colocalization

[Reinat Nevo]

Hi,

I am trying to do colocalization measurements of FluorescentCells (Image J - sample image). When I do it for the whole image I don't get any error massage and the values are:

Manders M1 (threshold): 0.974
Manders M2 (threshold): 0.954

When I mask the image to have only the cells (without the background), I get the following massages:
Warning! y-intercept high - The absolute y-intercept of the auto threshold regression line is high.
Maybe you should use a ROI, maybe do a background subtraction in both channels.
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.

Manders M1 (threshold): 0.320
Manders M2 (threshold): 0.369

What is the correct way to measure the colocalization in such images?

Thank you,
Reinat


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