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MuscleJ_1_0_2.ijm
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MuscleJ_1_0_2.ijm
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/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
// MuscleJ: A high content analysis method to study skeletal muscles.
// Copyright (C) 2018 Anne Danckaert – Alicia Mayeuf-Louchart
//
// This program is free software: you can redistribute it and/or modify
// it under the terms of the GNU General Public License as published by
// the Free Software Foundation, either version 3 of the License.
//
// This program is distributed in the hope that it will be useful,
// but WITHOUT ANY WARRANTY; without even the implied warranty of
// MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
// GNU General Public License for more details.
//
// You should have received a copy of the GNU General Public License
// along with this program. If not, see <https://www.gnu.org/licenses/>.
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
// MuscleJ: A high content analysis method to study skeletal muscles.
//
// The MuscleJ macro is a compilation of tools allowing for the analysis of fiber phenotypes.
//
//
// Authors: Anne Danckaert – Alicia Mayeuf-Louchart
//
// Features
// Fiber morphology – Centro Nuclei Fiber detection (CNF) – Vessel detection – Satellite Cell detection (Sat) – Fiber typing
//
// Citation : Mayeuf-Louchart et al. SkeletalMuscle (2018) 8:25
// Tutorial in Additional file 1: Tutorial
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
function AreaClassAttribution(Area,FiberAreaClassCount) {
if (Area < 250) {
FiberAreaClassCount[0]= FiberAreaClassCount[0]+1;
}
else if ((Area < 500) && (Area >= 250)) {
FiberAreaClassCount[1]= FiberAreaClassCount[1]+1;
}
else if ((Area < 750) && (Area >= 500)) {
FiberAreaClassCount[2]= FiberAreaClassCount[2]+1;
}
else if ((Area < 1000) && (Area >= 750)) {
FiberAreaClassCount[3]= FiberAreaClassCount[3]+1;
}
else if ((Area < 2000) && (Area >= 1000)) {
FiberAreaClassCount[4]= FiberAreaClassCount[4]+1;
}
else if ((Area < 3000) && (Area >= 2000)) {
FiberAreaClassCount[5]= FiberAreaClassCount[5]+1;
}
else if ((Area < 4000) && (Area >= 3000)) {
FiberAreaClassCount[6]= FiberAreaClassCount[6]+1;
}
else if ((Area < 5000) && (Area >= 4000)) {
FiberAreaClassCount[7]= FiberAreaClassCount[7]+1;
}
else if ((Area < 6000) && (Area >= 5000)) {
FiberAreaClassCount[8]= FiberAreaClassCount[8]+1;
}
else if (Area >= 6000) {
FiberAreaClassCount[9]= FiberAreaClassCount[9]+1;
}
}
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
function ChooseColorCartography(Area, nLast,NameAreaImage) {
Color = newArray(3);
selectWindow(NameAreaImage);
run("RGB Color");
roiManager("select", nLast);
if (Area < 250) {
Color[0]=255;
Color[1]=255;
Color[2]=255;
}
if ((Area < 500) && (Area >= 250)) {
Color[0]=221;
Color[1]=255;
Color[2]=221;
}
if ((Area < 750) && (Area >= 500)) {
Color[0]=189;
Color[1]=255;
Color[2]=189;
}
if ((Area < 1000) && (Area >= 750)) {
Color[0]=153;
Color[1]=255;
Color[2]=153;
}
if ((Area < 2000) && (Area >= 1000)) {
Color[0]=120;
Color[1]=255;
Color[2]=120;
}
if ((Area < 3000) && (Area >= 2000)) {
Color[0]=90;
Color[1]=255;
Color[2]=90;
}
if ((Area < 4000) && (Area >= 3000)) {
Color[0]=0;
Color[1]=200;
Color[2]=20;
}
if ((Area < 5000) && (Area >= 4000)) {
Color[0]=0;
Color[1]=150;
Color[2]=0;
}
if ((Area < 6000) && (Area >= 5000)) {
Color[0]=0;
Color[1]=100;
Color[2]=0;
}
if (Area >= 6000) {
Color[0]=0;
Color[1]=50;
Color[2]=10;
}
setForegroundColor(Color[0], Color[1], Color[2]);
