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SMG-get-Col4-BM-edge-AUC-peakHeight-by-line-scan.ijm
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SMG-get-Col4-BM-edge-AUC-peakHeight-by-line-scan.ijm
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inputFolder = getDirectory("Choose the folder containing images to process:");
// Create an output folder based on the inputFolder
parentFolder = getPath(inputFolder); inputFolderPrefix = getPathFilenamePrefix(inputFolder);
outputFolder = parentFolder + inputFolderPrefix + "-BM-line-scan-output" + File.separator;
if ( !(File.exists(outputFolder)) ) { File.makeDirectory(outputFolder); }
rawLinescanFolder = outputFolder + "raw-BM-linescan-profile-record" + File.separator;
if ( !(File.exists(rawLinescanFolder)) ) { File.makeDirectory(rawLinescanFolder); }
line_width = 3; // in microns; the script will use image-embedded pixel size information to convert into pixels
n_edges = 5; // number of edges to quantify per filed of view
cCol4 = 3; // the channel number of alpha9-integrin staining
cNLSmSL = 2; // the channel number of NLS-mSarlet-I
run("Close All"); run("Collect Garbage"); // Release occupied memory
run("Clear Results");
setBatchMode(false); // always needs user input (draw line)
// Create a text window to record the summary metrics of E-cadherin quantification
// Note: this is to create a separate holder to circumvent that the ImageJ macro only allows 1 open file handle at a time
sumTextWindow = "Ecad-quantification-Summary";
if ( isOpen(sumTextWindow) ) { print("["+sumTextWindow+"]", "\\Update:"); } // clears the window
else { run("Text Window...", "name=["+sumTextWindow+"]"); }
print("["+sumTextWindow+"]", "file_name" + "," + "id" + "," + "Col4_AUC" + "," + "Col4_peak_height" + "," + "z_position");
fileList = getFileList(inputFolder);
for (i=0; i<fileList.length; i++) {
//for (i=0; i<1; i++) {// uncomment to run only 1 image for testing
// loop through the file list
filename = inputFolder + fileList[i];
outputPrefix = getFilenamePrefix(fileList[i]);
if ( endsWith(filename, ".nd2") || endsWith(filename, ".czi") || endsWith(filename, ".tif") ) {
// only process image files that ends with nd2, czi or tif
open(filename); id = getImageID();
// adjust brightness
Stack.setChannel(cCol4);
satu=1.0; run("Enhance Contrast", "saturated="+satu);
Stack.setChannel(cNLSmSL);
satu=1.0; run("Enhance Contrast", "saturated="+satu);
// get pixel size and convert desired micron-unit of line width to pixel size
getPixelSize(unit, pixelWidth, pixelHeight);
if (unit == "microns") {
line_width_px = round(line_width / pixelWidth);
}
else {
print("WARNING: pixel size is not in microns. Check image scaling.");
line_width_px = 30; // assuming pixel size is ~0.1 micron, a close estimate for 60x high-resolution immunostaining images
}
for (id_edge=1; id_edge<n_edges+1; id_edge++) {
// ask user to draw a line to get the line scan profile
setTool("line"); run("Line Width...", "line="+line_width_px); // line width can be adjusted to what you think is appropriate
waitForUser("Draw a line across the bud edge from an NLSmSL positive cell");
// open a text file to store the line scan profile
textRecord = rawLinescanFolder + outputPrefix + "-" + id_edge + "-a6int.txt";
f = File.open(textRecord);
print(f, "Col4_intensity");
// Get raw data of the E-cadherin line scan profile
selectImage(id); Stack.setChannel(cCol4);
profile = getProfile();
for (j=0; j<profile.length; j++) {
print(f, profile[j]);
}
File.close(f); // Close the file after saving all line scan values
Col4_AUC = getAUC(profile);
Col4_peak_height = getPeakHeight(profile);
// open a text file to store the line scan profile
