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
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!