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Try what happens if you switch which stack you use for which channel.
I was just doing colocalization analyses this week, and I had a set,
that gave different thresholds when I switched channels - one was
negative in first case, and tM coefficients were 1, and after using
switched images for channels 1 and 2, both thresholds were positive, and
tM were low (the results should be low colocalization).
Also, it's better to use a ROI - make a freehand selection around your
cell(s).
The problem with auto thresholds may be if one of the images has high
background - correct this with Image-Adjust-Brightness&Contrast, and set
Minimum to a higher value, but not so high that you clip the low values,
Apply, Apply to all slices.
-Ales Kladnik
> I am new in Image J and I am trying to do a colocalization analysis using
> the plugin "Colocalization thresholds" using the z-stack of my images (2
> channels).
> In some analysis I have some negatives output values for the Ch1 thresh and
> Ch2 thresh or values like 13 and 21 that don't seem close to zero, but I
> read in a tutorial that they need to be close to zero.
> Besides, in some analysis the values for M1 and tM1 are the same.
> Can somebody help me to understand these values, please?
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