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