Dear all,

I have a few questions about the coloc2 plugin.

- What is the method used by coloc2 to define the “max threshold” for each channel?

I tried JaCoP on the same images, and the thresholds given by coloc2 are different from both the “automatic” thresholds and the Costes’ thresholds given by JaCoP.

- Are the “max thresholds” used for the calculation of the Manders’ coefficients by coloc2? Again, Manders’ coefficients from coloc2 are different from all the sets of Manders’s coefficients that I get with JaCoP.

And finally, a more general question: I have analyzed only a few images until now, but for all of them, and all my ROIs, I get a message saying that “The absolute y-intercept of the auto threshold regression line is too high”, and suggesting that I should use a ROI or do a background subtraction. However, the values of the y-intercept do not seem to me

excessively high; what I consider as background has a low mean value too, even inside ROIs. Also, I feel it strange to subtract a background before asking the plugin to calculate a threshold that is used to determine what “background” is. What is your opinion about that?

Thank you for your help !