// Paired down ND file processer for faster data exctraction. 

// This macro is an iteration of Bologna_AstroCaProcess_v3.ijm macro meant to be applied to 
// nd2 files thrown from the STORM microscope in the Nikon Imaging core at Vanderbilt. 

// Turns out it's REALLY REALLY SLOW for big image files. took almost 10min to process one video. 
// I'm making a new slimmer version to cut down on the processing time and speed things up. 

path = File.openDialog("Select .nd2 Image File")
par = File.getParent(path)
//filename = File.getName(path)
filename = File.nameWithoutExtension()
print(path)
print(filename)

Dialog.create("Experiment Info");
Dialog.addMessage("Input Image Info?");
Dialog.addNumber("Number of Images = ", 301)
Dialog.addString("File Header = ", filename)
Dialog.show() ;
stack = Dialog.getNumber();
header = Dialog.getString()

//stack = 200					// How many images in each file stack
run("Bio-Formats", "open=[&path]");
//run("Green Fire Blue");
rename(header)
orig = getTitle()			// Original imagge stack variable

File.makeDirectory(par+"\\processed")				// Make processed images directory in parent directory
File.makeDirectory(par+"\\processed\\!Final\\")		// !Final directory in 'processed' directory for final corrected stacks + time plots
File.makeDirectory(par+"\\processed\\"+orig+"\\")		// subfolder for each trial in the 'processed' folder for originals, IPs, masks, ROI lists, FFC factors, etc
processed_dir = par+"\\processed\\"
final_dir = par+"\\processed\\!Final\\"
pro_head_dir = par+"\\processed\\"+header+"\\"


getRawStatistics(mean)
mean1 = mean
t = nSlices()
run("Z Project...", "projection=[Max Intensity]");
run("Cyan Hot");
rename(orig+"_MaxIP")
MaxIP = getTitle()
getRawStatistics(mean)
mean2 = mean
selectWindow(orig)
run("Z Project...", "projection=[Median]");
MedIP = getTitle()
selectWindow(orig)
run("Z Project...", "projection=[Standard Deviation]");
run("!Orangeish")
stdIP = getTitle()

run("Calculator Plus", "i1=&MaxIP i2=&MedIP operation=[Subtract: i2 = (i1-i2) x k1 + k2] k1=1 k2=0 create");
rename(MaxIP+"_bkgdsub")
FFC = getTitle()
//run("Subtract Background...", "rolling=250");	// just subtract background, no mask generation
//run("Calculator Plus", "i1=&SIP i2=&FFC operation=[Subtract: i2 = (i1-i2) x k1 + k2] k1=1 k2=&mean2 create");
//rename(SIP+"_corr")
//selectWindow(FFC)
//run("Duplicate...", " ");
//rename(FFC+"_stack")
//FFC_stack = getTitle()
//run("Divide...", "value=&t");
//run("Calculator Plus", "i1=&orig i2=&FFC_stack operation=[Subtract: i2 = (i1-i2) x k1 + k2] k1=1 k2=&mean1 create");
//rename(orig+"_final")
//corr = getTitle()
//close(FFC)
//close(FFC_stack)
selectWindow(MaxIP)
save(pro_head_dir+MaxIP+".tif")
selectWindow(MedIP)
save(pro_head_dir+MedIP+".tif")
selectWindow(FFC)
save(pro_head_dir+FFC+".tif")
selectWindow(stdIP)
save(pro_head_dir+stdIP+".tif")
close(MaxIP)
close(MedIP)
//close(SIP)
//selectWindow(SIP+"_corr")
//SIPcorr = getTitle()

//selectWindow(corr)
//save(final_dir+corr+".tif")	

//selectWindow(corr)
//run("Z Project...", "start=10 projection=[Median]");  // Start from image 10 to get rid of photobleaching artifact. 
//run("Green Fire Blue");
//rename(orig+"_MedIP")
//MedIP = getTitle()
//save(pro_head_dir+MedIP+".tif")

selectWindow(FFC)
run("Enhance Contrast", "saturated=0.35");
roiManager("reset");
roiManager("show all with labels") 
setTool("ellipse");
run("Tile");
string = "Use the Elipse tool to make your own ROIs. Press 'Ctrl+T' to add each ROI to the ROI list. Click 'OK' when all ROIs are measured"
waitForUser(string)	// Wait for user input once ROIs are selected
setTool("oval");
string = "Add a background ROI with the oval tool, then press 'OK'"
waitForUser(string)	
//roiManager("Add")
roiManager("save", pro_head_dir+orig+"_ROImanual.zip")
// ROIs will be outputted in a fashion where each collumn is a cell/ROI, and the last collumn is a Background

// Measure ROIs and save output data
selectWindow(orig)
roiManager("show all with labels") 
run("Set Measurements...", "mean redirect=None decimal=6");
roiManager("multi measure one")
run("Input/Output...", "jpeg=85 gif=-1 file=.csv use_file");
saveAs("Results", final_dir+header+"_data");

//close(MedIP)
selectWindow(orig)
//save(pro_head_dir+header+".tif")	//Files too large, take up too much room. Downsample
//close()
//close(SIPcorr)
close("Results")

roiManager("open", pro_head_dir+orig+"_ROImanual.zip")
roiManager("show all with labels") 
//selectWindow(corr)
//run("Plot Z-axis Profile");
//close(SIP)

// Downsample for easy transport and revaluating. Comment out if necessary. 
// Set parameters: width, heigh, depth
w = 800
h = 798
d = 300
run("Size...", "width=&w height=&h depth=&d constrain average interpolation=Bilinear");
run("Green");
ds = getTitle()


save(pro_head_dir+orig+"_ds.tif")
print("Saved: "+pro_head_dir+orig+"_ds.tif")
close()

print(orig+" FINISHED")