roiManager("Fill");
}
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
function FillFiberCartography(LamininWindows,ROIFiberFile,NameAreaImage,FiberArea,LegendBox) {
selectWindow(LamininWindows);
run("Duplicate...", "title="+NameAreaImage);
run("Enhance Contrast...", "saturated=0.3");
run("Add to Manager");
roiManager("Open", ROIFiberFile);
run("Select None");
nbFibers=roiManager("count");
for (i=0 ; i<nbFibers; i++) {
ChooseColorCartography(FiberArea[i], i, NameAreaImage);
}
if (LegendBox=="Yes") {
RangeArea=newArray("<250","250-500","500-750","750-1000","1000-2000","2000-3000","3000-4000","4000-5000","5000-6000",">6000");
ColorArea=newArray(255,255,255,221,255,221,189,255,189,153,255,153,120,255,120,90,255,90,0,200,20,0,150,0,0,100,0,0,50,10);
roiManager("reset");
selectWindow(NameAreaImage);
getDimensions(width, height, channels, slices, frames);
for (i=0; i < 10; i++) {
makeRectangle(0, i*100, 200, 100);
roiManager("Add");
}
for (i=0; i < 10; i++) {
roiManager("select", i);
setForegroundColor(ColorArea[3*i],ColorArea[3*i+1],ColorArea[3*i+2]);
roiManager("Fill");
if (i<8) {
setColor("black");
}
else {
setColor("white");
}
setFont("Sanserif", 40);
if (i<4) {
drawString(RangeArea[i], 25, i*100+75);
}
else {
drawString(RangeArea[i], 10, i*100+75);
}
}
}
}
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
function FillCentroNucleiCartography(LamininWindows,ROICentroFiberFile,NameCentroImage,CentroNuclei,LegendBox) {
selectWindow(LamininWindows);
run("Duplicate...", "title="+NameCentroImage);
run("Enhance Contrast...", "saturated=0.3");
run("Add to Manager");
roiManager("Open", ROICentroFiberFile);
run("Select None");
nbFibers=roiManager("count");
for (i=0 ; i<nbFibers; i++) {
selectWindow(NameCentroImage);
run("RGB Color");
roiManager("select", i);
if (CentroNuclei[i] == 1) {
setForegroundColor(255, 255, 0);
}
if (CentroNuclei[i] == 2) {
setForegroundColor(255, 128, 0);
}
if (CentroNuclei[i] > 2) {
setForegroundColor(255, 0, 0);
}
if (CentroNuclei[i] == 0) {
setForegroundColor(255, 255, 255);
}
roiManager("Fill");
}
if (LegendBox=="Yes") {
roiManager("reset");
selectWindow(NameCentroImage);
getDimensions(width, height, channels, slices, frames);
BottomY=height-100;
BottomYMidle=height-25;
makeRectangle(0, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 0);
setForegroundColor(255, 255, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 50);
drawString("0", 50, BottomYMidle);
makeRectangle(100, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 1);
setForegroundColor(255, 255, 0);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 50);
drawString("1", 125, BottomYMidle);
makeRectangle(200, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 2);
setForegroundColor(255, 128, 0);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 50);
drawString("2", 225, BottomYMidle);
makeRectangle(300, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 3);
setForegroundColor(255, 0, 0);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 50);
drawString("3+", 325, BottomYMidle);
}
}
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
function FillSatCellCartography(LamininWindows,NameSatCellImage,SatCell,LegendBox) {
selectWindow(LamininWindows);
run("Duplicate...", "title="+NameSatCellImage);
run("Enhance Contrast...", "saturated=0.3");
run("Add to Manager");
roiManager("Open", ROIFiberFile);
run("Select None");
nbFibers=roiManager("count");
for (i=0 ; i<nbFibers; i++) {
selectWindow(NameSatCellImage);
run("RGB Color");
roiManager("select", i);
if (SatCell[i] == 1) {
