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Hi, Michael,

You're right, 2 runs would do it - but one of the users of our imaging facility has triple labelled preps ready to analyze. He is looking for the presence of 3 proteins in a cell but I am not sure about their proximity to each other. Visual observation at high magnification could give a clue. I think the user wants some numerical value for a grant. 

Thanks.
Judy


>>> Michael Weber <[log in to unmask]> 08/27/08 9:45 AM >>>
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Judy,

colocalization analysis is quite straight forward with ImageJ and the
"Colocalisation Threshold" plus "Colocalization Test" plugin according to
Costes.

Regarding triple labeling, which type of questions do you plan to answer?
I can think about a scenario with two marked structures and how they are
colocalizing with the nuclei - then you do two runs: nuclei vs. staining
1, nuclei vs. staining 2. I am not aware of an established three-color
colocalization equation. Or do I miss something here?

Michael


> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
>
> I have a triple labelled sample and would like to do colocalization
> analysis. What approaches are most people using? The plugins I have seen
> or used only handle double labelled specimen. A 3-D fluorogram perhaps?
>
> Thank you.
>
> Judy Trogadis
> Bio-Imaging Coordinator
> St. Michael's Hospital, 7Queen
> 30 Bond St.
> Toronto, ON M5B 1W8, Canada
> ph:  416-864-6060  x6337
> pager: 416-685-9219
> fax: 416-864-6043
> [log in to unmask]