/*
* Cell_proliferationHCS
* Authors: Pau Carrillo-Barberà, José M. Morante-Redolat, José F. Pertusa
* Department of Cellular & Functional Biology
* University of Valencia (Valencia, Spain)
*
* February 2018
* Last update: January 10, 2019
*/
//This macro is a high-content screening tool for cell proliferation assays of
//adherent cell cultures. It is based on nucleoside analogue pulse alone or in
//combination with up to two additional nuclear markers.
macro "Cell_proliferationHCS" {
//choose a macro mode and a directory
#@ String (label=" ", value="High Content Screening
Cell Proliferation", visibility=MESSAGE, persist=false) heading
#@ String(label="Select mode:", choices={"Analysis", "Pre-Analysis (parameter tweaking)", "Pre-Analysis (visualization)", "Filename Transformation"}, style="radioButtonVertical") mode
#@ File(label="Select a directory:", style="directory") dir
#@ String (label=" ", value="![](\"http://oi64.tinypic.com/ekrmvs.jpg\")
", visibility=MESSAGE, persist=false) logo
#@ String (label=" ", value="Neuromolecular Biology Lab
ERI BIOTECMED - Universitat de València (Spain)", visibility=MESSAGE, persist=false) message
requires("1.52e");
//Filename Transformation
if(mode=="Filename Transformation") {
formats=newArray("Operetta", "NIS Elements");
Dialog.create("Input Format");
Dialog.addRadioButtonGroup("Select:", formats, formats.length, 1, formats[0]);
Dialog.show()
inputFormat=Dialog.getRadioButton();
print("Initializing 'Filename Transformation' mode");
//Operetta
if(inputFormat=="Operetta") {
//create an array containing the names of the files in the directory path
list = getFileList(dir);
Array.sort(list);
tiffFiles=0;
//count the number of TIF files
for (i=0; i count+1) {
index1=indexOf(tiffArray[count], "wv ");
index2=lastIndexOf(tiffArray[count], " - ");
channels[count]=substring(tiffArray[count], index1+3, index2);
count++;
}
//add an 'Empty' option into the channels name array
for (i=0; i=imagesxfield) {
channels[i]="Empty";
}
}
//create a channel array without the 'Empty' option
channelsSlice=Array.slice(channels, 0, channels.length-1);
//‘Pre-Analysis (parameter tweaking)’ and ‘Analysis’ parameterization
//browse a parameter dataset file (optional) & define output folder and dataset file names
dirName="Output - " + File.getName(dir);
resultsName="ResultsTable - " + File.getName(dir);
radioButtonItems=newArray("Yes", "No");
//‘Input & Output’ dialog box
Dialog.create("Input & Output");
Dialog.addRadioButtonGroup("Browse a pre-established parameter dataset:", radioButtonItems, 1, 2, "No");
Dialog.addMessage("Output folder:");
Dialog.addString("", dirName, 40);
Dialog.addMessage("Output parameter dataset file (txt):");
Dialog.addString("", "parameter_dataset", 40);
if(mode=="Analysis") {
Dialog.addMessage("Results table:");
Dialog.addString("", resultsName, 40);
}
html = ""
+"Having generated a parameter dataset txt file using the
"
+"Pre-Analysis (parameter tweaking) mode it is possible to
"
+"browse the file to apply the pre-established parameters";
Dialog.addHelp(html);
Dialog.show()
browseDataset=Dialog.getRadioButton();
outputFolder=Dialog.getString();
datasetFile=Dialog.getString();
if(mode=="Analysis") {
resultsTableName=Dialog.getString();
}
//set some parameter menu arrays
enhanceContrastOptions=newArray("0", "0.1", "0.2", "0.3", "0.4", "None");
threshold=getList("threshold.methods");
pattern=newArray(4);
//Extract values from a parameter dataset file
if(browseDataset=="Yes") {
parametersDatasetPath=File.openDialog("Choose the parameter dataset file to Open:");
//parameter selection (pre-established)
parametersString=File.openAsString(parametersDatasetPath);
parameterRows=split(parametersString, "\n");
parameters=newArray(parameterRows.length);
for(i=0; i"
+"
"
+""
+"Funded by the Galactic Empire
"
+"
"
+""
+"In ImageJ 1.46b or later, dialog boxes
"
+"can have a Help button that displays
"
+"HTML formatted text.
