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

Sounds to me like a TIRF microscope might help...

Cheers Mark Cannell

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