53bp1_count.cppipe (10.4 KB)
I have acquired images with 3-channels: DAPI, EdU (S phase marker), 53BP1. In regards to the microscope used, these images were taken on an advanced spinning disk confocal microscope, 40x/1.3NA objective, with 16-bit depth camera and 1x1 binning. I have acquired a Z-stack (~11 planes, a total of 5um), for which I have done a maximum intensity Z projection so it is easier for analysis.
I wish to identify the number of 53BP1 foci for each cell. However, the number and size of these foci differ between cell cycle stages. For this reason I also wish to measure the intensity of DAPI and EdU channel for each cell, to create a cell cycle profile, from which I can separate the 53bp1 foci between G1/S/G2 stages.
I have created a pipeline using online guides which can identify the 53BP1 foci, however I found that it is very inconsistent between individual images. In one image, the foci identification would be close to perfect, how I would count them if I were to do it manually (see two good examples I have attached). However, when I tried to check another image set, the foci identification was less good (I have included two bad examples). I tried to change some of the thresholding parameters, but they would apply just to one image set, then they would be useless when I would move to another image set. I cannot figure why this is the case, as the images were all acquired using the same microscope settings.
I have attached the pipeline and two original sets of images: Set 1 worked nicely in identifying the foci, whilst set 2 did not.
The original images were in Slidebook files (.sld), which I have used to make the Max intensity Z projection and then to export as .tif files.
If anyone has any clues it would be great help!