Help with batch analysis of Fluorescence Proximity ligation assay images (2D PLA Foci)

Hi everyone!

I am new to ImageJ and am using it to quantify 2D foci and number of nuclei in cell immunofluorescence images.

In situ proximity ligation assay was used to detect receptor dimers in coverslips grown cells. Each dimer interaction is visualised as a red fluorescent spot and nuclei counterstained with DAPI.

From reading the ImageJ guide, what I have done was:

  1. composite image split into red and blue channels
  2. On the DAPI-channel, the threshold was adjusted to attain the best visualisation of the nuclei.
  3. Touching nuclei were separated using the “Watershed” function followed by “Analyze particle” to get nuclei count
  4. On the Texas-Red channel, the focis were quantitated using “Find maxima” function.

These steps were individually macro-ed and shortcut-keyed. But now I have 3-4 folders with about 200 images each and these process is too slow and unpractical…

I like to get some help/advise with designing a more streamline macro that can batch analyse the entire folder, giving a txt result readout: Filename| Maxima count| Nuclei count.

Following up my first post with my current workflow (which I like to streamlined into a batch-analytic macro):

‘Split composite into Foci and Nuclei channels’
run("Split Channels");

‘For Foci counting- red channel’
run("Find Maxima...", "noise=80 output=Count");

'Threshold nuclei for counting’
setThreshold(0, 70);
//setThreshold(0, 70);
setOption(“BlackBackground”, false);
run(“Convert to Mask”);
run(“Convert to Mask”);
run(“Convert to Mask”);
run(“Convert to Mask”);
run(“Make Binary”);

‘For nuclei counting- Blue channel’
run("Analyze Particles...", "size=0.10-Infinity circularity=0.50-1.00 show=[Overlay Masks] display in_situ");

each were shortcut into F1,F2,F3,F4 keys


your getting there :slight_smile:

This wiki page should help you for the next step:

Also there are many examples in the forum, that make a good starting point:



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