I am analyzing the colocalization of PSD95 and synapsin-1 using ImageJ coloc2 plugin, but I am not sure how to treat the images, such as background subtraction, and the threshold of pictures in using coloc2. Also, there's a PSF parameter, I don't know what does that mean. What number should I choose? I choose 3 or 10 to run, and got warnings like this:

!!! WARNINGS !!!

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.

Warning! Threshold of ch. 1 too high - Too few pixels are taken into account for above-threshold calculations. The threshold is above the channel's mean.

Warning! Threshold of ch. 2 too high - Too few pixels are taken into account for above-threshold calculations. The threshold is above the channel's mean.

Who can teach me step by step? Thank you so much.