On Thu, 6 Aug 1992, Keith Bartels wrote:
 
> >
> >   I agree, but what it is happening there is not improving the
> >   "geometry" of the system but simply ignoring the weaker fluorescence
> >   at the top and at the botton that makes the bead appear longer.
> >   I believe that if the x-y-z resolutions are matched, then the amount
> >   of elongation indicates how "confocal" the system is.
> >
> >
>
> I agree, so please allow me to make the original question of Shanti more
 clear.
> When looking at an x-z section through a 10um flourescent bead, we see an
> ellipse that measures 13um(z) and 10um(x) on its major and minor axes
> respectively.  Doing a similar experiment in a straight reflectance
> (non-fluorescent) mode, this elongation is not apparent.  Obviously, as
> mentioned above, the saystem is less "confocal" in this fluorescent mode. This
> is to be expected from theory (Several sources Wilson et al.) but not to
> this degree, I don't think. Has anyone else noticed such a difference in
 fluor.
> and non-fluor. confocal modes?  The 3um elongation is simply not caused by
> the fact that the xy res. is 150nm and the z res. being 200nm. It looks more
> like the z res. is on the order of 1um!!
>
>   -Keith Bartels.
 
Perhaps my confusion is that I don't get that result on my system, if I
use a high n.a. objective. On a 100 nm bead, the resolution explains it.
On a 10 um bead, I would say that the scope is not very confocal. On my
MRC-600, I would expect, based on other samples, that I would get
10um X 10 um X 10.1 um. When I have tested the Zeiss confocal in the past,
I have gotten results that bad. On the BioRad and the Sarastro systems,
the numbers are nowhere near that.
 
Paul Goodwin