Colocalisation of zymosan particles with nanoparticles

Hi all,
I have imaged the zymosan particles (Green), to calculate the Pearson coefficient, colocalisation with nanoparticles (Red)
The process is:
1- Subtract the background from both channels (Red and green)
2-Analyse —> colocalisation ------> Coloc 2
3- I put Red channel ana green channel
4- Threshold regression ----Costes
5- then OK

However, the merge images show that the particles phagocytosed with macrophage with nanoparticles, the result of colocalisation shows weak colocalisation?

Is the image processing in the right way?

Thanks

You may want to look into how the different colocalization measurements work,

https://journals.physiology.org/doi/full/10.1152/ajpcell.00462.2010
but I would expect to find low colocalization for this image as most of the green is not in the same pixels with the red, and most of the red is not in the same pixels with the green.
As indicated in the papers, Pearson’s has no threshold, so you probably want to be careful over what area you apply it to. Manders might more easily exclude all of the empty background space.

It may also be possible that colocalization is the wrong approach for your analysis, and I have run into this before:


Looking at a couple of locations where the green and red are “interacting,” it seems like the green specifically excludes the red, as, somewhat obviously once you think about it, if the green is a solid particle, the red CANNOT go inside of it.
So everywhere you see colocalization is actually red above or below the green… and depending on whether you are using widefield or confocal, that could be a frequent occurrence, or almost never happen.

Thanks alot.
That’s really valuable

Best regards

Reem

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