How is the kohler illumination maintained in epi-fluorescence microscope ?

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Bo - 05 Jul 2007 17:24 GMT

Hi all,

In the Kohler illumination, the image of the light source should be
ultimately projected onto the rear focal plane of the objective. But
when the objective moves up and down to acquire a stack of images, its
rear focal plane also moves. So what is the mechanism that allows the
image of the light source always synhronized with the movement of the
rear focal plane of the objective ? Does the illuminator of the
microscope automatically adjust the positions of its lens system
according to the movement of the objective ?

Thanks.

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rene - 05 Jul 2007 20:42 GMT

Bo, it's basically not that sensitive, movement will be in the order
of tens of micron. Besides, in epifluorescence there's only one
requirement: a concentrated spot of light, as even as possible over
the field of view. Nothing fancier then that.

Rene.

> Hi all,
>
[quoted text clipped - 8 lines]
>
> Thanks.

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Kevin Cunningham - 06 Jul 2007 12:28 GMT

> Hi all,
>
[quoted text clipped - 8 lines]
>
> Thanks.

There hasn't been a micrscope equiped with real Kohler illumination since
about 1968. Most micrscopes use a ground glass as the initiation point so
critical illumination is about as good as you can get.

If you refocus you would have to check illumination by closing the field
diaphragm and refocusing however most folks just ignore it.

Thanks,

Kevin Cunningham
SMS

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Victor - 28 Jul 2007 22:16 GMT

Fluorescence illumination does not apply to the principles of Kohler,
as you are not directly illuminating the sample, rather you are
bombarding the sample with photons of one wavelength, and allowing it
to emit it's own photons of another. It's neither direct nor
reflected illumination, rather it's more of a "radiation'
technique.

Having said that, evenness of bombardment is necessary to the function
of the fluorescent microscope, as if you bombard the sample with a
greater number of photons in one area and less in another, then the
emission will follow a similar pattern.

The collector lens system of an epi fluorescent microscope isn't that
precise, and the beam convergence point is long enough to cover the
focal distance of the microscope to provide substantially even
bombardment of the sample.

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Natural Science Forum / Physics / Optics / July 2007

How is the kohler illumination maintained in epi-fluorescence microscope ?