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At 10:11 AM 9/9/2005, you wrote:
>Search the CONFOCAL archive at 
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>Dear all,
>
>I have some (basic) questions about microscopy optics:

These questions are deceptively basic- not so simple to answer!



>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?

As has been pointed out, this makes no difference.  As far as the 
microscope is concerned, the light path is the same as (near-monochromatic) 
brightfield: the object emits light at a certain wavelength, and the optics 
correct for color as normal.  Now, there are two kids of color correction: 
transverse and longitudinal.  Transverse makes white stars look like small 
circular rainbows, while longitudinal will make the point change color 
depending on where the optics are focused in 'z'.  Confocal microscopy 
demands very low longitudinal color aberration, and higher-quality lenses 
will gain an addition "CS" designation (or something similar) to indicate 
that they are superior performing lenses.

>
>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?

No- parfocal means that the distance from the shoulder of the lens housing 
to the focal plane is a constant from lens to lens.  This is why (in 
theory) one can switch objectives and stay in focus.  And why (in theory), 
once the condenser lens is brought into alignment for Kohler, the lens does 
not need to be readjusted for every objective lens. In practice, small 
adjustments are necessary for parcentration and manufacturing tolerances. 
Lens designers have a lot of freedom to adjust the various parameters of 
the individual elements (mainly curvature and spacing) to make the total 
lens 'fit' into the allowed space.    Now, as one focuses through a thick 
sample, Kohler illumination will be lost as the condenser is not moving to 
compensate for the change in focal plane.

>
>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?

Oof- this is a complicated question.  It gets into the reasons why Kohler 
(or critical!) illumination is preferred over simply shooting light into 
the sample.  Simply put, Kohler and critical illumination maximize the 
information-carrying potential of the illumination light.  Critical 
illumination focuses the source onto the sample, and Kohler focuses the 
(spatial) Fourier transform of the source onto the sample.  Now, as soon as 
you move off the focal plane, the illumination profile is rather complex to 
describe.  Qualitatively, the regions just prior to and just past the focal 
plane are blurred images of the focal plane.  Often, the numerical aperture 
of the condenser is so much lower than the NA of the objective, the range 
of 'z' that one looks at is always near the focal plane of the condenser lens.

  Hope this helps. I'd be happy to discuss this further with you, if you 
wish- email me directly.

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