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>Hi Michael,
>
>Thanks for your response.
>
>>  From my experience,
>>  Koehler focusing indeed produces optimal illumination of a
>>  slide, although most manuals recommend slight defocusing of
>>  the collector. The problem is that an image of the arc may
>>  not be big enough to fill the back focal plane of the
>>  objective, so one may have to compromise here.
>
>I'm not sure, though, why the image of the arc should be filling the
>objective back aperture in widefield epi-Koehler illumination? The
>illumination light is completely defocussed at the sample (focus at
>infinity), contrary to confocal microscopy where the point source should be
>focused at the sample. Then I understand that the laserbeam should be
>(over)filling the back aperture to get a diffraction limited spot according
>to the objective's full NA.
>
>Kevin

Hi Kevin,

The answer lies in the "partial confocal" nature of widefield 
fluorescence imaging (see ref below). This becomes particularly 
evident when you view a small object (such as a nucleus) and close 
the epi-illumination field diaphragm so that only the nucleus is 
excited (say a diameter or 5 microns in the focus plane).  It we 
assume that the NA 1 objective is fully illuminated and that it 
converges at 45 degrees, then 5 microns above or below the focus 
plane, the diameter will be 15 microns and the intensity 
(photons/s/cn2) will be 9x lower. Any feature that is 5 microns out 
of focus will be 9x less bright than it would be if the field 
diaphragm were opened fully. From this one can see that the Widefield 
fluorescence PSF depends on the filling of the BFP and the size of 
the field diaphragm.

Things are more complex if the image of the arc and its reflection 
are actually present in the BFP as this means that the brightness of 
an out of focus feature depends on where it is with respect to the 
"inhomogenous cone" of light coming towards (or away from) the focus 
plane. (i.e., Areas of the out of focus light field corresponding to 
rays from the images of the arc will brighter than rays that come 
from darker areas of the BFP.)

As a result, companies that really work hard at deconvolution such as 
API, not only provide a fiber-scrambled light source that does fill 
the BFP, they also suggest that you collect a different PSF for each 
size of field aperture setting.

And by the way, it is hard to have the arc 'completely defocused' at 
the focus plane because the arc is more or less spherical. you can 
set things up so that the plane in the center of it is "out of focus" 
at the focus plane but parts of the arc in front of and behind will 
be in-focus and the illumination will not be as uniform as you might 
wish. The much-despised ground glass surface may reduce excitation 
brightness considerably (10x?) but at least it is planar that can be 
focused into another plane and hence avoid this problem.

Cheers,

Jim P.


Hiraoka, Y., Sedat, J.W., and Agard, D.A. (1990). Determination of 
the three-dimensional imaging properties of an optical microscope 
system: partial confocal behavior in epi-fluorescence microscopy. 
Biophys. J., 57: 325-333.
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Prof. James B. Pawley,               		   Ph.  608-263-3147 
Room 223, Zoology Research Building,                         FAX  608-262-9083
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"A scientist is not one who can answer questions but one who can
question answers."  Theodore Schick Jr., Skeptical Enquirer, 21-2:39