Re: question regarding Colocalization Threshold in Fiji - Coloc2

Posted by jo on Aug 02, 2011; 7:59am
URL: http://imagej.273.s1.nabble.com/Re-question-regarding-Colocalization-Threshold-in-Fiji-Coloc2-tp3683647p3683649.html

>>> Dan White <[hidden email]> 1.8.2011 20:40 >>>
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, Íve 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...
>Sorry the p values are Ok actually.My mistake:I mis-spelled..
I meant the r(rand) not the p values.
My problem now is just that I can´t use coloc2 and I can´t
rely too much on Colocalization threshold plugin that actually
provides
too low thresholds in some cases..

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 [phone]

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

[phone] (German Mobile)
+49  (0)351 210 2627 (Work phone at MPI-CBG)
+49  (0)351 210 1078 (Fax MPI-CBG LMF)

http://www.bioimagexd.netBioImageXD 
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[hidden email]
( [hidden email] )