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@glynnelson
glynnelson authored Jul 8, 2020
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81 changes: 81 additions & 0 deletions Eva_foci_counting_wholedir_20x_moments_thresh.txt
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//Assumes the image is a tif with DAPI in channel1, H2AX in channel2
// It looks for nuclei then counts respective spots for each nucleus.
//All results get stored in a concatenating results file.
//Plus the mask images are stored (all 2 original images converted to masks)
// so you can see what it did.
// altered so find maxima works on raw data,not thresholded.
// var tolerance = 30; removed this and set manually in find maxima.
run("Set Measurements...", "area integrated limit display redirect=None decimal=0");

dir1 = getDirectory("Choose folder with tif files ");
list = getFileList(dir1);

setBatchMode(true);

// create folders for the tifs
dir1parent = File.getParent(dir1);
dir1name = File.getName(dir1);
dir2 = dir1parent+File.separator+dir1name+"_masks";
if (File.exists(dir2)==false) {
File.makeDirectory(dir2); // new directory for mask images
}

for (i=0; i<list.length; i++) {
showProgress(i+1, list.length);
print("processing ... "+i+1+"/"+list.length+"\n "+list[i]);
filename = dir1 + list[i];
if (endsWith(filename, "tif")) {
path=dir1+list[i];
run("Bio-Formats Importer", "open=["+filename+"] autoscale color_mode=Default view=Hyperstack stack_order=XYCZT");
originalImage = getTitle();
roiManager("Reset");
selectWindow(originalImage);
run("Duplicate...", "title=nuc duplicate channels=2"); //Change this to the DAPI channel slice*******
wait(1000);
setAutoThreshold("Moments dark");
run("Threshold");
run("Watershed");
run("Analyze Particles...", "size=48-2500 circularity=0.04-1.00 exclude add");
run("Blue");
run("16-bit");
//nuclei now added to ROI manager

//next extract h2ax channel
selectWindow(originalImage);
run("Duplicate...", "title=h2ax duplicate channels=1");
wait(1000);

//next find H2AX foci per nuc
selectWindow("h2ax");
rename(originalImage+"_h2ax");
for(j=0; j<roiManager("count"); j++) {
roiManager("select", j);
run("Find Maxima...", "noise=100 output=[Count]");
// run("Find Maxima...", "noise="+tolerance+" output=[Point Selection]");
run("Add Selection...");
}
saveAs("Results", dir2+File.separator+originalImage+"_h2ax.xls"); //This is saving as it goes along in case it crashes
selectWindow(originalImage+"_h2ax");
rename("h2ax2");
run("Enhance Contrast", "saturated=0.35");
selectWindow(originalImage);
wait(1000);
close();

run("Merge Channels...", "c1=h2ax2 c2=nuc create");
rename("masks");

saveAs("TIFF", dir2+File.separator+originalImage+"_ThresholdMasks.tif");


close();
}
setBatchMode(false);
}

showMessage(" Well, I think we gave them a damn good thrashing there, what what??");

// macro



223 changes: 223 additions & 0 deletions Foci_counting_wholedir_Choose_thresh.ijm
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/* Macro to count spots inside other spots
* Updated March 2020
* It now asks whichc hannels the nuclear counterstain and the spots are in (can work with more than 2 channel images)
* It still assumes the image is a tif
* It looks for nuclei then counts respective spots for each nucleus.
* All results get stored in a concatenating results file.
* Plus the mask images are stored (all 2 original images converted to masks)
* so you can see what it did.
*
* Input:
* Assumes the image is a tif (any number of channels but it has to be 1 z, not a stack)
* It asks for the channel numbers for nuclear counterstain and sub nculear spots
*
* Note- tifs can be produced via another macro to convert from proprietary file format
*
* Then you are asked to review the threshold stack it has created and choose the best method.
* NOTE- It does NOT work if you choose Huang2!!!! Some weird choice of 2 different lists in ImageJ.
*
* Output:
* All results get stored in a concatenating results file.
* Plus the mask images are stored (all 2 original images converted to masks) so you can check on accuracy.
*
* To do: convert to python- way easier than this clunky thing, and would be able to deal with 3D datasets.
* Add to run from bioformats and only use the chosen filetype (easy enough)
*
*
* Glyn Nelson, Newcastle University Bioimaging Unit
*/

