Re: More on A.pellucida

DF reduces resolution over bright field or DIC. With a DF system the NA
of the objective and condenser is quite low in comparision to bright field
and DIC. NA is resolution, resolution is NA. Thats why DF is yesterday's
news.


Kevin:

Yes, DF reduces resolution over BF and DIC due to the closed iris located
inside the oil immersion objective. We both know that it must be closed to
around 1.10 but not more than 1.20 in order to obtain decent darkfield
illumination. The condenser for DF does not lower resolution since any
decent DF condenser using oil immersion provides a light cone that
illuminates between NA 1.20 to 1.40. Hence the resolution decrease is purely
due to the objective's iris (or stop).

However, the problem to distinguish certain, tiny diatom structures, is not
just a lack of resolution; it is the availability of sufficient contrast. In
DF, I can detect particles that are way below the resolution limit of BF and
DIC. But these particles appear in DF larger than their physical dimensions.
It is clear from diffraction theory of light that when the size of a
particle or feature falls below the minimal particle size that can be
resolved with a given optical setup, the size of the image of said particle
is no longer smaller than the minimal particle size, but its contribution to
the image contrast decreases as the diameter of the subresolution particle
gets smaller.

DF is recommended for this application in circles that study diatoms
intensively using LM, while DIC isn't due to its image artifacts that make
particle/feature interpretations difficult. Amateur microscopists tend to
use DIC and PC to produce pretty pictures. Personally, I prefer COL for
diatoms, which happens to decrease contrast when compared to DF but, of
course, offers the full resolution of the objective/condenser combination.

Cheers,

Gregor


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