Posted by
Daniel James White on
Aug 01, 2011; 6:40pm
URL: http://imagej.273.s1.nabble.com/Re-question-regarding-Colocalization-Threshold-in-Fiji-Coloc2-tp3683647p3683648.html
Hi Rob,
On Aug 1, 2011, at 6:00 AM, IMAGEJ automatic digest system wrote:
>
> Date: Sun, 31 Jul 2011 19:38:53 -0500
> From: Robert Baer <
[hidden email]>
> Subject: Re: question regarding Colocalization Threshold in Fiji - Coloc2
>
> I applied the Colocalization threshold plugin on 2 background-subtracted
non specific background is still real signal and should not be subtracted,
else the image info becomes non linear.
ccd camera bias/offset (0 light image on a ddc does not give zero in the pixel values)
confocal PMTs must be set so the offset gives just above zero on average in areas with no signal.
> images for example one “positive” image where colocalization is
> expected/”visible” and one “negative” image with no colocalized signals.
> Then, I´ve tested the results with the Colocalization test plugin and all
> the p values were much lower than 0.
the coloc test plugin randomises the image many times then compares pearsons r for each with the original.
we expect there to be lower pearsons r values than the real image pair when there is true coloc,
but about the same if the apparent coloc is no better than random.
read the Costes 2004 paper to get a full explanation.
Can you send me the images for testing?
>
> Do you really mean negative or is this a mis-speak? The theoretical range
> of p is 0 to 1.0
no, the range for Pearsons r is -1 to +1
> Thus, values lower than zero suggest that the code is
> broken or improperly applied.
negative values are certainly possible,
but perhaps the issue here is that there is another related problem...
let me have the images and i will investigate...
did you use a region of interest?
cheers
Dan
>
> Rob
>
>
>
>
>
> ------------------------------------------
> Robert W. Baer, Ph.D.
> Professor of Physiology
> Kirksville College of Osteopathic Medicine
> A. T. Still University of Health Sciences
> 800 W. Jefferson St.
> Kirksville, MO 63501
> 660-626-2322
> FAX 660-626-2965
Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Processing and Analysis
Light Microscopy Facility
Max Planck Institute of Molecular Cell Biology and Genetics
Pfotenhauerstrasse 108
01307 DRESDEN
Germany
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http://www.chalkie.org.uk[hidden email]
(
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