"
+"";
Dialog.addHelp(html);
Dialog.show()
pattern[0]=Dialog.getChoice();
pattern[1]=Dialog.getChoice();
pattern[2]=Dialog.getChoice();
pattern[3]=Dialog.getChoice();
rollingNuclei=Dialog.getNumber();
enhanceNuclei=Dialog.getChoice();
gaussianNuclei=Dialog.getNumber();
thresholdNuclei=Dialog.getChoice();
erodeNuclei=Dialog.getNumber();
openNuclei=Dialog.getNumber();
watershedNuclei=Dialog.getCheckbox();
minNuclei=Dialog.getNumber();
maxNuclei=Dialog.getNumber();
rollingNucleoside=Dialog.getNumber();
enhanceNucleoside=Dialog.getChoice();
gaussianNucleoside=Dialog.getNumber();
thresholdNucleoside=Dialog.getChoice();
erodeNucleoside=Dialog.getNumber();
openNucleoside=Dialog.getNumber();
watershedNucleoside=Dialog.getCheckbox();
minNucleoside=Dialog.getNumber();
maxNucleoside=Dialog.getNumber();
//check the channel selection
if(pattern[0]==pattern[1]) {
beep();
exit("Nuclei ["+pattern[0]+"] and nucleoside analogue ["+pattern[1]+"] channels can not be the same")
} else if (pattern[0]==pattern[2] || pattern[0]==pattern[3]) {
beep();
exit("Nuclei ["+pattern[0]+"] and addititional marker ["+pattern[2]+"]/["+pattern[3]+"] channels can not be the same")
} else if (pattern[1]==pattern[2] || pattern[1]==pattern[3]) {
beep();
exit("Nucleoside analogue ["+pattern[1]+"] and addititional marker ["+pattern[2]+"]/["+pattern[3]+"] channels can not be the same")
} else if (pattern[2]==pattern[3] && pattern[2]!="Empty") {
beep();
exit("Addititional marker ["+pattern[2]+"]/["+pattern[3]+"] channels can not be the same")
}
//Create an output folder
outputFolderPath=dir+"\\"+outputFolder;
File.makeDirectory(outputFolderPath);
//Create a parameter dataset file
title1 = "Parameter dataset";
title2 = "["+title1+"]";
f = title2;
run("Table...", "name="+title2+" width=500 height=500");
print(f, "Nuclei\t" + pattern[0]);
print(f, "Nucleoside analogue\t" + pattern[1]);
print(f, "Marker1\t" + pattern[2]);
print(f, "Marker2\t" + pattern[3]);
print(f, "Rolling (nuclei)\t" + rollingNuclei);
print(f, "Enhance (nuclei)\t" + enhanceNuclei);
print(f, "Gaussian (nuclei)\t" + gaussianNuclei);
print(f, "Threshold (nuclei)\t" + thresholdNuclei);
print(f, "Erode (nuclei)\t" + erodeNuclei);
print(f, "Open (nuclei)\t" + openNuclei);
print(f, "Watershed (nuclei)\t" + watershedNuclei);
print(f, "Rolling (nucleoside)\t" + rollingNucleoside);
print(f, "Enhance (nucleoside)\t" + enhanceNucleoside);
print(f, "Gaussian (nucleoside)\t" + gaussianNucleoside);
print(f, "Threshold (nucleoside)\t" + thresholdNucleoside);
print(f, "Erode (nucleoside)\t" + erodeNucleoside);
print(f, "Open (nucleoside)\t" + openNucleoside);
print(f, "Watershed (nucleoside)\t" + watershedNucleoside);
print(f, "Size-Min (nuclei)\t" + minNuclei);
print(f, "Size-Max (nuclei)\t" + maxNuclei);
print(f, "Size-Min (nucleoside)\t" + minNucleoside);
print(f, "Size-Max (nucleoside)\t" + maxNucleoside);
//save as TXT
saveAs("txt", outputFolderPath+"\\"+datasetFile);
selectWindow(title1);
run("Close");
//create an array containing the well codes
for (i=0; i=10) {
maxRandomFields=10;
} else {
maxRandomFields=fieldsxwell;
}
Dialog.addMessage("Random fields per well:");
Dialog.addSlider("", 1, maxRandomFields, maxRandomFields);
}
Dialog.show();
selectAll=Dialog.getCheckbox();
for (i=0; i=0; i--) {
if (number==randomArray[i]) {
recurrent=true;
}
}
if(recurrent==false || count==0) {
//Open images
for (i=0; i10 && aproxN<=255) { //maxima filter IF statement beginning