//Variables need to be defined if used between functions and the main macro, not just for sharing between macros.
var fred;
var FociChannel =1;
var DapiChannel = 2;
run("Set Measurements...", "area integrated limit display add redirect=None decimal=0");

setChannelNames();

dir1 = getDirectory("Choose folder with tif files ");
list = getFileList(dir1);

setBatchMode(true);

// create folders for the tifs
dir1parent = File.getParent(dir1);
dir1name = File.getName(dir1);
dir2 = dir1parent+File.separator+dir1name+"_masks";
if (File.exists(dir2)==false) {
File.makeDirectory(dir2); // new directory for mask images
}
for (i=0; i<list.length; i++) {
showProgress(i+1, list.length);
print("processing ... "+i+1+"/"+list.length+"\n "+list[i]);
filename = dir1 + list[i];
if (endsWith(filename, "tif")) {
path=dir1+list[i];
run("Bio-Formats Importer", "open=["+filename+"] autoscale color_mode=Default view=Hyperstack stack_order=XYCZT");
originalImage = getTitle();
roiManager("Reset");
selectWindow(originalImage);
run("Make Composite", "display=Composite");
// Stack.getDimensions(width, height, channels, slices, frames);
// run("Stack to Hyperstack...", "order=xyczt(default) channels=channels slices=slices frames=frames display=Color");
run("Duplicate...", "title=nuc duplicate channels=" +DapiChannel);
selectWindow("nuc");
run("Grays");
BuildThreshStack(); //I am absolutely damned if I can get this to work. it insists that it the variable ThresholdChoice doesn't exist once I go through this as a function.
selectWindow("nuc");
setOption("BlackBackground", true);
setAutoThreshold(fred);
run("Threshold");
run("Watershed");
run("Analyze Particles...", "size=48-2500 circularity=0.04-1.00 exclude add");
run("Blue");
run("16-bit");
//nuclei now added to ROI manager

//next extract h2ax channel
selectWindow(originalImage);
run("Duplicate...", "title=h2ax duplicate channels=FociChannel");
run("16-bit");
wait(1000);

//next find H2AX foci per nuc
selectWindow("h2ax");
rename(originalImage+"_h2ax");
for(j=0; j<roiManager("count"); j++) {
roiManager("select", j);
run("Find Maxima...", "noise=100 output=[Count]");
// run("Find Maxima...", "noise="+tolerance+" output=[Point Selection]");
run("Add Selection...");
}
saveAs("Results", dir2+File.separator+originalImage+"_h2ax.xls"); //This is saving as it goes along in case it crashes
selectWindow(originalImage+"_h2ax");
rename("h2ax2");
run("Enhance Contrast", "saturated=0.35");
selectWindow(originalImage);
wait(1000);
close();
run("Merge Channels...", "c1=h2ax2 c2=nuc create");
rename("masks");
saveAs("TIFF", dir2+File.separator+originalImage+"_ThresholdMasks.tif");
close();
}
setBatchMode(false);
}

showMessage(" Well, I think we gave them a damn good thrashing there, what what??");

