Dealing with multiple images#

We have a two-channel image, and we would like to perform some quantification on these images. Let's say we're interested in two things:

What area of the bax staining is inside and outside the nucleus
Whether the average intensity of the staining inside the nucleus is higher than the intensity outside the nucleus

First, we need to split the channels.

Make a macro that opens the raw image and splits the two channels

open("path/bax_DAPI_overlay.tif"); // (1)!
run("Split Channels");

Replace path/bax_DAPI_overlay.tif with the path to the image.

Now we have two files. Unfortunately, they are not named very intuitively. Let's fix that.

Add lines to your script that renames C1-bax_DAPI_overlay.tif to nuclei and C2-bax_DAPI_overlay.tif to bax.

open("path/to/file/bax_DAPI_overlay.tif");
run("Split Channels");
selectWindow("C1-bax_DAPI_overlay.tif");
rename("nuclei"); // (1)!
selectWindow("C2-bax_DAPI_overlay.tif");
rename("bax");

Generally when working with multiple images in a script, it's a good idea to rename them so that we can see what is what more easily later in our script.

Now we can select the windows using their new names nuclei and bax. Handy!