setForegroundColor(255, 200, 255);
}
if (SatCell[i] >= 2) {
setForegroundColor(255, 50, 255);
}
if (SatCell[i] == 0) {
setForegroundColor(255, 255, 255);
}
roiManager("Fill");
}
if (LegendBox=="Yes") {
roiManager("reset");
selectWindow(NameSatCellImage);
getDimensions(width, height, channels, slices, frames);
BottomY=height-100;
BottomYMidle=height-25;
makeRectangle(0, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 0);
setForegroundColor(255, 255, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 50);
drawString("0", 50, BottomYMidle);
makeRectangle(100, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 1);
setForegroundColor(255, 200, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 50);
drawString("1", 125, BottomYMidle);
makeRectangle(200, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 2);
setForegroundColor(255, 50, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 50);
drawString("2+", 225, BottomYMidle);
}
}
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
function FillVesselsCartography(LamininWindows,NameVesselImage,CountVessel,LegendBox) {
selectWindow(LamininWindows);
run("Duplicate...", "title="+NameVesselImage);
run("Enhance Contrast...", "saturated=0.3");
run("Add to Manager");
roiManager("Open", ROIFiberFile);
run("Select None");
nbFibers=roiManager("count");
for (i=0 ; i<nbFibers; i++) {
selectWindow(NameVesselImage);
run("RGB Color");
roiManager("select", i);
if (CountVessel[i]<=1) setForegroundColor(255, 255, 255);
else
if ((CountVessel[i]>= 2) && (CountVessel[i]<4)) setForegroundColor(200, 150, 255);
else
if ((CountVessel[i]>= 4) && (CountVessel[i]<6)) setForegroundColor(200, 100, 255);
else
if ((CountVessel[i]>= 6) && (CountVessel[i]<8)) setForegroundColor(200, 0, 255);
else
if ((CountVessel[i]>= 8) && (CountVessel[i]<10)) setForegroundColor(100, 0, 150);
else
if (CountVessel[i]>= 10) setForegroundColor(100, 0, 100);
roiManager("Fill");
}
if (LegendBox=="Yes") {
roiManager("reset");
selectWindow(NameVesselImage);
getDimensions(width, height, channels, slices, frames);
BottomY=height-100;
BottomYMidle=height-25;
makeRectangle(0, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 0);
setForegroundColor(255, 255, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 40);
drawString("0-1", 25, BottomYMidle);
makeRectangle(100, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 1);
setForegroundColor(200, 150, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 40);
drawString("2-3", 125, BottomYMidle);
makeRectangle(200, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 2);
setForegroundColor(200, 100, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 40);
drawString("4-5", 225, BottomYMidle);
makeRectangle(300, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 3);
setForegroundColor(200, 0, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 40);
drawString("6-7", 325, BottomYMidle);
makeRectangle(400, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 4);
setForegroundColor(100, 0, 150);
roiManager("Fill");
setColor("white");
setFont("Sanserif", 40);
drawString("8-9", 425, BottomYMidle);
makeRectangle(500, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 5);
setForegroundColor(100, 0, 100);
roiManager("Fill");
setColor("white");
setFont("Sanserif", 40);
drawString("10+", 525, BottomYMidle);
}
}
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
function TypeCartographyFill(NameTypeImage,FiberPhenoType1,FiberPhenoType2a,FiberPhenoType2b,FiberPhenoType2x,LegendBox)
{
Color = newArray(3);
selectWindow(NameTypeImage);
