Colocalization troubleshooting/guidance in Image J

Hi everybody,

I have TUNEL (Green) / DAPI (Blue) stained cells.

The goal is to get apoptotic index (%) which is defined as (# of TUNEL stained cells/ # of total cells).

So approach is;

First, count total number of cells by DAPI siginal

Second, count TUNEL staining (Green).

For the first part, I did thresholding - watershed - analyze particle, then I got the total number of DAPI positive cells. I did same thing on TUNEL stained cells to count it.

However, it cannot completely exclude TUNEL staining that does not colocalize with DAPI staining.

In that, I used another approach where

1.Apply threshold on TUNEL staining and watershed
2. Create selection on the binary TUNEL image.
3. Restored selection on the original (non-binary) DAPI image.

Image from #3 shows that you see some noise from TUNEL staining. So I would like to only count DAPI that colocalized with TUNEL.

Could you please help me to get the number of colocalized cells or % of colocalized cells from step 3 please? If there is any easier method to get number or percentage of colocalization, I am opened for other suggestions. Thanks!

p.s) I do not want to use Jacop or Coloc2 since it does not seem to spit out # of cells.