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Hi Kathy,

How about trying a Di dye?

They are more ubiquitous in the membrane and you could get them in
somewhat similar (DiI ex/em at 549/565)spectra.

No Commercial Interest

Best,

 

Gary Laevsky, Ph.D.

Imaging Application Specialist

 

Andor Technology

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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Kathryn Spencer
Sent: Wednesday, August 06, 2008 4:44 PM
To: [log in to unmask]
Subject: co-planar localization

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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
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10550 N. Torrey Pines Road
La Jolla, CA  92037
(858) 784-8437
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