Measuring signal distribution - is this right?



I need to analyse the distribution of Endoplasmic Reticulum (ER) signal from the nucleus towards the plasma membrane. I think I do it right but not fully sure, so will be grateful if you can check the pipeline before I include it in a paper.
In short:

  • I have DAPI and use that as primary object (attached image ending _0002).
  • I use actin as a whole cell label (attached image ending _0000).
  • I use one ER marker (attached image (_0001).
  • I noticed that I get best segmentation when I use a merged image excluding the nuclei (attached image ending_0003).
  • I identify the whole cell (secondary object) by using the Nuclei as primary object and the merged image (actin +ER).
  • I then measure the ER distribution (within the secondary object = whole cell) using the thresholded ER image, starting from the edge of the nuclei, across 10 scaled bins, to the cell membrane. I use the Mean Fraction as a readout.

I attach the pipeline and an example of image, analysed. At the end of analysis, I check every image for proper segmentation, and exclude the cells that are not properly segmented.

Many thanks for your help.
Sorry, i just found out I cannot attach files as I am a new user.


Hello Viorica,
Just respond to this reply and you will be able to attach files.