// macro


//FUNCTION- determine channel order
function setChannelNames() {
Dialog.create("Set which channels are which"); //enable use interactivity
Dialog.addMessage("Which channel is DAPI/ nuclear counterstain in?");
Dialog.addString(" DAPI Channel", DapiChannel); //add extension
Dialog.addMessage("Which channel are the sub-nuclear spots labelled in?");
Dialog.addString("Foci Channel", FociChannel); //add extension
Dialog.addMessage("Use channel numbers (eg 1,2) to set the channels to use for determining the nuclei and spots");
Dialog.show();
//StringDapiChannel = Dialog.getString(DapiChannel); ******Apparently don't need to convert a number from a string- it figures it out itself.
//Dapi = parseFloat(StringDapiChannel);
//StringFociChannel = Dialog.getString(FociChannel);
//Foci = parseFloat(StringFociChannel);
}
//need to set var Foci and var Dapi
//End of function setChannelNames




//FUNCTION Choose global Threshold Method
function BuildThreshStack() {
run("Auto Threshold", "method=[Try all] white show");
selectWindow("Montage");
run("Montage to Stack...", "images_per_row=5 images_per_column=4 border=0");
run("RGB Color");
run("Colors...", "foreground=green background=black selection=red");
style = 4; // Font.BOLD
call("ij.gui.TextRoi.setFont", "Sans", 32, style)

//This is nuts, but I've spent so long trying to use arrays or lists to run through this labelling that I've given up.
//Writting the next 34 lines took less than 10 minutes. Grrrrrrrrr
//run("Label...", "format=0000 x=14 y=20 font=32 text=Method use use_text");
setSlice(1);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Default] range=1-1");
setSlice(2);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Huang] range=2-2");
setSlice(3);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Huang2] range=3-3");
setSlice(4);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Intermodes] range=4-4");
setSlice(5);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[IsoData] range=5-5");
setSlice(6);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[li] range=6-6");
setSlice(7);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[MaxEntropy] range=7-7");
setSlice(8);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Mean] range=8-8");
setSlice(9);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[MinError] range=9-9");
setSlice(10);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Minimum] range=10-10");
setSlice(11);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Moments] range=11-11");
setSlice(12);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Otsu] range=12-12");
setSlice(13);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Percentile] range=13-13");
setSlice(14);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[RenyiEntropy] range=14-14");
setSlice(15);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Shahbhag] range=15-15");
setSlice(16);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Triangle] range=16-16");
setSlice(17);
run("Label...", "format=Text starting=0 interval=1 x=10 y=30 font=32 text=[Yen] range=17-17");


waitForUser("Browse the Stack to choose the best Method \n then click OK to input your choice");
Dialog.create("Choose your desired threshold"); //enable use interactivity
Dialog.addChoice("Best threshold is:", newArray("Default", "Huang","Huang2","Intermodes","IsoData","Li","MaxEntropy","Mean","MinError","Minimum","Moments","Otsu","Percentile","RenyiEntropy","Shanbhag","Triangle","Yen"));
Dialog.show();
fred = Dialog.getChoice();

selectWindow("Stack");
close();

selectWindow("Montage");
close();
}

//ensure var fred is set
//End of function BuildThreshStack


//Neither of the functions belwo are in use in this version.

//FUNCTION- choose autoThreshold method
function choose_me() {
waitForUser("Browse the Stack to choose the best Method \n then click OK to input your choice");
Dialog.create("Choose your desired threshold"); //enable use interactivity
Dialog.addChoice("Best threshold is:", newArray("Default", "Huang","Huang2","Intermodes","IsoData","Li","MaxEntropy","Mean","MinError","Minimum","Moments","Otsu","Percentile","RenyiEntropy","Shanbhag","Triangle","Yen"));
Dialog.show();
fred = Dialog.getChoice();

//print("diaolog gives fred is ", fred);

}
//need to set var fred
//End of function choose_me


//FUNCTION Call_Choice
function Call_Choice(){
waitForUser("Browse the Stack to choose the best Method \n number then click OK to input your choice");
Desired_Threshold_Method = getNumber("Choose your Preferred Threshold Number", 0);
//Stack.getPosition(slice);
//print ("Your Choice is...", Desired_Threshold_Method);
}
//ensure global var Desired_Threshold_Method is set
//End of Function Call_Choices
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