Re: colocalization analysis

Posted by Daniel James White on Mar 13, 2011; 8:54pm
URL: http://imagej.273.s1.nabble.com/JACoP-parameter-and-PSF-tp3685080p3685094.html

Hi Gauri,

dont forget to cc me directly... or i might miss message to addressed to me but only sent to the mailing list.

On Mar 13, 2011, at 6:00 AM, IMAGEJ automatic digest system wrote:

> Date:    Sat, 12 Mar 2011 14:42:51 -0800
> From:    gauri <[hidden email]>
> Subject: Re: colocalization analysis
>
> Thanks Dan
> I had another question about analysis of colocalization using JACoP.
> i make substack of 4-5 slices for both the channels. Do i just use those
> substack as such for colocalization or do i merge these 4-5 slices in one
> image before i use them in JACoP.

if you merge z slices from the same xy field of view,
you lose the z resolution, and can then get false coloc of objects in different z planes.
Don't do that!

However, if the slices are from different x,y areas of the sample, then its ok.

> If i use 4-5 slices for each substack, how does JACoP calculate the r value?

Pearsons r is a global statistic calculated from all the pixels included in the calculation.
You would get the same r if the same pixels were in many z slices or only one 2D image...
or even in a different order. Its a pixel by pixel calculation, and the result is summed over all pixels counted.

> Does it look at r value of each slice and then average it, or does it
> average out the pixel. i am so confused about how it works.

Look at the maths on the Manders and Costes articles.
You will be able to convince yourself by understanding the
simple equation used to calculate Pearson's r :

http://en.wikipedia.org/wiki/Pearson_product-moment_correlation_coefficient

https://ifn.mpi-cbg.de/wiki/ifn/images/0/0d/QuantitativeColocAnalysis-12-2010.pdf

r =  ( sum of (channel1 pixel intensity - average ch1 intensity) x (channel2 pixel intensity - average ch1 intensity) ) / sqrt of square of top half of eqn

...that either way gives the same result or not....

In practice, one typically takes all pixels from all slices to be analysed and uses the mean of those...
and does not do it slice by slice then average the results.... there is no point in doing the latter...? Right?

There are 2 (or maybe more) ways to implement Pearsons r in computer code.
1) The "fast" way used in the colocalization thershold plugin, using a rearrangement of the equation
2) the "classic" way using the equation as written.

The new Coloc_2 plugin can do both... and we notice that the classic way is not really slower and less susceptible to numerical problems... eg when the means of both images are the same.

Not sure which JaCOP uses without looking at the code...

cheers

Dan






Dr. Daniel James White BSc. (Hons.) PhD
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Light Microscopy Facility
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