//Merge channels
run("Merge Channels...", "c1="+pattern[1]+" c3="+pattern[0]+" create keep");
run("Stack to RGB");
rename("RGB");
//Draw the outlines of the nuclei and nucleoside analogue binary masks
//Nuclei segmentation
segmentationPreAnalysis(pattern[0], rollingNuclei, enhanceNuclei, gaussianNuclei, thresholdNuclei, erodeNuclei, openNuclei, watershedNuclei, "RGB", minNuclei, maxNuclei, 0, 255, 255);
//Nucleoside analogue segmentation
segmentationPreAnalysis(pattern[1], rollingNucleoside, enhanceNucleoside, gaussianNucleoside, thresholdNucleoside, erodeNucleoside, openNucleoside, watershedNucleoside, "RGB", minNucleoside, maxNucleoside, 255, 105, 0);
//Save the image
saveAs("tiff", outputFolderPath+"\\"+wellName[z]+" fld "+field[(z*fieldsxwell*imagesxfield)+(number*imagesxfield)]);
//Clean up
cleanUp();
randomArray[count]=number;
count++;
} else { //maxima filter ELSE statement
beep();
cleanUp();
} //maxima filter IF-ELSE statement ending
cleanUp();
} else { //nImages ELSE statement
beep();
cleanUp();
} //nImages IF-ELSE statement ending
}
}
}
} //nWells FOR statement ending
setBatchMode(false);
print("End of process");
print("Find the output at:");
print(outputFolderPath);
//Visualization
setVisualization(outputFolderPath);
}
//Analysis workflow
if(mode=="Analysis") {
print("Initializing 'Analysis' mode");
wellsToAnalyze=0;
for(i=0; i=i) {
progressBar+="*";
} else {
progressBar+="-";
}
}
progressBar+="|";
print(progressBar, progressString, "%");
//Channel images checkpoint
if (nImages==imagesxfield) { //nImages IF statement beginning
//Channel identification
channelIdentification(imagesxfield, pattern);
//8-bits conversion (nuclei & nucleoside analogue)
selectImage(pattern[0]);
run("8-bit");
selectImage(pattern[1]);
run("8-bit");
//maximaFilter checkpoint
aproxN=maximaFilter(pattern[0]);
if (aproxN>10 && aproxN<=255) { //maxima filter IF statement beginning
//Nuclei segmentation
segmentation(pattern[0], rollingNuclei, enhanceNuclei, gaussianNuclei, thresholdNuclei, erodeNuclei, openNuclei, watershedNuclei);
//Nucleoside analogue segmentation
segmentation(pattern[1], rollingNucleoside, enhanceNucleoside, gaussianNucleoside, thresholdNucleoside, erodeNucleoside, openNucleoside, watershedNucleoside);
rename("Segmented");
//Nucleoside analogue size selection
run("Analyze Particles...", "size="+minNucleoside+"-"+maxNucleoside+" show=Masks");
rename(pattern[1]);
close("Segmented");
//One by one nuclei analysis
//Create a nuclei ‘Count Masks’ image
nFeat=createCountMasks(pattern[0], minNuclei, maxNuclei);
imageResults=newArray(nFeat);
meanResult1=newArray(nFeat);
meanResult2=newArray(nFeat);
imageDataname=newArray(nFeat);
//Single nucleus segmentation
for (i=1; i<=nFeat; ++1) { //nucleus by nucleus analysis FOR statement beginning
nameNucleous=pattern[0]+"-"+i;
nameM1=pattern[1]+"-"+i;
//Nucleoside analogue analysis
imageResults[i-1]=nucleosideAnalysis(pattern[0], nameNucleous, i, pattern[1], nameM1);
//Additional marker 1 analysis
meanResult1[i-1]=markerAnalysis(pattern[2], nameNucleous);
//Additional marker 2 analysis
meanResult2[i-1]=markerAnalysis(pattern[3], nameNucleous);
//Close binary images of nucleoside analogue nucleous-by-nucleous loop
close(nameM1);
close(nameNucleous);
//Results storage
imageDataname[i-1]=wellAndFieldName;
if(i==nFeat) {
if(firstRound==true) {
nucleosideAnalogue=imageResults;
marker1=meanResult1;
marker2=meanResult2;
