrackerPluginforImageJ

2)openGFPCESA3par/clesdatasetinImageJ3)openthePar/cleTrackerPlugin(Plugins>Par/cleDetector&Tracker>Par/cleTracker)4)setRadiusto“2”(thisdefinestheradiusofthepar/clestodetect)5)keepCutoffatdefault“3.0”6)setPercen/leto“10.0”

7)press“PreviewDetected”

1)downloadImageJandGFPCESA3/melapsedataat:bit.ly/2eOl5cu

UsingthePar/cleTrackerPluginforImageJ1)downloadImageJanddataat:bit.ly/2eOl5cu2)openGFPCESA3par/clesdatasetinImageJ3)openthePar/cleTrackerPlugin(Plugins>Par/cleDetector&Tracker>Par/cleTracker)4)setRadiusto“2”(thisdefinestheradiusofthepar/clestodetect)5)keepCutoffatdefault“3.0”

6)setPercen/leto“10.0”

7)press“PreviewDetected”

8)keepLinkRangeat“2”

9)setDisplacementto“1”10)click“OK”

UsingthePar/cleTrackerPluginforImageJ1)downloadImageJanddataat:bit.ly/2eOl5cu2)openGFPCESA3par/clesdatasetinImageJ3)openthePar/cleTrackerPlugin(Plugins>Par/cleDetector&Tracker>Par/cleTracker)4)setRadiusto“2”(thisdefinestheradiusofthepar/clestodetect)5)keepCutoffatdefault“3.0”

6)setPercen/leto“10.0”

7)press“PreviewDetected”

8)keepLinkRangeat“2”

9)setDisplacementto“1”10)click“OK”

11)click“VisualizeAllTrajectories”

12)click“FilterOp/ons”,setto7frames

13)click“DisplayFullReport”

ReportDataContainsTrajectoryInfoandCoordinates%%Trajectories:1stcolumn:framenumber2ndcolumn:xcoordinatetop-down3rdcolumn:ycoordinatele1-right4thcolumn:zero-orderintensitymomentm05thcolumn:second-orderintensitymomentm26thcolumn:non-par/clediscrimina/onscore

-thisinforma/oncanbeusedtocalculatepar/clespeedanddirec/on

%%Trajectory1

00.07119712.7215090.9078820.811220350.50698910.18907512.9844380.8566060.716557294.65496830.18278912.3344991.1363840.747800309.06677240.23789012.8136001.4393520.726033246.69104050.20082712.4840681.2978320.921498521.51715160.25605112.5828801.3035030.989276618.61145070.19797512.6616421.2563340.690050234.66691680.39522012.6069971.7062050.891610401.780334

-otherop/onsforpar/cletrackingincludeBitplane’sImarissoQware(allowsfor3Dpar/cledetec/onandtracking)

-par/clespeedcanalsobecalculatedfromkymographs