Hello everyone,

I am trying to use FIJI Coloc2 plugin for my below 16-bit fluorescence images of 2 proteins (png files below). I am interested in calculating colocalization, especially at the clusters/puncta in 2 images.

In theory, these clustered proteins should co-localize well, resulting in high Pearson’s and Mander’s coefficients. I subtracted background and tried Coloc2 with below settings: (I calculated PSF using 1.22λ/NA formula to get the value of approx. 3)

I got below results:

*Warning! y-intercept far from zero - The ratio of the y-intercept of the auto threshold regression*

*line to the mean value of Channel 2 is high. This means the y-intercept is far from zero, implying a*

*significant positive or negative zero offset in the image data intensities. Maybe you should use a*

*ROI. Maybe do a background subtraction in both channels. Make sure you didn’t clip off the low*

*intensities to zero. This might not affect Pearson’s correlation values very much, but might harm*

*other results.*

*Pearson’s R value (no threshold): 0.54*

*Pearson’s R value (below threshold): 0.00*

*Pearson’s R value (above threshold): 0.31*

*Manders’ M1 (Above zero intensity of Ch2): 1.000*

*Manders’ M2 (Above zero intensity of Ch1): 0.982*

*Manders’ tM1 (Above autothreshold of Ch2): 0.773*

*Manders’ tM2 (Above autothreshold of Ch1): 0.591*

*Costes P-Value: 1.00*

Can anyone figure out why I have the **warning message** though I subtracted background and selected ROI for calculation? And I am not sure if **Pearson’s R value (above threshold): 0.31** is too low? I also tried using 8-bit binary mask but it was even worse.

Any help is much appreciated. Thank you in advance!