dataname=imageDataname;
firstRound=false;
} else {
nucleosideAnalogue=Array.concat(nucleosideAnalogue, imageResults);
marker1=Array.concat(marker1, meanResult1);
marker2=Array.concat(marker2, meanResult2);
dataname=Array.concat(dataname, imageDataname);
}
}
} //nucleus by nucleus analysis FOR statement ending
cleanUp();
} else { //maxima filter ELSE statement
beep();
cleanUp();
} //maxima filter IF-ELSE statement ending
cleanUp();
} else { //nImages ELSE statement
beep();
cleanUp();
} //nImages IF-ELSE statement ending
} else { //checkbox ELSE statement
count += imagesxwell;
count2 = fieldsxwell;
} //checkbox IF-ELSE statement ending
} //count2 WHILE statement ending
} //nWells FOR statement ending
setBatchMode(false);
//results table
resultsTable("Results table", pattern[2], pattern[3], dataname, nucleosideAnalogue, marker1, marker2);
//save as TXT
saveAs("txt", outputFolderPath+"\\"+resultsTableName);
selectWindow("Results table");
run("Close");
// From ImageJ website (macro examples GetDateAndTime.txt)
// This macro demonstrates how to use the getDateAndTime()
// function, available in ImageJ 1.34n or later.
MonthNames = newArray("Jan","Feb","Mar","Apr","May","Jun","Jul","Aug","Sep","Oct","Nov","Dec");
DayNames = newArray("Sun", "Mon","Tue","Wed","Thu","Fri","Sat");
getDateAndTime(year, month, dayOfWeek, dayOfMonth, hour, minute, second, msec);
TimeString ="Date: "+DayNames[dayOfWeek]+" ";
if (dayOfMonth<10) {TimeString = TimeString+"0";}
TimeString = TimeString+dayOfMonth+"-"+MonthNames[month]+"-"+year+"\nTime: ";
if (hour<10) {TimeString = TimeString+"0";}
TimeString = TimeString+hour+":";
if (minute<10) {TimeString = TimeString+"0";}
TimeString = TimeString+minute+":";
if (second<10) {TimeString = TimeString+"0";}
TimeString = TimeString+second;
title1 = "Info";
title2 = "["+title1+"]";
fInfo = title2;
run("Table...", "name="+title2+" width=500 height=500");
print(fInfo, "ImageJ "+getVersion());
print(fInfo, TimeString);
saveAs("txt", outputFolderPath+"\\Info");
selectWindow("Info");
run("Close");
print("\\Clear");
print("ImageJ "+getVersion());
print(TimeString);
print("");
print("End of process");
print("Find the results table at:");
print(outputFolderPath);
print("");
}
if(mode=="Pre-Analysis (visualization)") {
open(dir+"\\"+"Multi-image.tif");
}
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
//User-defined functions
function channelIdentification(imageNumber, channels) { //CHANNELIDENTIFICATION function beginning
fileArray=newArray(imageNumber);
for (i=1; i<=imageNumber; i++) {
selectImage(i);
ima1=getTitle();
fileArray[i-1]=ima1;
}
for (j=1; j<=imageNumber; j++) {
for (i=1; i<=imageNumber; i++) {
if (indexOf (fileArray[i-1], channels[j-1])>0) {
selectImage(fileArray[i-1]);
rename (channels[j-1]);
}
}
}
} //CHANNELIDENTIFICATION function ending
function maximaFilter(image) { //MAXIMAFILTER function beginning
selectImage(image);
run("Duplicate...", "title="+image+"-MaximaFilter");
run("Subtract Background...", "rolling=50");
run("Enhance Contrast...", "saturated=0.4 normalize");
run("Find Maxima...", "noise=100 output=[Count]");
localMaxima=getResult("Count", 0);
run("Clear Results");
close(image+"-MaximaFilter");
return localMaxima;
} //MAXIMAFILTER function ending
function segmentation(image, rolling, enhance, gaussian, threshold, erode, openArg, watershed) { //SEGMENTATION function beginning
selectImage(image);
run("Subtract Background...", rolling);