run("RGB Color");
nbFibers= roiManager("count")- 1;
for (i=0 ; i<nbFibers; i++) {
selectWindow(NameTypeImage);
// Ambiguous Type
Color[0]=255;
Color[1]=255;
Color[2]=255;
// Type I
if ((FiberPhenoType1[i] ==1) && (FiberPhenoType2a[i]==0) && (FiberPhenoType2b[i]==0)) {
Color[0]=255;
Color[1]=0;
Color[2]=0;
}
else
// Type IIA
if ((FiberPhenoType1[i] ==0) && (FiberPhenoType2a[i]==1) && (FiberPhenoType2b[i]==0)) {
Color[0]=185;
Color[1]=0;
Color[2]=255;
}
else
// Type IIB
if ((FiberPhenoType1[i] ==0) && (FiberPhenoType2a[i]==0) && (FiberPhenoType2b[i]==1)) {
Color[0]=0;
Color[1]=0;
Color[2]=255;
}
else
// Type IIB ambig
if ((FiberPhenoType1[i] ==0) && (FiberPhenoType2a[i]==0) && (FiberPhenoType2b[i]==2)) {
Color[0]=0;
Color[1]=0;
Color[2]=255;
}
else
// Type I or IIA
if ((FiberPhenoType1[i] ==1) && (FiberPhenoType2a[i]==1) && (FiberPhenoType2b[i]==0)) {
Color[0]=255;
Color[1]=0;
Color[2]=255;
}
else
// Type I or IIB
if ((FiberPhenoType1[i] ==1) && (FiberPhenoType2a[i]==0) && (FiberPhenoType2b[i]>0)) {
Color[0]=255;
Color[1]=140;
Color[2]=255;
}
else
// Type IIA or IIB
if ((FiberPhenoType1[i] ==0) && (FiberPhenoType2a[i]==1) && (FiberPhenoType2b[i]>0)) {
Color[0]=150;
Color[1]=150;
Color[2]=255;
}
else
// Type IIX
if (FiberPhenoType2x[i] ==1) {
Color[0]=0;
Color[1]=30;
Color[2]=139;
}
roiManager("select", i);
setForegroundColor(Color[0], Color[1], Color[2]);
roiManager("Fill");
}
// Add legend on bottom left
if (LegendBox=="Yes") {
roiManager("reset");
selectWindow(NameTypeImage);
getDimensions(width, height, channels, slices, frames);
BottomY=height-225;
BottomYMidle=height-150;
BottomYHyb=height-100;
BottomYHybMidle=height-25;
makeRectangle(0, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 0);
setForegroundColor(255, 0, 0);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 50);
drawString("I", 50, BottomYMidle);
makeRectangle(100, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 1);
setForegroundColor(185, 0, 255);
roiManager("Fill");
setColor("white");
setFont("Sanserif", 50);
drawString("IIA", 125, BottomYMidle);
makeRectangle(200, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 2);
setForegroundColor(0, 30, 139);
roiManager("Fill");
setColor("white");
setFont("Sanserif", 50);
drawString("IIX", 225, BottomYMidle);
makeRectangle(300, BottomY, 100, 100);
roiManager("Add");
roiManager("select", 3);
setForegroundColor(0, 0, 255);
roiManager("Fill");
setColor("white");
setFont("Sanserif", 50);
drawString("IIB", 325, BottomYMidle);
drawString("Pure Types", 425, BottomYMidle);
// Hybrid
makeRectangle(0, BottomYHyb, 100, 100);
roiManager("Add");
roiManager("select", 4);
setForegroundColor(255, 0, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 35);
drawString("I-IIA", 25, BottomYHybMidle);
makeRectangle(100, BottomYHyb, 100, 100);
roiManager("Add");
roiManager("select", 5);
setForegroundColor(255, 140, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 35);
drawString("I-IIB", 125, BottomYHybMidle);
makeRectangle(200, BottomYHyb, 100, 100);
roiManager("Add");
roiManager("select", 6);
setForegroundColor(150, 150, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 35);
drawString("IIA-IIB", 205, BottomYHybMidle);
makeRectangle(300, BottomYHyb, 100, 100);
roiManager("Add");
roiManager("select", 7);
setForegroundColor(255, 255, 255);
roiManager("Fill");
setColor("black");
setFont("Sanserif", 35);
drawString("ND", 325, BottomYHybMidle);
setColor("white");
setFont("Sanserif", 50);
drawString("Hybrid Types", 425, BottomYHybMidle);
roiManager("reset");
}
}
