I am trying to make a cellprofiler pipeline that allows me to analyze the degree of mitochondrial clearance by autophagy in a high throughput format.
As a readout the cells express a double-tagged (mCherry-EGFP) mitochondrial localization signal (seen as yellow). Once the mitochondria reaches the lysosome - and due to the acidic environment- EGFP gets quenched (and degrading structures can be seen as mCherry only).
I am trying to measure these mCherry only structures in an automated way that avoids having to manually feed it any parameters in the process (as I am doing now).
In the attached image you can find two example images (right and left panels show the same images, I just changed red to magenta in case someone is colorblind). The upper row shows active degradation whereas the bottom row shows no degradation.
I have some working cellprofiler pipeline already, but I am not completely happy with the result and I was wondering if you have any suggestion that could help me improve the pipeline.
What I currently do is:
- to make a mask image that combines all structures in mCherry and EGFP channels.
- to measure the intensities of mCherry and EGFP within the segmented structures.
- to calculate the EGFP/mCherry ratio (I avoid mCherry/EGFP as it would give NaN in case EGFP is quenched) within the structures
- THIS IS THE STEP I WOULD LIKE TO AVOID: by using the DisplayDataOnImage module on cellprofiler I check what ratio value corresponds to mCherry-only or yellow structures and I use this value to filter the objects, binning them between mCherry-only or yellow.
- I can then count and measure objects in each category.
The fact that in both situations there is a vast extension of mitochondrial network that is not being degraded makes the analysis difficult… It would be great if you could suggest a way of measuring the amount of mCherry-only (either red or magenta in the image) structures.
Thank you very much in advance for your time and help!
I would like to thank @Christian_Tischer for his help. He has also been the one suggesting me to post this question here and see if you guys could give us some nice suggestion to approach the problem.