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/**
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* Intensity Ratio Nuclei Cytoplasm Tool
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* Collaborators:
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* Olivier Coux
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*
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* Subtract the background intensity and measure the ratio of the intensity in the nuclei and the intensity in
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* the cytoplasm.
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*
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* (c) 2014, INSERM
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* written by Volker Baecker at Montpellier RIO Imaging (www.mri.cnrs.fr)
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*
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*/
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var _SUBTRACT_BACKGROUND_RADIUS = 1;
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var _SUBTRACT_BACKGROUND_OFFSET = 1;
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var _SUBTRACT_BACKGROUND_ITERATIONS = 2;
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var _SUBTRACT_BACKGROUND_SKIP_LIMIT = 0.05;
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var _NUCLEI_CHANNEL = "Hoechst";
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var _CYTOPLASM_CHANNEL = "GFP";
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var _MAX_PERCENT_SATURATED = 0.5;
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var _THRESHOLD_METHOD = "Huang";
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var _MIN_NUCLEUS_AREA = 20;
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var _helpURL = "http://dev.mri.cnrs.fr/wiki/imagej-macros/Intensity_Ratio_Nuclei_Cytoplasm_Tool";
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var _FILENAME;
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var _DIR;
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var _ID_CYTOPLASM_IMAGE;
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var _ID_NUCLEI_IMAGE;
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macro "Unused Tool - C037" { }
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macro 'Intensity Ratio Nuclei Cytoplasm Help (f1) Action Tool - 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'{
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intensityRatioForSingleImageHelp();
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}
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macro "correct background (f2) Action Tool - C000T4b12c" {
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findAndSubtractBackground(_SUBTRACT_BACKGROUND_RADIUS, _SUBTRACT_BACKGROUND_OFFSET, _SUBTRACT_BACKGROUND_ITERATIONS, _SUBTRACT_BACKGROUND_SKIP_LIMIT);
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}
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macro "correct background (f2) Action Tool Options" {
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Dialog.create("Correct Background Options");
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Dialog.addNumber("radius", _SUBTRACT_BACKGROUND_RADIUS);
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Dialog.addNumber("offset", _SUBTRACT_BACKGROUND_OFFSET);
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Dialog.addNumber("iterations", _SUBTRACT_BACKGROUND_ITERATIONS);
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Dialog.addNumber("skip limit", _SUBTRACT_BACKGROUND_SKIP_LIMIT);
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Dialog.show();
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_SUBTRACT_BACKGROUND_RADIUS = Dialog.getNumber();
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_SUBTRACT_BACKGROUND_OFFSET = Dialog.getNumber();
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_SUBTRACT_BACKGROUND_ITERATIONS = Dialog.getNumber();
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_SUBTRACT_BACKGROUND_SKIP_LIMIT = Dialog.getNumber();
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}
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macro "Intensity Ratio For Single Image (f3) Action Tool - C000T4b12s" {
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intensityRatioForSingleImage();
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}
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macro "Intensity Ratio For Single Image (f3) Action Tool Options" {
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createOptionsDialog();
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}
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macro "Intensity Ratio Batch (f4) Action Tool - C000T4b12b" {
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intensityRatioBatch();
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}
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macro "Intensity Ratio Batch (f4) Action Tool Options" {
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createOptionsDialog();
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}
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function createOptionsDialog() {
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Dialog.create("Intensity Ratio Options");
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Dialog.addString("nuclei channel: ", _NUCLEI_CHANNEL);
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Dialog.addString("cytoplasm channel: ", _CYTOPLASM_CHANNEL);
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Dialog.addNumber("max. % saturation: ", _MAX_PERCENT_SATURATED);
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Dialog.addChoice("thresholding method", newArray("Default", "Huang", "Intermodes", "IsoData", "IJ_IsoData", "Li", "MaxEntropy", "Mean", "MinError", "Minimum", "Moments", "Otsu", "Percentile", "RenyiEntropy", "Shanbhag", "Triangle", "Yen"), _THRESHOLD_METHOD);
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Dialog.addNumber("min. nucleus area", _MIN_NUCLEUS_AREA);
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Dialog.show();
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_NUCLEI_CHANNEL = Dialog.getString();
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_CYTOPLASM_CHANNEL = Dialog.getString();
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_MAX_PERCENT_SATURATED = Dialog.getNumber();
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_THRESHOLD_METHOD = Dialog.getChoice();
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_MIN_NUCLEUS_AREA = Dialog.getNumber();
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}
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macro "intensityRatioNucleiCytoplasmHelp [f1]" {
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intensityRatioForSingleImageHelp();
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}
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macro "correctBackground [f2]" {
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findAndSubtractBackground(_SUBTRACT_BACKGROUND_RADIUS, _SUBTRACT_BACKGROUND_OFFSET, _SUBTRACT_BACKGROUND_ITERATIONS, _SUBTRACT_BACKGROUND_SKIP_LIMIT);
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}
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macro "intensityRatioForSingleImage [f3]" {
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intensityRatioForSingleImage();
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}
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macro "intensityRatioBatch [f4]" {
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intensityRatioBatch();
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}
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function intensityRatioForSingleImageHelp() {
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run('URL...', 'url='+_helpURL);
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}
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function intensityRatioForSingleImage() {
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measureIntensityRatioForOpenImage();
