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:
- composite image split into red and blue channels
- On the DAPI-channel, the threshold was adjusted to attain the best visualisation of the nuclei.
- Touching nuclei were separated using the “Watershed” function followed by “Analyze particle” to get nuclei count
- 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.