ImageJ macros for processing ratiometric imaging movies with a static marker channel

ratio_alternating.ijm

Dialog.create("Process interleaved ratiometric movie");
Dialog.addString("Experiment name:", "expt");
Dialog.addNumber("Background", 1600)
Dialog.show()
expt_name = Dialog.getString();
background = Dialog.getNumber();
 
expt_dir = getDirectory("Choose a save directory");
 
id = getImageID();
rename(expt_name + "_interleaved.tif");
Stack.getDimensions(w, h, c, s, f);
saveAs("Tiff", expt_dir + File.separator + expt_name + "_interleaved.tif");
 
odd_str = "1-"+(f-1)+"-2";
even_str = "2-"+f+"-2";
 
run("Make Substack...", "  slices="+odd_str);
rename(expt_name + "_odd.tif");
 
selectImage(id);
run("Make Substack...", "  slices="+even_str);
rename(expt_name + "_even.tif");
 
run("Ratio Plus", "image1=["+expt_name+"_odd.tif] image2=["+expt_name+"_even.tif] background1="+background+" clipping_value1=0 background2="+background+" clipping_value2=0 multiplication=1");
rename(expt_name + "_ratio.tif");
saveAs("Tiff", expt_dir + File.separator + expt_name + "_ratio.tif");

ratio_bgsub.ijm

Dialog.create("Process interleaved ratiometric movie");
Dialog.addString("Experiment name:", "expt");
Dialog.addNumber("Background filter radius", 150)
Dialog.show()
expt_name = Dialog.getString();
bg_radius = Dialog.getNumber();

expt_dir = getDirectory("Choose a save directory");

id = getImageID();
rename(expt_name + "_interleaved.tif");
Stack.getDimensions(w, h, c, s, f);
saveAs("Tiff", expt_dir + File.separator + expt_name + "_interleaved.tif");
run("Subtract Background...", "rolling="+bg_radius+" stack");
 
odd_str = "1-"+(f-1)+"-2";
even_str = "2-"+f+"-2";

run("Make Substack...", "  slices="+odd_str);
rename(expt_name + "_odd.tif");

selectImage(id);
run("Make Substack...", "  slices="+even_str);
rename(expt_name + "_even.tif");
 
run("Ratio Plus", "image1=["+expt_name+"_odd.tif] image2=["+expt_name+"_even.tif] background1=0 clipping_value1=0 background2=0 clipping_value2=0 multiplication=1");
rename(expt_name + "_ratio.tif");
saveAs("Tiff", expt_dir + File.separator + expt_name + "_ratio.tif");

segmentation.ijm

// Split into individual channels
selectWindow("example.tif");
run("Deinterleave", "how=2 keep");

// Segment all cells in a single frame
selectWindow("example.tif #2");
run("Make Substack...", "  slices=1");
run("Subtract Background...", "rolling=100");
setAutoThreshold("Otsu dark");
run("Convert to Mask");
cell_mask = getImageID();

// Segment the YFP image
selectWindow("ypf_example.tif");
run("Subtract Background...", "rolling=100");
setAutoThreshold("Otsu dark");
run("Convert to Mask");
yfp_mask = getImageID();

// Make the YFP+ mask
imageCalculator("AND create", cell_mask, yfp_mask);

// Make the YFP- mask
selectWindow(yfp_mask);
run("Invert");
yfp_inv_mask = getImageID();
imageCalculator("AND create"), cell_mask, yfp_inv_mask);

// TODO ratio image

// WIP: apply ROIs to ratio
run("Create Selection");
selectWindow("exaple.tif #1");
run("Restore Selection");
run("ROI Manager...");
roiManager("Add");
roiManager("Split")
roiManager("Show All");
roiManager("Multi Measure");
//For FURA-YFP cells
run("Invert");
imageCalculator("AND create", "Substack (1)","Mask");