Colic 2, warnings

imagej

#1

Hello everyone,
I am Morris and I m writing from Italy,
Here I found already a lot of useful answers but now I have a question.
I m doing a co-localization analysis using two channels green and red, but I have this warnings coming out from the result:

!!! 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.

how can I subtract the background?

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.

how can I adjust the threshold?

Thank you guys