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
function NucleiDetectionOnDAPI(System,DAPIWindows) {
//open nuclei image
selectWindow(DAPIWindows);
run("Duplicate...", "title=[DAPI Temp]");
// Segmentation of nuclei
if (System=="Spinning Disk") {
run("Enhance Contrast...", "saturated=0.1");
}
else {
run("Enhance Contrast...", "saturated=0.1 normalize");
}
run("Subtract Background...", "rolling=50");
setAutoThreshold("Otsu dark");
setOption("BlackBackground", true);
run("Convert to Mask");
run("Options...", "iterations=1 count=2 black do=Erode");
run("Watershed");
run("Ultimate Points");
setThreshold(1, 255);
run("Convert to Mask");
run("Options...", "iterations=2 count=1 black do=Dilate");
run("Analyze Particles...", "size=1-200 pixel display clear");
nbNuclei= nResults - 1;
roiManager("reset");
selectWindow("Results");
run("Close");
return nbNuclei;
}
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
function ArtefactDetectionOnLaminin(FiberImageWindows, bCrop, LimitDetectionFiberArea, bArtefacts) {
selectWindow(FiberImageWindows);
run("Duplicate...", "title=[Fiber Temp]");
getStatistics(area, mean, min, max, std, histogram);
//MinArea=1000;
MinArea=0.05*area;
// Segmentation of entire section
run("Enhance Contrast", "saturated=0.60 normalize equalize");
if (bCrop)
setAutoThreshold("Li dark");
else
setAutoThreshold("Otsu dark");
run("Convert to Mask");
run("Fill Holes");
run("Analyze Particles...", "size="+MinArea+"-Infinity add display clear");
MaxSurf=0;
// Max Area has been kept
if (nResults >0) {
selectWindow("Results");
MaxIndex=0;
for (i=0; i<nResults; i++) {
CurrentArea=getResult("Area", i);
if (MaxSurf < CurrentArea) {
MaxIndex=i;
MaxSurf=CurrentArea;
}
}
run("Close");
selectWindow("ROI Manager");
if (roiManager("count") > 1) {
for (i=0; i < roiManager("count"); i++) {
if (i != MaxIndex) {
roiManager("Select", i);
roiManager("Delete");
}
}
}
roiManager("Set Color", "red");
roiManager("Set Line Width", 0);
// Quality Check
selectWindow(FiberImageWindows);
run("Duplicate...", "title=[Fiber Quality Check]");
selectWindow("Fiber Quality Check");
run("Enhance Contrast...", "saturated=10 normalize equalize");
run("Gaussian Blur...", "sigma=2");
run("Enhance Contrast...", "saturated=1 normalize");
run("Subtract Background...", "rolling=20");
if (bCrop == false) {
run("Gaussian Blur...", "sigma=10");
run("Subtract Background...", "rolling=50");
}
getStatistics(area, mean, min, max, std, histogram);
if (bCrop == false) {
ThresholdF=mean+std/3.0;
}
else {
ThresholdF=mean;
}
run("Find Maxima...", "noise="+ThresholdF+" output=[Segmented Particles] light");
selectWindow("Fiber Quality Check Segmented");
run("Invert");
run("Options...", "iterations=2 count=1 black do=Dilate");
run("Options...", "iterations=3 count=1 black do=Close");
run("Invert");
selectWindow("ROI Manager");
roiManager("Select", 0); // ROI selected as max area
run("Analyze Particles...", "size=0-infinity circularity=0.45-1.00 display add in_situ");
selectWindow("Results");
TotFiberArea=0;
for (i=1; i < roiManager("count")-1; i++) {
TotFiberArea=TotFiberArea+getResult("Area", i);
}
selectWindow("Results");
run("Close");
selectWindow("Fiber Quality Check Segmented");
close();
selectWindow("Fiber Quality Check");
close();
FibersAreaPercent= TotFiberArea/MaxSurf;
FibersAreaPercent= 100*FibersAreaPercent;
if (FibersAreaPercent < LimitDetectionFiberArea) {
bArtefacts=true;
}
AllROI=roiManager("count")-1;
i=1;
while (i <= AllROI) {
roiManager("Select", 1);
roiManager("Delete");
i=i+1;
}
}
selectWindow("Fiber Temp");
close();
return MaxSurf;
