Quantify Colocalization

Quantify Colocalization is a free, stand-alone Windows program. Current version is v0.5. It is provided “as-is”, without any express or implied warranty.

Quantify Colocalization is to quantify fluorescent signals from multiple channels to test whether there is a correlation between them. It is really simple (It took only three days to write from scratch.) but it does the job quite well. The idea is that you can open multiple (up to three) images, click to create simple, same-size ROIs, and the program will quantify the average signals in those ROIs.


  • Single click to create ROIs.
  • ROIs can be moved with mouse, or deleted by right clicking.
  • ROI size can be changed at any time, it applies to all ROIs, including the existing ones.
  • ROIs can be square or circular. Changes applies to all ROIs, including the existing ones.
  • All channels can be hidden, providing a way to do things blindly.
  • Has built-in global background subtraction and it is intuitive. Simple click the set background icon (5th from right) and use the mouse to select a rectangle. The average intensity will be used for background subtraction.
  • Final results can be copied to the clipboard and paste to Excel for statistical analysis.

How to use

To try using Quantify Colocalization, download the program with sample images and extract the files to a folder. The files are CountSignal.exe and three images files: Green (MLC).tif, Red (Tf).tif and Blue (DAPI).tif.

  1. Double click to run CountSignal.exe.
  2. Drag and drop Green (MLC).tif.
  3. Drag and drop Red (Tf).tif.
  4. Start clicking on particles.
  5. Click the copy button to copy the results to the clipboard.
  6. Open Excel and paste the result in to it.

Note, you can drag and drop Blue (DAPI).tif to the program at any time, even after creating ROIs. The program will recalculate accordingly.