//Please set following parameters:
//(Remeber to save changes)

//The value should be an area [number in pixel^2 e.g. 20]

area_of_the_smallest_region_of_interest = 20;


//Choose "auto" (with quotes) for auto local threshold processing, or "manual" (with quotes) for manual adjustment of global threshold

threshold_type = "manual";









/////////////////////////////////////////////////////Do not change code below this line////////////////////////////////////////////////////////////////////////////////

run("Set Measurements...", "area display redirect=None decimal=3");
macro_path = getInfo("macro.filepath");
upplim = area_of_the_smallest_region_of_interest*500;
choice = "Analyze next image";
while(choice == "Analyze next image" ){
run("Close All");
run("Open...");
path=getDirectory("image");
name=getTitle;
output="Drawing of "+name;
newfolder = path+substring(path,lengthOf(path)-1,lengthOf(path))+"results"+substring(path,lengthOf(path)-1,lengthOf(path));

selectWindow(name);
run("Set Scale...", "distance=0 known=0 pixel=1 unit=pixel");
setLocation(1097, 122);
run("Duplicate...", " ");
duplicate = substring(name,0,lengthOf(name)-4)+"-1.tif";
run("Subtract Background...", "rolling=50 sliding");
if(threshold_type == "manual"){
run("Threshold...");
call("ij.plugin.frame.ThresholdAdjuster.setMode", "B&W");
wait(500);
setThreshold(30, 255);
waitForUser("Set threshold");
getThreshold(lower, upper);
run("Analyze Particles...", "size=area_of_the_smallest_region_of_interest-upplim pixel show=Outlines clear summarize");
selectWindow(name);
selectWindow("Drawing of "+duplicate);
run("Add Image...", "image=[name] x=0 y=0 opacity=70");
waitForUser("Compare images");
Dialog.create("Is correction needed?");
Dialog.addCheckbox("Is correction needed?", false);
Dialog.show();
correction = Dialog.getCheckbox();
while (correction) {
selectWindow("Drawing of "+duplicate);
close();
selectWindow(duplicate);
run("Threshold...");
setThreshold(lower, 255);
waitForUser("Set threshold");
IJ.deleteRows(0, 0);
getThreshold(lower, upper);
run("Analyze Particles...", "size=area_of_the_smallest_region_of_interest-upplim pixel show=Outlines clear summarize");
selectWindow(name);
selectWindow("Drawing of "+duplicate);
run("Add Image...", "image=[name] x=0 y=0 opacity=70");
waitForUser("Compare images");
Dialog.create("Is correction needed?");
Dialog.addCheckbox("Is correction needed?", false);
Dialog.show();
correction = Dialog.getCheckbox();
}
}
if(threshold_type == "auto"){
run("Auto Local Threshold...", "method=Phansalkar radius=15 parameter_1=0 parameter_2=0 white");
run("Invert");
run("Analyze Particles...", "size=area_of_the_smallest_region_of_interest-upplim pixel show=Outlines clear summarize");
}
if (!File.exists(newfolder)){
File.makeDirectory(newfolder);
}
selectWindow(name);
selectWindow("Drawing of "+duplicate);
run("Add Image...", "image=[name] x=0 y=0 opacity=70");
selectWindow("Drawing of "+duplicate);
saveAs("Tiff", newfolder+output);
if (threshold_type == "manual") {print(output); print("Lower threshold value:"); print(lower); print(" ");}
selectWindow(duplicate);
close();
Dialog.create("Work finished.");
Dialog.addMessage("Remeber to copy and store data from 'Summary' window");
if (threshold_type == "manual") {Dialog.addMessage("Lower threshold value is displayed in 'Log' window");}
Dialog.addChoice("What would you do next?:", newArray("Analyze next image", "Set FA size boundaries","Nothing - Close this window", "Change threshold type", "Quit"));
Dialog.show();
choice = Dialog.getChoice();

if(choice == "Set FA size boundaries"){
if (threshold_type == "manual"){
selectWindow("Log");
run("Close");
selectWindow("Threshold");
run("Close");}
selectWindow("Summary");
run("Close");
selectWindow("Drawing of "+name);
close();
run("Edit...", "open=["+macro_path+"]");
selectWindow(name);
run("ROI Manager...");
//setTool("polygon");
waitForUser("Measure area of smallests focal adhesion sites");
}

if(choice == "Change threshold type"){
if (threshold_type == "manual"){
selectWindow("Log");
run("Close");
selectWindow("Threshold");
run("Close");}
run("Close All");
run("Edit...", "open=["+macro_path+"]");
}

if(choice == "Quit"){
if (threshold_type == "manual"){
selectWindow("Log");
run("Close");
selectWindow("Threshold");
run("Close");}
run("Close All");
}

}