SpotCountPLA

This toolbox counts spots per cell from images obtained from proximity ligation assays (PLA).

This function counts spots per cell from images obtained from proximity ligation assays (PLA). It is particularly adapted to batch process the images.

The toolbox contains two functions:

pla_analysis: A function that obtains the number of spots per cell based on a nuclear image and the corresponding spot-image. The are processed separately. Both the nuclear image and the spot image are (i) normalized to maximum intensity, (ii) contrast-enhanced using adaptive histogram equalization, (iii) and a threshold applied to them to eliminate background. To segment the cells, the function uses an extended-maxima transform followed by a watershed transform to define a region of influence for each cell. The spot image is sharpened using the unsharp masking technique, and the spots identified by labeling connected components in a binary version of the image. The images with the regions of influence and with the spots are overlaid and each spot assigned to the corresponding cell provided it is at a distance shorter than 150 pixels from the nucleus of the cell.

SpotCountPLA_Example_Red. Example of a script to batch process the analysis of 4 fields (easily scalable) obtained from a PLA assay performed to measure Foxp3 acetylation in mouse regulatory T cells.

The toolbox also contains example images.