textRecord = rawLinescanFolder + outputPrefix + "-" + id_edge + "-NLSmSL.txt";
f = File.open(textRecord);
print(f, "NLSmSL_intensity");
// Get raw data of the NLSmSL line scan profile
selectImage(id); Stack.setChannel(cNLSmSL);
profile = getProfile();
for (j=0; j<profile.length; j++) {
print(f, profile[j]);
}
File.close(f); // Close the file after saving all line scan values
// NLSmSL_cell_1 = getNLSmSL1(profile);
// NLSmSL_cell_2 = getNLSmSL2(profile);
Stack.getPosition(channel, z_position, frame);
print("["+sumTextWindow+"]", "\n" + outputPrefix + "," + id_edge + "," + Col4_AUC + "," + Col4_peak_height + "," + z_position);
run("Draw", "slice"); // Draw ROI on the image to avoid repeat measurements
}
// Duplicate the single slice image and save for record (or re-run line scan on a smaller data set if necessary)
selectImage(id); run("Close");
}
}
// Save and the summary text window
selectWindow(sumTextWindow);
inputFolderPrefix = getPathFilenamePrefix(inputFolder);
timeFinish = getTime(); timeStamp = timeFinish % 10000;// time stamp for saving log and summary text files
sumOutFile = outputFolder + inputFolderPrefix + "-" + sumTextWindow + "-" + timeStamp + ".txt";
saveAs("txt", sumOutFile); run("Close");
setTool("rectangle");
run("Close All");
run("Collect Garbage");
run("Clear Results");
function getAUC(profile) {
background_length = floor(profile.length/10);
background_profile = Array.trim(profile, background_length);
Array.getStatistics(background_profile, min, max, mean_background, stdDev);
Array.getStatistics(profile, min, max, mean_raw, stdDev);
AUC = profile.length * (mean_raw - mean_background);
return AUC;
}
function getPeakHeight(profile) {
background_length = floor(profile.length/10);
background_profile = Array.trim(profile, background_length);
Array.getStatistics(background_profile, min, max, mean_background, stdDev);
Array.getStatistics(profile, min, max_raw, mean, stdDev);
peak_height = max_raw - mean_background;
return peak_height;
}
function getNLSmSL1(profile) {
NLSmSL1_length = floor(profile.length/3);
NLSmSL1_profile = Array.trim(profile, NLSmSL1_length);
Array.getStatistics(NLSmSL1_profile, min, max, mean, stdDev);
return mean;
}
function getNLSmSL2(profile) {
NLSmSL2_length = floor(profile.length/3);
NLSmSL2_profile = Array.slice(profile, profile.length-NLSmSL2_length);
Array.getStatistics(NLSmSL2_profile, min, max, mean, stdDev);
return mean;
}
function getPath(pathFileOrFolder) {
// this one takes full path of the file (input can also be a folder) and returns the parent folder path
temp = split(pathFileOrFolder, File.separator);
if ( File.separator == "/" ) {
// Mac and unix system
pathTemp = File.separator;
for (i=0; i<temp.length-1; i++) {pathTemp = pathTemp + temp[i] + File.separator;}
}
if ( File.separator == "\\" ) {
// Windows system
pathTemp = temp[0] + File.separator;
for (i=1; i<temp.length-1; i++) {pathTemp = pathTemp + temp[i] + File.separator;}
}
return pathTemp;
}
function getPathFilenamePrefix(pathFileOrFolder) {
// this one takes full path of the file
temp = split(pathFileOrFolder, File.separator);
temp = temp[temp.length-1];
temp = split(temp, ".");
return temp[0];
}
function getFilenamePrefix(filename) {
// this one takes just the file name without folder path
temp = split(filename, ".");
return temp[0];
}
function deleteFolder(folder) {
// Delete all the files inside the folder, then the folder itself
list = getFileList(folder);
// Delete the files and the folder
for (i=0; i<list.length; i++){
ok = File.delete(folder+list[i]);
}
ok = File.delete(folder);
if (File.exists(folder))
exit("Unable to delete the folder: " + folder);
else
print("Successfully deleted: " + folder);
}