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Kevin wrote:
> 1. Chromatic aberration in Zeiss microscopes is not (entirely?) corrected
> at
> the objective lens, but is done at the tube lens. Then how is chromatic
> aberration addressed in the illumination path when doing epi-fluorescence?

It isn't.  The focus of the illuminating light does not
make any difference to the image since fluorescence is an
incoherent process.  In fact, when using UV excitation the
focus will be quite a way off, since objectives aren't
usually corrected for uv anyway.

> 2. Since objective lenses are designed to be parfocal, it seems that the
> position in space of the back focal plane must be different for objective
> lenses of different magnification. However, for Koehler illumination, a
> real
> image of the light source has to be projected on this back focal plane,
> which is done by the collector lens of the lamp housing. It therefore
> seems
> to me that the collector lens has to be refocused to maintain Koehler
> illumination every time a different objective lens is selected? I've
> looked
> at a few different texts on Koehler illumination, but none of them says
> something about this issue. So maybe I'm just mistaking, or perhaps it
> doesn't matter a lot?

You are confused here.  The position of the BFP will indeed vary
but since both the condenser and the objective are focussed on the
specimen the objective will still image the field iris at the
image plane and therefore the lamp filament and condenser iris at
its BFP.  The important thing is where each lens is focussed
not the position of the planes.  (In principle this means you
should refocus your lamp system if you change to a long WD
condenser but I bet nobody does.)

> 3. Most texts on Koehler illumination mention 'homogeneous illumination'
> and
> 'improved optical sectioning' as one of the advantages. I was wandering,
> though, about the axial illumination profile. Could someone point me to a
> text which discusses the (axial) intensity distribution of the
> illumination
> beam in Koehler configuration?
>

The field will be uniform at the plane of focus and
progressively change to a filament image as you move
away from there.  "Improved optical sectioning" really
is just a function of improved resolution and therefore
reduced depth of field with large aperture illumination.
Close the condenser aperture down and you'll see this
clearly enough.

                                              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