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}
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function intensityRatioBatch() {
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print("\\Clear");
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setBatchMode(true);
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getDateAndTime(year, month, dayOfWeek, dayOfMonth, hour, minute, second, msec);
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print(dayOfMonth + "-" + (month+1) + "-" + year + " " + hour + ":" + minute + ":" + second + "." + msec);
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dir = getDirectory("Select the folder containing the images");
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files = getFileList(dir);
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numberOfImages = 0;
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for (i=0; i<files.length; i++) {
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file = files[i];
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if (isInputImage(file)) {
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numberOfImages++;
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}
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}
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counter = 1;
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for (i=0; i<files.length; i++) {
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file = files[i];
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if (isInputImage(file)) {
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print("\\Update1:Processing file " + (counter) + " of " + numberOfImages);
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open(dir+"/"+file);
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measureIntensityRatioForOpenImage();
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outDir = dir + "/" + "control-images";
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if (!File.exists(outDir)) {
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File.makeDirectory(outDir);
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}
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save(outDir + "/" + _FILENAME);
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close();
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counter++;
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}
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}
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selectWindow("INTENSITY-RATIO");
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saveAs("Text", _DIR + "/INTENSITY-RATIO" + ".xls");
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print("FINISHED");
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getDateAndTime(year, month, dayOfWeek, dayOfMonth, hour, minute, second, msec);
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print(dayOfMonth + "-" + (month+1) + "-" + year + " " + hour + ":" + minute + ":" + second + "." + msec);
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setBatchMode("exit and display");
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}
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function isInputImage(name) {
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if (!endsWith(name, ".tif") && !endsWith(name, ".TIF")) return false;
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if (indexOf(name, _CYTOPLASM_CHANNEL)==-1) return false;
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return true;
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}
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function measureIntensityRatioForOpenImage() {
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run("Set Measurements...", "area mean integrated area_fraction display redirect=None decimal=3");
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_FILENAME = getInfo("image.filename");
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_DIR= getInfo("image.directory");
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if (isImageSaturated(_MAX_PERCENT_SATURATED)) {
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print("skipped saturated image: " + _DIR + "/" + _FILENAME);
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return;
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}
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loadOtherChannel();
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selectImage(_ID_CYTOPLASM_IMAGE);
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findAndSubtractBackground(_SUBTRACT_BACKGROUND_RADIUS, _SUBTRACT_BACKGROUND_OFFSET, _SUBTRACT_BACKGROUND_ITERATIONS, _SUBTRACT_BACKGROUND_SKIP_LIMIT);
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selectImage(_ID_NUCLEI_IMAGE);
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setAutoThreshold(_THRESHOLD_METHOD + " dark");
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setOption("BlackBackground", true);
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run("Convert to Mask");
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run("Analyze Particles...", "size="+_MIN_NUCLEUS_AREA+"-Infinity show=Masks in_situ");
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run("Fill Holes");
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run("Create Selection");
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resetThreshold();
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selectImage(_ID_CYTOPLASM_IMAGE);
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run("Restore Selection");
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run("Measure");
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run("Make Inverse");
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run("Measure");
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totalIntensityNuclei = getResult("IntDen", nResults-2);
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meanIntensityNuclei = getResult("Mean", nResults-2);
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areaNuclei = getResult("Area", nResults-2);
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totalIntensityCytoplasm = getResult("IntDen", nResults-1);
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areaOutside = getResult("Area", nResults-1);
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percentAreaAboveZero = getResult("%Area", nResults-1);
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areaCytoplasm = (areaOutside * percentAreaAboveZero ) / 100;
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meanIntensityCytoplasm = totalIntensityCytoplasm / areaCytoplasm;
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tableTitle="INTENSITY-RATIO";
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table="["+tableTitle+"]";
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if (!isOpen(tableTitle)) {
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run("Table...", "name="+tableTitle+" width=550 height=250");
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print(table, "\\Headings:n\timage\ticn factor\t% nuclei\t% cytoplasm\tav. nuclei intensity\tav. cytoplasm intensity\tnuclei area\tcytoplasm area\tt. nuclei intensity\tt. cytoplasm intensity\tfolder");
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}
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selectWindow(tableTitle);
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info = split(getInfo("window.contents"), "\n");
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index = lengthOf(info);
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percentIntensityNuclei = 100 * (totalIntensityNuclei / ((totalIntensityCytoplasm + totalIntensityNuclei) * 1.0));
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percentIntensityCytoplasm = 100 * (totalIntensityCytoplasm / ((totalIntensityCytoplasm + totalIntensityNuclei) * 1.0));
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icnFactor = percentIntensityNuclei / (percentIntensityCytoplasm * 1.0);
