// AstroSeg.ijm

// Trying to build a better astrocyte segmentation algorithm for Ca Imaging analysis. Doing it by hand sucks. 
// Development of this final processing pipeline is iterated and tested in AstroSeg_dev.ijm
// This it the final working product of AstroSeg for user use. 

// Wilson R Adams | Vanderbilt Biophotonics Center | 1 July 2019

stk = getTitle()
	//filepath conventions
path = File.getName(stk)
par = File.directory
print(par); print(path)
fn = File.nameWithoutExtension()
savedir = par+"bkgd\\"
File.makeDirectory(savedir)		// Make processed images directory in parent directory
print(savedir)

	//sum bkgd sub
stk = getTitle()
fn = File.nameWithoutExtension(); print(fn)
run("Z Project...", "stop=50 projection=[Sum Slices]");
rename(fn+"_50sumIP")
sumIP = getTitle()
run("Duplicate...", fn+"_bkgd ");
sumBkgd = getTitle()
run("Gaussian Blur...", "sigma=50");
run("Calculator Plus", "i1=&sumIP i2=&sumBkgd operation=[Subtract: i2 = (i1-i2) x k1 + k2] k1=1 k2=0 create");
rename(fn+"_sumbkdgsub")
sumBkgdsub = getTitle()
close(sumIP); close(sumBkgd);

	//Stack bkgd sub (avgIP)
selectWindow(stk)
run("Z Project...", "stop=50 projection=[Average Intensity]");
avgBkgd = getTitle()
run("Gaussian Blur...", "sigma=50");
getStatistics(area, mean, min, max, std, histogram)	// only need mean
run("Calculator Plus", "i1=&stk i2=&avgBkgd operation=[Subtract: i2 = (i1-i2) x k1 + k2] k1=1 k2=&mean create");
rename("_bkgdSub")
stk_bkgdSub = getTitle()
close(avgBkgd)
run("Enhance Contrast", "saturated=0.35"); run("!Blueish")
close(avgBkgd)

//Z projections for Activation maps
selectWindow(stk_bkgdSub)
run("Z Project...", "projection=[Max Intensity]"); rename(fn+"_maxIP")
stk_maxIP = getTitle()
run("Enhance Contrast", "saturated=0.35"); run("!DarkPurpish")

selectWindow(stk_bkgdSub)
run("Z Project...", "projection=[Standard Deviation]"); rename(fn+"_stdIP")
stk_stdIP = getTitle()
run("Enhance Contrast", "saturated=0.35"); run("!Orangeish")

// Saving stuff
selectWindow(stk_bkgd)
save(savedir+stk_bkgdSub)
selectWindow(sumBkgdsub)
save(savedir+sumBkgdsub)