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Hello,
I am trying to use Coloc 2 to quantify colocalization between red and green mitochondria. I am using the most current version of Fiji/Coloc2. I open my images and convert them to 8-bit followed by a round of background subtraction using a rolling ball radius of 200 pixels and the sliding paraboloid parameter. I use the free-hand tool to draw an ROI and then I execute the Coloc 2 analysis with a PSF of 10 and 100 Costes randomizations. I very often receive these three error messages.
The error states:
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.
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.
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.
What do these errors ultimately mean and how can I remedy them?
Also can I still utilize the values given for Pearson's coefficient, manders 1 and 2 and li's ICQ when my coloc 2 analysis is showing these errors?
Any input would be greatly appreciated as I'm very new to using this software. Thank you!
-A
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