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

Hi all,

Thanks again, Raghu, Julio, Richard.  I went back to lab to determine again
what my microscope can do.  Raghu is right, the minimum bleach spot I can
get is actually about 23 microns, which is indeed the size of the field
diaphragm when fully stopped down and focused through a 100x lens.  The
mistake I had made earlier was to measure a spot on a bilayer that I had
bleached previously with the diaphragm fully open.  So it appears that
modifying the Nikon scope with a custom aperture is not necessary. 

Also, thanks for the suggestions about how exactly to go about measuring
diffusion.  My original intention was to simply follow the average intensity
over the whole spot with time and use that to calculate D.  I have done this
previously (on a different microscope and setup).  One way was to simply use
the recovery half-time to calculate an order of magnitude estimate for D. 
The other was to solve the 1-D diffusion equation analytically and fit the
solution to the recovery curve to extract D (following the famous Axelrod
paper, Biophys J 1976 if I'm not mistaken).  I have not tried
modeling/fitting the solution only to the time-varying edge profile - it may
have some advantages I suppose.  

Apart from an estimate of D, I want to see if the fluorescence recovers
completely - this is because I am studying diffusion of lipopeptides that
are anchored in the bilayer - and it may be interesting to see if they have
a 100% 'mobile fraction'.  Any suggestions/insight on this?

Once again, thanks for your help.  
best
badri