}
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
function FiberShapeDetectionOnLaminin(FiberImageWindows,ROIFiberFile,ROICentroFiberFile,ROISatCellFiberFile,ROIVesselFiberFile,FiberArea,FiberFeret,MinFiberFeret,FiberAreaClassCount,nThreshold, bCrop) {
// Segmentation of Fibers
selectWindow(FiberImageWindows);
run("Duplicate...", "title=[Fiber Temp]");
selectWindow("Fiber Temp");
run("Enhance Contrast...", "saturated=10 normalize equalize");
run("Gaussian Blur...", "sigma=2");
run("Enhance Contrast...", "saturated=1 normalize");
run("Subtract Background...", "rolling=20");
if (bCrop == false) {
run("Gaussian Blur...", "sigma=10");
run("Subtract Background...", "rolling=50");
}
getStatistics(area, mean, min, max, std, histogram);
if (bCrop == false) {
ThresholdF=mean+std/3.0;
}
else {
ThresholdF=mean;
}
run("Find Maxima...", "noise="+ThresholdF+" output=[Segmented Particles] light");
nThreshold[0]=ThresholdF;
selectWindow("Fiber Temp Segmented");
run("Invert");
run("Options...", "iterations=2 count=1 black do=Dilate");
run("Options...", "iterations=3 count=1 black do=Close");
run("Invert");
roiManager("Select", 0); // ROI selected as max area
run("Analyze Particles...", "size=0-infinity circularity=0.00-1.00 display add in_situ");
selectWindow("Results");
AreaMean=Variance=0;
for (i=1; i <roiManager("count")-1; i++) {
AreaMean=AreaMean+getResult("Area", i);
}
AreaMean=AreaMean/(roiManager("count")-2);
for (i=1; i <roiManager("count")-1; i++) {
Variance=Variance+((getResult("Area", i)-AreaMean)*(getResult("Area", i)-AreaMean));
}
selectWindow("Results");
run("Close");
Variance=Variance/(roiManager("count")-2);
StdDev=sqrt(Variance);
MinArea=100;
if (bCrop) {
MaxArea=AreaMean+4*StdDev;
}
else
MaxArea=AreaMean+3*StdDev;
AllROI=roiManager("count")-1;
i=1;
while (i <= AllROI) {
roiManager("Select", 1);
roiManager("Delete");
i=i+1;
}
TotSurfaceFibers =0;
nbFibers=nbFibersLast=0;
// second quality check to eliminate outliers
roiManager("Select", 0); // ROI selected as max area
run("Analyze Particles...", "size=MinArea-MaxArea circularity=0.45-1.00 display add in_situ");
nbFibers= roiManager("count")-1;
selectWindow("Results");
for (j=0; j<nbFibers; j++) {
FiberArea[j]=getResult("Area", j);
AreaClassAttribution(FiberArea[j],FiberAreaClassCount);
FiberFeret[j]=getResult("Feret", j);
MinFiberFeret[j]=getResult("MinFeret", j);
TotSurfaceFibers=TotSurfaceFibers+FiberArea[j];
}
selectWindow("ROI Manager");
roiManager("Select", 0); // ROI selected as max area
roiManager("Delete");
roiManager("Show All without labels");
roiManager("Set Color", "green");
roiManager("Set Line Width", 0);
roiManager("Save", ROIFiberFile);
selectWindow("Results");
run("Close");
// Save ROIs for Fiber Morphology Analysis
if (nbFibers > 0) {
selectWindow("ROI Manager");
// Save ROIs for FCN Analysis
for (i=0 ; i<nbFibers; i++) {
roiManager("select", i);
//1/5 of Feret Diameter Length
RealFNCROIwithFeret=MinFiberFeret[i]/5; // previousversion 8
run("Enlarge...", "enlarge=-"+RealFNCROIwithFeret);
//run("Enlarge...", "enlarge=-15");
roiManager("Add");
}
for (i=0 ; i<nbFibers; i++) {
roiManager("Select", 0);
roiManager("Delete");
}
roiManager("Set Color", "blue");
roiManager("Set Line Width", 0);
roiManager("Save", ROICentroFiberFile);
roiManager("reset");
// Save ROIs for Satellite Cell Analysis and Peri nuclei counting
roiManager("Open", ROIFiberFile);
nbSelectedFibers=roiManager("count");
roiManager("Open", ROICentroFiberFile);
AllFibersROI=roiManager("count");
selectWindow("ROI Manager");
roiManager("Show All without labels");
