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Hi,

In my opinion there is not really a problem here, or at least no more of a
problem than there would be if the patches would be overlapping. The fact
that you see both types of patches in the same focal plane means obviously
that they are in the same plane within the microscope's resolution. If the
patches would be visually overlapping you would still have the same
uncertainty with regard to the z-position of both. It just looks a bit
nicer, that's all. Colocalisation based on the coincidence of colors in
still images is always limited by the spatial resolution.

Best regards,

Kevin


> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[log in to unmask]]
> Namens Kathryn Spencer
> Verzonden: woensdag 6 augustus 2008 22:44
> Aan: [log in to unmask]
> Onderwerp: co-planar localization
> 
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> 
> Hello all;
> 	Interesting volume localization and quantitation question for
> you.
> 	One of my users wants to show that her marker moves to the
> plasma membrane after stimulation. We have used Alexa 568 WGA on her
> cells to label the membrane. Her channel protein is eGFP-labeled. Both
> signals are not continuous, but are punctate. After stimulation, the
> signal appears to move to the membrane, but the WGA and her eGFP do not
> overlap, i.e., the signals are found in exclusive patches, as shown by
> LSM confocal, 60x, zoom x2, 1.4NA in Z-stacks. I have thresholded the
> WGA signal in MetaMorph (with a variety of values), made a binary mask,
> and overlaid this on the GFP signal. What we see is the suggestion that
> the channel protein is at the membrane in the same plane as the WGA,
> but
> there is minimal overlap (as determined by line-scans). How to quantify
> this? She likes my reference to the patches on a soccer ball...they are
> obviously in the same spherical plane, but do not overlap. Would volume
> rendering and modeling help?
> 	Thanks.
> 	Kathy
> 
> 
> 
> Kathryn Spencer, Ph.D.
> The Scripps Research Institute
> ICND 210
> 10550 N. Torrey Pines Road
> La Jolla, CA  92037
> (858) 784-8437
> [log in to unmask]