if(enhance!="None") {
run("Enhance Contrast...", "saturated="+enhance+" normalize");
}
run("Gaussian Blur...", "sigma="+gaussian);
setAutoThreshold(threshold+" dark");
run("Convert to Mask");
run("Fill Holes");
run("Options...", "iterations="+erode+" count=1 do=Erode");
run("Options...", "iterations="+openArg+" count=1 do=Open");
if(watershed) {
run("Watershed");
}
} //SEGMENTATION function ending
function createCountMasks(image, min, max) { //CREATECOUNTMASKS function beginning
selectImage(image);
run("Analyze Particles...", " size="+min+"-"+max+" show=[Count Masks] display clear");
rename(image+"-Count Masks");
close(image);
output=nResults;
run("Clear Results");
return output;
} //CREATECOUNTMASKS function ending
function nucleosideAnalysis(nuclei, nucleusIter, iter, nucleoside, nucleosideIter) { //NUCLEOSIDEANALYSIS function beginning
run("Set Measurements...", "display redirect=None decimal=2");
selectImage(nuclei+"-Count Masks");
run("Duplicate...", "title="+nucleusIter);
setThreshold(iter, iter);
run("Convert to Mask");
run("Make Binary");
//binary reconstruct of nucleoside analogue
run("BinaryReconstruct ", "mask="+nucleoside+ " seed="+nucleusIter+" create white");
rename(nucleosideIter);
run("Analyze Particles...", " size=0-Infinity show=Nothing display clear");
results=nResults;
if (results>0) {
outcome=1;
} else {
outcome=0;
}
run("Clear Results");
return outcome;
} //NUCLEOSIDEANALYSIS function ending
function markerAnalysis(marker, nucleusIter) { //MARKER function beginning
if (marker!="Empty") {
selectImage(nucleusIter);
run("Set Measurements...", "mean redirect="+marker+" decimal=2");
run("Analyze Particles...", " size=0-Infinity show=Nothing display clear");
output=getResult("Mean", 0);
run("Clear Results");
} else {
output=0;
}
return output;
} //MARKER function ending
function resultsTable(title, m1, m2, nameArray, nucleosideArray, m1Array, m2Array) { //RESULTSTABLE function beginning
title1 = title;
title2 = "["+title1+"]";
f = title2;
run("Table...", "name="+title2+" width=500 height=500");
print(f, "\\Headings:n\tImage\tS-phase\t"+m1+"\t"+m2);
for (i=0; i0) {
selectImage(nImages());
run("Close");
}
} //CLEAN-UP function ending
function setVisualization(folder) {
setBatchMode(true);
run("Image Sequence...", "open=["+folder+"] file=tif sort");
width=getWidth();
height=getHeight();
setBackgroundColor(255, 255, 255);
run("Canvas Size...", "width="+width+width*0.05+" height="+height+" position=Center");
width=getWidth();
run("Canvas Size...", "width="+width+" height="+height+height*0.025+" position=Bottom-Center");
height=getHeight();
run("Canvas Size...", "width="+width+" height="+height+height*0.10+" position=Top-Center");
Stack.getDimensions(width, finalHeight, channels, slices, frames);
for (i=1; i<=slices; i++) {
Stack.setSlice(i);
setForegroundColor(0, 255, 255);
setLineWidth(height*0.005);
drawLine(width*0.1, height+height*0.033, width*0.2, height+height*0.033);
setForegroundColor(255, 105, 0);
drawLine(width*0.1, height+height*0.066, width*0.2, height+height*0.066);
setForegroundColor(0, 0, 0);
setFont("SansSerif", height*0.025);
setJustification("left");
drawString("Nuclei segmentation outlines", width*0.25, height+(height*0.033)+(height*0.01));
drawString("Nucleoside analogue segmentation outlines", width*0.25, height+(height*0.066)+(height*0.01));
}
Stack.setSlice(1);
setBatchMode(false);
saveAs("tiff", folder+"\\Multi-image");
}
}