Re: Colocalization analysis w/ ICA and JACoP - No intensity saturation please

Posted by Daniel James White on Jun 04, 2010; 12:00pm
URL: http://imagej.273.s1.nabble.com/Re-Colocalization-analysis-w-ICA-and-JACoP-No-intensity-saturation-please-tp3688067p3688068.html

Hi, Bryan

Begin forwarded message:

>
> Certainly these images could not be quantified, they were not taken with
> such intent.  At this point they are the only ones I had on hand to begin
> testing methods of analysis.  I wanted to make sure I could analyze the
> images properly before collecting them.

Phew, thats ok then!


>
> I did not think poor image quality would result in differences between
> quantification methods, but perhaps they handle saturated and empty pixels
> differently?  This would be important to know.

For sure you must avoid:
1) detector Saturation
2) incorrect zero offset
3) noise

all three of these defeat the methods of the coloc plugins/methods.
1 and 2 make the intensity info non linear vs signal in the sample
3 makes the coloclaisation look weaker than it really is,
since it decreases the correlation between the 2 signals.

>
> I've found a few sample colocalization image sets online and I have begun
> using those to test various methods of analysis.  It appears that there are
> still some differences between the ICA and JACoP plugins, although primarily
> in how they apply thresholds.  

its likely (and I need to investigate further)
that these plugins implement the Costes auto threshold method in different ways.
Specifically how the implementation does the search for the thresholds below whioch there is 0 pearsons correlation.
You can start high and move down incrementally until you hit the first place where pearsons is 0 below thresholds,
or you could try to get there faster using a bisection or other minimization method to find it.
These might give slightly different thresholds on the same image, and thus different thresholded Pearsons and Manders numbers (and ICQ , is that thresholded??)

I plan to fing a fast robust way of doing the autothreshold and using that in the next generation coloc plugin we are designing.

> I would like to understand better what is
> going on;

me too.

> I think this has important implications for interpreting the
> results.  For example, Li et al. emphasize that 0-0 pixel pairs should not
> be counted in ICQ analysis.  

yes, and in the coloc plugins in Fiji, you can toggle on and off to include zero zero pixels.
It makes sense to ignore them if the image has many, as they falsely increase the correlation of the whole image.
thats why the offset zero value should be set do there are only a few if any zero values in the image.
If a alge area of the image is zero zero, then you definately need to use a region of interest to measure only where there is something to look at.
Better still take images with very few zeros - but withoiut a large positive offset either (don't waste dynamic range).

> The ICA documentation states that the lower
> threshold must be >0 and the upper threshold must be set to 255, but I do
> not know how JACoP addresses this.

Maybe Fabrice can fill us in?


Daniel James White wrote:
>
>
> For a tutorial on how to get it right look here:
> http://pacific.mpi-cbg.de/wiki/index.php/Colocalization_Analysis
>
>

I appreciate the link, I have also found the following article to be very
helpful in describing imaging requirements and various methods of
quantification.

http://labs.pbrc.edu/cellbiology/documents/Colocalizationtutorial.pdf

Yes thats the article that goes with JACoP plugin.

I hope to work together with those authors to design the next generation colc tool for ImageJ2.


cheers

Dan



Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Visualisation, Processing and Analysis
Light Microscopy and Image Processing Facilities
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany

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