// OHSU Ca Imaging - Morphological Segmentation

// This is temporary while I figure out how to implement CNMF into a scriptable format. 

// ================================================================
// Written by Wilson Adams | Siciliano Lab | Sept 2021
// ================================================================

// Assumed stack to process is already open
stk = getTitle(); 

Stack.getDimensions(width, height, channels, slices, frames);
f_avg = 50;
if (frames<50) {
	f_avg = frames;
} else {}

// Make an average intensity projection
run("Z Project...", "stop=&f_avg projection=[Average Intensity]");
avgip = getTitle(); 
run("Enhance Contrast", "saturated=0.35");

// Generate a copy of image to bandpass filter
run("Duplicate...", avgip+"_filt");
filt = getTitle(); 
run("Gaussian Blur...", "sigma=3");
run("Subtract Background...", "rolling=12");
//run("Bandpass Filter...", "filter_large=15 filter_small=8 suppress=None tolerance=5 autoscale saturate");
run("Morphological Filters", "operation=[White Top Hat] element=Disk radius=9");
filt = getTitle(); 
run("Duplicate...", avgip+"_mask");

setAutoThreshold("Huang dark");
run("Threshold...");
run("Convert to Mask");
mask = getTitle();
run("Duplicate...", avgip+"_dist");
run("Distance Map");
dist = getTitle(); 

selectWindow(filt);
run("Find Maxima...", "prominence=80 output=[Point Selection]");
roiManager("Add");
roiManager("show none");
newImage(avgip+"_max", "8-bit black", 1024, 1024, 1);
maxima = getTitle(); 
selectWindow(maxima);
roiManager("Select", 0);
roiManager("Fill");
run("Marker-controlled Watershed", "input=&dist marker=&maxima mask=&mask binary calculate use");
rename(avgip+"_roimap");
map = getTitle(); 
run("Duplicate...", avgip+"_roiext");
rois = getTitle(); 
setThreshold(1, 65535);
run("Threshold...");
run("Convert to Mask");

roiManager("reset");
run("Analyze Particles...", "clear add");

run("Tile");

//run("Bandpass Filter...", "filter_large=15 filter_small=3 suppress=None tolerance=5 autoscale saturate");

//close("\\Others");
// run("TIFF Virtual Stack...", "open=[Z:/Collaborations/2021_09 OHSU/Data/E_drive_final_copy/2021_09 OHSU2021/2021_09_21 Animal 11/A11_S1 (fluo4)/Spontaneous/A10_S5_006.tif]");