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

//Area of the smallest region of interest (cell) [number in pixel^2 e.g 18000]
// starting value for calibration = 0;

the_smallest_cell = 0;

//Area of the biggest region of interest (cell) [number in pixel^2 e.g 100000]
//starting value for calibration  = "infinity";

the_biggest_cell =  "infinity";




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


run("Set Measurements...", "area perimeter circularity display redirect=None decimal=3");
macro_path = getInfo("macro.filepath");
choice = "Analyze next image";
while(choice == "Analyze next image" ){
print("macro has started");
run("Close All");
run("Open...");
path=getDirectory("image");
newfolder = path+substring(path,lengthOf(path)-1,lengthOf(path))+"results"+substring(path,lengthOf(path)-1,lengthOf(path));
name=getTitle;
selectWindow(name);
run("Set Scale...", "distance=0");
if (roiManager("Count") > 0){
roiManager("Select", 0);
run("Select All");
roiManager("Deselect");
roiManager("Delete");
}
selectWindow(name);
run("Extend Image Borders", "left=10 right=10 top=10 bottom=10 fill=Black");
extented=getTitle;
selectWindow(name);
close();
closing = extented+"-Closing";
run("Morphological Filters", "operation=Closing element=Disk radius=9");
iterator=0;
while(!isOpen(closing)){
wait(500);
iterator++;
print(iterator);
}
watershed=closing+"-watershed-lines";
run("Morphological Segmentation");
iterator=0;
while(!isOpen("Morphological Segmentation")){
wait(500);
iterator++;
print(iterator);
}
selectWindow("Morphological Segmentation");
//setTool("point");
selectWindow("Morphological Segmentation");
call("inra.ijpb.plugins.MorphologicalSegmentation.setDisplayFormat", "Watershed lines");
selectWindow("Morphological Segmentation");
call("inra.ijpb.plugins.MorphologicalSegmentation.segment", "tolerance=8.0", "calculateDams=true", "connectivity=4");
selectWindow("Morphological Segmentation");
iterator = 0;
while(!isOpen(watershed)){
wait(500);
iterator++;
print(iterator);
selectWindow("Morphological Segmentation");
call("inra.ijpb.plugins.MorphologicalSegmentation.createResultImage");
wait(500);
selectWindow(closing);
close();
}
selectWindow("Morphological Segmentation");
close();
selectWindow(watershed);
run("Dilate");
run("Invert");
run("Analyze Particles...", "size=the_smallest_cell-the_biggest_cell circularity=0.01-1.00 show=Nothing display exclude clear add");
selectWindow(watershed);
close();
selectWindow(extented);
selectWindow("Log");
run("Close");
roiManager("Show None");
roiManager("Show All");
run("Brightness/Contrast...");
setMinAndMax(0, 50);
if (roiManager("Count") > 0){
selectWindow(extented);
if (!File.exists(newfolder)){
File.makeDirectory(newfolder);
}
run("From ROI Manager");
saveAs("Tiff", newfolder+extented);
selectWindow("Results");
} else {
waitForUser("No ROI found, adjust cell size threshold values");
}
macro_path = getInfo("macro.filepath");
String.copyResults();
Dialog.create("Work finished.");
Dialog.addMessage("If you intend to modify selections, now its the time");
Dialog.addMessage("Your results have been copied to clipborad");
Dialog.addChoice("What would you do next?:", newArray("Analyze next image", "Set cell size boundaries", "Modify selections or brightness","Nothing - Close this window", "Quit"));
Dialog.show();
choice = Dialog.getChoice();

if(choice == "Set cell size boundaries"){
run("Edit...", "open=["+macro_path+"]");
run("Clear Results");
selectWindow(extented+".tif");
selectWindow("ROI Manager");
waitForUser("Measure area of few smallest and largest cells in the image");
}

while(choice == "Modify selections or brightness"){
selectWindow("B&C");
selectWindow("ROI Manager");
waitForUser("Modify selections or brightness");
run("Clear Results");
if (roiManager("Count") > 0){
roiManager("Select", 0);
roiManager("Deselect");
roiManager("Measure");
selectWindow(extented+".tif");
if (!File.exists(newfolder)){
File.makeDirectory(newfolder);
}
run("From ROI Manager");
saveAs("Tiff", newfolder+extented);
selectWindow("Results");
} else {
waitForUser("No ROI found, adjust cell size threshold values");
}
String.copyResults();
Dialog.create("Work finished.");
Dialog.addMessage("If you intend to modify selections, now its the time");
Dialog.addMessage("Your results have been copied to clipborad");
Dialog.addChoice("What would you do next?:", newArray("Analyze next image", "Set cell size boundaries", "Modify selections or brightness","Nothing - Close this window", "Quit"));
Dialog.show();
choice = Dialog.getChoice();

}

if(choice == "Quit"){
run("Close All");
selectWindow("ROI Manager");
run("Close");
selectWindow("B&C");
run("Close");
selectWindow("Results");
run("Close");
}
}