for (i=0 ; i<nbSelectedFibers; i++) {
iEnlarge=i+nbSelectedFibers;
MultiSelect= newArray(i,iEnlarge);
roiManager("Select", MultiSelect);
roiManager("XOR");
roiManager("Add");
}
selectWindow("ROI Manager");
for (i=0; i < AllFibersROI; i++) {
roiManager("Select", 0);
roiManager("Delete");
}
roiManager("Set Color", "#ffc800");
roiManager("Set Line Width", 0);
roiManager("Save", ROISatCellFiberFile);
roiManager("reset");
// Save ROIs for Vessel counting
roiManager("Open", ROIFiberFile);
nbSelectedFibers=roiManager("count");
roiManager("Show All without labels");
for (i=0 ; i<nbSelectedFibers; i++) {
roiManager("Select", i);
RealVROIwithFeret=MinFiberFeret[i]/8;
run("Enlarge...", "enlarge="+RealVROIwithFeret);
roiManager("Add");
}
AllFibersROI=roiManager("count");
for (i=0 ; i<nbSelectedFibers; i++) {
iEnlarge=i+nbSelectedFibers;
MultiSelect= newArray(i,iEnlarge);
roiManager("Select", MultiSelect);
roiManager("XOR");
roiManager("Add");
}
selectWindow("ROI Manager");
for (i=0; i < AllFibersROI; i++) {
roiManager("Select", 0);
roiManager("Delete");
}
roiManager("Set Color", "cyan");
roiManager("Set Line Width", 0);
roiManager("Save", ROIVesselFiberFile);
roiManager("reset");
}
selectWindow("Fiber Temp Segmented");
run("Close");
selectWindow("Fiber Temp");
run("Close");
return TotSurfaceFibers;
}
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
function CentroNucleiFiberTracking(ROIFiberFile,ROICentroFiberFile,ROIPeriFiberFile,DAPIWindows,CentroNuclei,PeriNuclei) {
selectWindow("DAPI Temp");
run("Make Binary");
run("Add to Manager");
roiManager("Open", ROICentroFiberFile);
nbFibers=roiManager("count");
NbCentroNucleiCount = 0;
roiManager("Show None");
for (i=0; i<nbFibers; i++) {
selectWindow("DAPI Temp");
roiManager("select", i);
run("Analyze Particles...", "size=1-100 pixel circularity=0.45-1.00 display clear");
CentroNuclei[i] = nResults;
if (CentroNuclei[i] > 0) {
NbCentroNucleiCount = NbCentroNucleiCount+1;
}
}
roiManager("reset");
roiManager("Open", ROIPeriFiberFile);
nbFibers=roiManager("count");
roiManager("Show None");
for (i=0; i<nbFibers; i++) {
selectWindow("DAPI Temp");
roiManager("select", i);
run("Analyze Particles...", "size=1-100 pixel circularity=0.45-1.00 display clear");
PeriNuclei[i] = nResults;
}
roiManager("reset");
return NbCentroNucleiCount;
}
/////////////////////////////////////////A REVOIR//////////////////////////////////////////////////////////
function SatCellDetectionOnPax7(System,Pax7Windows, DAPIWindows, nThreshold) {
selectWindow(Pax7Windows);
run("Duplicate...", "title=[Sat Temp]");
selectWindow("Sat Temp");
// Segmentation of cell
if (System=="Spinning Disk") {
run("Enhance Contrast...", "saturated=0.1");
}
else {
run("Enhance Contrast...", "saturated=0.1 normalize");
}
run("Subtract Background...", "rolling=50");
setAutoThreshold("Yen dark");
setOption("BlackBackground", true);
run("Convert to Mask");
run("Fill Holes");
run("Analyze Particles...", "size=3-100 circularity=0.45-1.00 display add clear"); // in microns!!
nbSatCells= nResults;
NbPosSatCells=0;
if (nbSatCells>0) {
selectWindow("Results");
run("Close");
// Algo without threshold
selectWindow("DAPI Temp");
run("Make Binary");
NbPosSatCells = 0;
roiManager("Show None");
for (i=0 ; i<nbSatCells; i++) {
selectWindow("DAPI Temp");
roiManager("select", i);
run("Analyze Particles...", "size=1-100 pixel circularity=0.45-1.00 display clear");
if (nResults > 0) {
NbPosSatCells = NbPosSatCells+1;
}
else {
roiManager("select", i);
roiManager("Delete");
i = i-1;
nbSatCells = nbSatCells-1;
}