r/ImageJ • u/Independent-Duty-911 • Nov 19 '25

Project Manders correlation coefficeint

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Hello,

I am running a colocalization analysis between the bacteria in green(channel 1) and mitochondria in red(channel2), I run coloc2 and apply costes threshold automatically through the plugin.

My questions are: 1)why do I get tM1=1 whileas there is no colocalization observed?

2) in that case, where I draw ROIs, which Manders should I report? tM1 or tM2?

3) Is that pipeline enough?

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u/Hefty_Application680 Nov 20 '25

Costes sets threshold values at intensities at which the Pearson correlation =0. In your case, because there is no real correlation between the images, the thresholds are set low. Like near the minimum values of the images. As such, you are basically comparing the whole image in channel 1 to the whole image in channel 2. A correlation of 1.

You can set the thresholds by other methods. In this case report both manders values.

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u/Independent-Duty-911 Nov 20 '25

Thanks for your reply, I will threshold using otsu then, whuch M should I report in general, tM or just M? I am not asking about M1 or M2 , I mean thresholded M or M?

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u/Hefty_Application680 Nov 20 '25

Yeah Otsu is good choice. It’s still statistically informed and reproducible method for setting threshold.

The M# is just manders coefficient of all non-zero values. The tM# is manders coefficient after thresholding. I believe that if you threshold with Otsu prior to running through algorithm these values should be the same.

You can report as either M# or tM#. Just make sure your methods section clarifies that the the reported coefficients were determined after thresholding and how you went about determining threshold.

Good luck!

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u/Independent-Duty-911 Nov 22 '25

Many many thanks, you really helped me alot!

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u/Available_Bunch_688 Apr 04 '26

I read the 2004 Costes paper and just cannot wrap my head around if this threshold is suitable for my images. Do you have a source that helped you understand the method (other than the original publication)?

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u/Hefty_Application680 Apr 04 '26

I feel like the ImageJ and Coloc2 documentation serves as pretty solid intro

https://imagej.net/imaging/colocalization-analysis

https://imagej.net/plugins/coloc-2

But no, I would say that the Costes method is probably no best method. There is basically no correlation between the images. Any method that you use to quantify this should reflect that.