//Jan Wisniewski Confocal Microscopy Core at Experimental Immunology Branch NCI NIH Bethesda MD

//Creates rotating 3D projection of cells selected from a Z-stack
//Applies false-color LUT to visualize areas containing different amounts of target

//  specify folders
res=getDirectory("_Choose a Destination for Results");
myDir1 = res+"Selected Cells"+File.separator;
			File.makeDirectory(myDir1);
myDir2 = res+"3D Cells"+File.separator;
			File.makeDirectory(myDir2);
input=getDirectory("Select a Source Directory");
NAMES=getFileList(input); 


//functions
function setChannel() {	Dialog.addString("Name This Channel", " ");
	items = newArray("B&W", "Blue", "Cyan", "Green", "Yellow", "Red", "Magenta");
	Dialog.addRadioButtonGroup("In the Final Composite, Display This Channel as:", items, 1, 7, "B&W");
	Dialog.show(); }
	
//channel setup

open();
getDimensions(width, height, channels, slices, frames);
rename("y");
run("Z Project...", "projection=[Max Intensity]");
rename("x");
close("y");

if(channels==1) {run("Enhance Contrast", "saturated=0.10");
	Dialog.create("Set Channel");
	setChannel();
	chnname1=Dialog.getString();
	chncolor1=Dialog.getRadioButton();
	print(chnname1, "   ", chncolor1);
	close("x");	}

if(channels>1) {run("Split Channels");
run("Tile");
selectWindow("C1-x");
run("Enhance Contrast", "saturated=0.10");
selectWindow("C2-x");
 
run("Enhance Contrast", "saturated=0.10");
if(channels>2) {selectWindow("C3-x");
run("Enhance Contrast", "saturated=0.10");
 }	


//multiple channel selection  
  rows = 1;
  columns = 4;
  n = channels;
  labels = newArray(n);
  defaults = newArray(n);
  for (i=0; i<n; i++) {
    labels[i] = "Channel_"+i+1;
    defaults[i] = false; }
  Dialog.create("Which channels would you like to use?");
  Dialog.addCheckboxGroup(rows,columns,labels,defaults);
  Dialog.show();
		cx1=Dialog.getCheckbox();
		cx2=Dialog.getCheckbox();
		if(channels>2) {cx3=Dialog.getCheckbox(); 	}

selectWindow("C1-x");
if(cx1==0) {close("C1-x"); }
else {run("Enhance Contrast", "saturated=0.10");
	Dialog.create("Set Channel");
	setChannel();
	chnname1=Dialog.getString();
	chncolor1=Dialog.getRadioButton();
	print(chnname1, "   ", chncolor1);
	close("C1-x"); }
selectWindow("C2-x");
if(cx2==0) {close("C2-x"); }
else {run("Enhance Contrast", "saturated=0.10");
	Dialog.create("Set Channel");
	setChannel();
	chnname2=Dialog.getString();
	chncolor2=Dialog.getRadioButton();
		print(chnname2, "   ", chncolor2);
	close("C2-x"); }
if(channels>2) {selectWindow("C3-x");
if(cx3==0) {close("C3-x"); }
else {run("Enhance Contrast", "saturated=0.10");
	Dialog.create("Set Channel");
	setChannel();
	chnname3=Dialog.getString();
	chncolor3=Dialog.getRadioButton();
	print(chnname3, "   ", chncolor3);
	close("C3-x"); }	}
	}

// open files
for (q=0; q<NAMES.length; q++) {
open(input+NAMES[q]);
name=File.nameWithoutExtension;
getDimensions(width, height, channels, slices, frames);
print("Original Image:", name);
print("channels:", channels, "   ", "slices", slices);
rename("z");
run("Brightness/Contrast...");
waitForUser("Move to the center of the stack and adjust B&C for the selevant channel.\nCheck how many cells would you like to select !");
Dialog.create("...")
Dialog.addNumber("How Many Cells Will Be Selected?", 1);
Dialog.show();
rois=Dialog.getNumber();
print(rois);
if(rois==0) {close("z"); }
if(rois>0) {
	for (i = 0; i < rois; i++) {j=i+1;
		run("Duplicate...", "duplicate");
		setTool("rectangle");
		waitForUser("Select Cell");
		run("Crop");
		saveAs("Tiff", myDir1+name+"_cell_"+j);
		close(); }




close("z"); }
	}

//fetch selected regions
NAMES2=getFileList(myDir1); 
for (k=0; k<NAMES2.length; k++) {
open(myDir1+NAMES2[k]);
name3=File.nameWithoutExtension;
getDimensions(width, height, channels, slices, frames);
print("Selected cell:", name3);
print("channels:", channels, "   ", "slices", slices);
rename("x");
if(channels==1) {if(chncolor1=="B&W") {run("Grays"); }
else {run(chncolor1);  }
run("Median...", "radius=1 stack");
waitForUser("Adjust B&C - Check across whole stack!");
run("16 colors");
run("Gamma...", "value=0.50 stack");
run("Apply LUT", "stack");
run("3D Project...", "projection=[Brightest Point] axis=Y-Axis slice=1 initial=0 total=360 rotation=3 lower=1 upper=255 opacity=0 surface=100 interior=50 interpolate");
saveAs("Tiff", myDir2+name3+"-"+chncolor1);
close();
close();}
else {run("Split Channels");
selectWindow("C1-x");
if(cx1==0) {close("C1-x"); }
else {if(chncolor1=="B&W") {run("Grays"); }
else {run(chncolor1);
  }
run("Median...", "radius=1 stack");
waitForUser("Adjust B&C - Check across whole stack!");
run("16 colors");
run("Gamma...", "value=0.50 stack");
run("Apply LUT", "stack");
run("3D Project...", "projection=[Brightest Point] axis=Y-Axis slice=1 initial=0 total=360 rotation=3 lower=1 upper=255 opacity=0 surface=100 interior=50 interpolate");
saveAs("Tiff", myDir2+name3+"-"+chncolor1);
close();
close();}
selectWindow("C2-x");
if(cx2==0) {close("C2-x"); }
else {if(chncolor2=="B&W") {run("Grays"); }
else {run(chncolor2);
run("Median...", "radius=1 stack");
waitForUser("Adjust B&C - Check across whole stack!");
run("16 colors");
run("Gamma...", "value=0.50 stack");
run("Apply LUT", "stack");
run("3D Project...", "projection=[Brightest Point] axis=Y-Axis slice=1 initial=0 total=360 rotation=3 lower=1 upper=255 opacity=0 surface=100 interior=50 interpolate");
saveAs("Tiff", myDir2+name3+"-"+chncolor2);
close();
close();
 }		}
if(channels>2) {selectWindow("C3-x");
if(cx3==0) {close("C3-x"); }
else {if(chncolor3=="B&W") {run("Grays"); }
else {run(chncolor3);run("Median...", "radius=1 stack");
waitForUser("Adjust B&C - Check across whole stack!");
run("16 colors");
run("Gamma...", "value=0.50 stack");
run("Apply LUT", "stack");
run("3D Project...", "projection=[Brightest Point] axis=Y-Axis slice=1 initial=0 total=360 rotation=3 lower=1 upper=255 opacity=0 surface=100 interior=50 interpolate");
saveAs("Tiff", myDir2+name3+"-"+chncolor3);
close();
close();
  }
   }
  }
	}
}