Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

What is that you want to quantify, Kathy?

If it is the spatial relations between the labeled structures in the 
plane of the membrane, there is a range of spatial statistics that let 
you decide if they are really associated or not (eg as compared with a 
uniform or random distribution). Doing this in 3D can be a bit of a 
trick, but there are several approaches that are not too difficult, as 
long as you can get some quantitative data out  of your image sets 
(typically x-y-z coordinates and grey-scale values of the features of 
interest.)

In my experience, this proviso is surprisingly difficult to extract 
easily from imaging programs that must have the data embedded within 
them. I haven't looked at MetaMorph for a while, so I can't offer any 
clues there. May be someone else can?

If this approach is what you are after, let me know, and I can give you 
some references.

IAN


On Thursday, August 7, 2008, at 06:14  AM, Kathryn Spencer wrote:

> 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]
>
>

* * * * * * * * * * *
Prof Ian Gibbins
Anatomy & Histology
Flinders University
GPO Box 2100
Adelaide SA 5001
AUSTRALIA

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