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line = "" + index + "\t" + _FILENAME + "\t" + icnFactor + "\t" + percentIntensityNuclei + "\t" + percentIntensityCytoplasm + "\t" + meanIntensityNuclei + "\t" + meanIntensityCytoplasm + "\t" + areaNuclei + "\t" + areaCytoplasm + "\t" + totalIntensityNuclei + "\t" + totalIntensityCytoplasm + "\t" + _DIR;
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print (table, line);
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createControlImage();
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}
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function createControlImage() {
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selectImage(_ID_NUCLEI_IMAGE);
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nucleiTitle = getTitle();
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run("RGB Color");
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selectImage(_ID_CYTOPLASM_IMAGE);
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cytoplasmTitle = getTitle();
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run("RGB Color");
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run("Make Inverse");
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foreground = getValue("color.foreground");
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run("Line Width...", "line=3");
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setForegroundColor(255, 0, 229);
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run("Draw");
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setForegroundColor(foreground);
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run("Line Width...", "line=1");
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run("Select None");
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run("Combine...", "stack1=" + cytoplasmTitle + " stack2="+ nucleiTitle+ " combine");
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}
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function loadOtherChannel() {
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if (indexOf(_FILENAME, _NUCLEI_CHANNEL)!=-1) {
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otherFilename = replace(_FILENAME, _NUCLEI_CHANNEL, _CYTOPLASM_CHANNEL);
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_ID_NUCLEI_IMAGE = getImageID();
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}
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else {
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otherFilename = replace(_FILENAME, _CYTOPLASM_CHANNEL, _NUCLEI_CHANNEL);
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_ID_CYTOPLASM_IMAGE = getImageID();
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}
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open(_DIR + "/" + otherFilename);
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if (indexOf(_FILENAME, _NUCLEI_CHANNEL)!=-1) {
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_ID_CYTOPLASM_IMAGE = getImageID();
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}
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else {
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_ID_NUCLEI_IMAGE = getImageID();
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}
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}
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function isImageSaturated(maxPercentSaturated) {
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getStatistics(area, mean, min, max, std, histogram);
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maxValue = 255;
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theBitDepth = bitDepth();
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width = getWidth();
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height = getHeight();
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if (theBitDepth==16) {
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maxValue = 65535;
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getHistogram(0, counts, 65536);
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histogram = counts;
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}
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totalNumberOfPixels = width * height;
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numberOfSaturatedPixels = histogram[maxValue];
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percent = (numberOfSaturatedPixels * 100) / (totalNumberOfPixels * 1.0);
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result = (percent>maxPercentSaturated);
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return result;
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}
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function findAndSubtractBackground(radius, offset, iterations, skipLimit) {
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width = getWidth();
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height = getHeight();
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getStatistics(area, mean, min, max, std, histogram);
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ratio = histogram[0] / ((width * height) * 1.0);
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if (ratio>skipLimit) {
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run("HiLo");
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run("Enhance Contrast", "saturated=0.35");
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return;
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}
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for(i=0; i<iterations; i++) {
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getStatistics(area, mean, min, max, std, histogram);
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minPlusOffset = min + offset;
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currentMax = 0;
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for(x=0; x<width; x++) {
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for(y=0; y<height; y++) {
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intensity = getPixel(x,y);
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if (intensity<=minPlusOffset) {
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value = getMaxIntensityAround(x, y, mean, radius, width, height);
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if (value>currentMax) currentMax = value;
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}
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}
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}
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result = currentMax / (i+1);
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run("Subtract...", "value=" + result);
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}
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run("HiLo");
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run("Enhance Contrast", "saturated=0.35");
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}
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function getMaxIntensityAround(x, y, mean, radius, width, height) {
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max = 0;
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for(i=x-radius; i<=x+radius; i++) {
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if (i>=0 && i<width) {
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for(j=y-radius; j<=y+radius; j++) {
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if (j>=0 && j<height) {
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value = getPixel(i,j);
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if (value<mean && value>max) max = value;
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}
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}
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}
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}
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return max;
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}
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