CellCountingProcedure

CreatePhotoshopfileofCompleteRawData
1. Mergeindividual.pict(or.tif)filesofrawdataintoacomplete.psdPhotoshopfile.
Thecompiledimagewillserveasthebackgroundlayerfortheprocesseddata.
2. Makesurethattherawdataimagewithallofthechannels(red=probe,green=brdu,
blue=dapi)presentistheBackgroundlayerundertheLayerstab.
CreateLevelsLayer
1. Createabackgroundcopyofthebackground.ClickonLayer,DuplicateLayer,As
BackgroundCopy.
2. RenamebackgroundcopylayertoLevelslayer:ClickonLayer,LayerProperties,
andtypeinLevels.
3. Inthislayer,beginbyadjustingthedataintheindividualchannelsbyGaussian
blurring:
GaussianBlur:ClickonFilter,Blur,GaussianBlur.AdjustRadiusto2.0
pixelsforgreenBrduchannelor1.0pixelsforredchannel(maychange
settingsdependingonimage).
4. Next,adjustthelevelsofbrightness/contrast/levelsoftheindividualchannelsto
optimizedataappearance.Thismaybedoneeithermanuallyorbyautomethods.
Usejudgement,andaccountforincreaseinbackgroundlevelswhenusingautolevels.
Useoneofthefollowing:
LevelAdjustment:ClickonImage,Levels,Adjust.Adjustlevelsto
minimizebackgroundwhilestillpreservingauthenticdata.
AutoLevels:ClickonImage,Adjust.AutoLevels.
Brightness/Contrast:ClickonImage,Adjust,Brighness/Contrast.Adjust
levelsasneeded.
CreateCellCountlayer
1. Createalevelscopyofthebackground:ClickonLayer,DuplicateLayer,AsLevels
Copy.
2. RenameLevelscopylayertoCellCountlayer:ClickonLayer,LayerProperties,
andtypeinCellCount.
3. DeleteDAPIfromCellCountLayer:Channels,BlueChannelonly,Selectall,and
cut.
4. Adjustthelevelsofbrightness/contrast/levelsoftheindividualchannelstofacilitate
counts.
Levels:ClickonImage,Adjust,Levels.Manuallyadjustlevels.
5. FormRNA,labelcellnucleiinbluechannel(pencil/paintbrushsizeof13pixels).
CreatePlotlayer
1.CreateaNewlayer:ClickonLayer,New,Layer.Changelayernametoplot.
3. Inthislayer,plotthedifferentcelllabels(green,red,oryellow),usingtheappropriate
color.SetthepaintbrushsizeatHardRound9pixels.
4. Toselectthecolorofthepaintbrush,clicktwiceontopcolorsquareatthebottomof
thetoolbar.Forred,enter255nexttor(red),and0forg(green)andb(blue).For
green,enter255nexttog,and0nexttoothers.Foryellow,enter255nexttorandg,
and0nexttoblue.

CellCountingProcedure
5. Ifuncertain,viewbackgroundorlevelslayertoseeifcellappearstobelabeled.
Countred/green/yellowcellsseparately.OnlycountBrdUgreencellsiftheyareofat
least3/4sizeofafullsizeBrdUcellnucleus.Todetermineifacellisdouble
labeled,itmustbelabeledyellowofmorethan1/2ofbothgreenandredcells.
6. Foreaseofviewingplot,createanewfilllayer:ClickLayer,New,Layer,andtitle
itfill.Next,clickonEdit,Fill.MakesureunderContents,Use,itissetto
black.ClickOk.
CreateGridlayer
1. Withallofthechannelson(workinginallofthechannels),clickonCellCountlayer.
2. Usingthelinetool,drawastraightlinedowncenterofareaoftheimagethatwillbe
countedbyholdingdowntheshiftkeyanddraggingthetoolacrosstheimage.This
createsanewlayercalledShape1.
3. Rasterizetheshapelayer:clickonlayer,rasterize,layer.
4. Usingthepencil/paintbrush,createaborderatthetopandbottomofthecortex
(excludingmeninges).UseDAPIstainasguidetocortexboundaries.
5. Create10binsbymeasuringthethicknessofthecortexateachedgeandonthecenter
lineanddividingby10ineachcase.Uselinetooltodrawbins.
6. Increatinglines,sometimesseveraldifferentshapelayersappear.Tokeepthemas
onelayer,clickonthesecondbox(wherethepaintbrushis)oftheadditionallayersto
getlinks.ClickonarrowabovethePathandLayersTabs;Mergelinked.
7. RenameShapelayertoGridlayer:ClickonLayer,LayerProperties,andtypein
Grid.
Count!
1. UseNIHImage/ScionImagetodocellcount(refertoNIHImageProcedure).
2. Enterdataonsheet.Forcontrols,recordcorticallayeraswellasbinnumber.
3. Enterdataintodatabase.
AreaMeasurement
1. SelectGridlayerandcopytonewimage.Makesuredimensionsare1600x1200and
backgroundisblack.Saveimageas.tiffilewithidentifyingname(e.g.12.5nlgrid.tif)
2. OpenimageinImageJandhitF3toinvertcolorsandsettheproperscale.
3. UsemagicwandtoselectanareaofabinandhitF1.Resultswindowwillpopup
withareameasurement.SelectnextpartofbinandhitF1.Resultswindowwilladd
upareastogivethetotalarea.
4. Onceallareashavebeenmeasured,recordtotalareaforbin.(Recordto4decimal
places)
5.
PressF2toclearResultswindowandproceedtonextbin.

CellCountingProcedure

FiveLayersofCellCounting
BackgroundLayer

RawData(unprocessed)

LevelsLayer
Optimalbrightnessand
contrast

CellCountLayer

foractualcellcounting

Adjustlevelsforeachchannel

1. Blur
2. Setlevelsforhighcontrast
(Thresholdlike)

PlotLayer

PlotofLabeledcellsforcounting

GridLayer

(boundriesattopandbottomofcortex,
10bins)

Drawcortexandbinboundrares