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Guy,

These are all valid points, perhaps I was not clear in my meaning: the 
depth of Kohler illumination is set by the condenser NA, not the objective 
NA.  The maximum resolution obtainable in a trans illuminated system is set 
by a combination of the condenser and objective NA.  My only comment was 
that worrying about maintaining Kohler illumination in a typical 
transillumination setup (immersion objective, moderate condenser NA) is 
pointless as Kohler illumination is maintained throughout a (relatively) 
large range of objective motion.

Andy

At 08:06 PM 9/12/2005, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Andrew wrote:
>
> > Finally, as for the axial illumination profile, the total intensity in any
> > plane is a constant (or nearly so, if the sample is partially
> > absorbing).  As for setting absolute limits on the transverse homogeneity
> > of the illumination, I can't say.  But, for a condenser NA of 0.55 (that's
> > what we have on our Axiovert 200), the depth of field is around 3 microns-
> > this means that one has true Kohler illumination over a 3 micron thickness
> > at the sample.
>
>A condenser NA that is much smaller than the objective NA
>will seriously reduce the resolution obtainable in widefield
>illumination.  To meet the Abbe condition your condenser NA
>needs to match the objective NA.  Only then will you obtain
>r = 0.5 lambda / NA (the Abbe resolution).  Of course if you
>only want high resolution in fluorescence and your widefield
>is just for finding the sample this is irrelevant.  But
>Kevin's queries were presumably aimed at high resolution.
>
>The question of focussing through the sample depends on how
>you do your focussing.  The ideal is to move the slide
>between fixed objective and condenser, as with the elegant
>(but fragile) Leica galvo stage.  Then your Koehler illumination
>will stay just the same throughout the focal series.
>However I doubt if many microscopists focus their
>illuminators to that sort of accuracy even if they do
>focus their condenser carefully.  And many microscopes
>have diffusers which prevent careful focussing of the
>illumination anyway.  With epi-illumination this is anyway
>a non-issue since lens to focus distant remains constant.
>But accurately focussing a lamp filament image at the BFP of
>the objective doesn't seem an easy task ...
>
>                                                      Guy
>
>
>--
>Associate Professor Guy Cox
>Electron Microscope Unit,
>University of Sydney,
>NSW 2006, Australia
>
>Phone:+61 2 9351 3176    Fax:+61 2 9351 7682
>http://www.guycox.net

Andrew Resnick, Ph. D.
Instructor
Department of Physiology and Biophysics
Case Western Reserve University
216-368-6899 (V)
216-368-4223 (F)