http://confocal-microscopy-list.275.s1.nabble.com/Boosting-bright-field-resolution-with-dichroic-filters-tp7583983p7583989.html
(Fig. 2). You can find their paper on
On Fri, Jul 10, 2015 at 9:59 AM, Smith, Benjamin E. <
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>
> Hey microscopists,
> I had a student ask if the department had a 1.4NA condenser for high
> resolution imaging of diatoms. This is a pretty specialized piece of
> equipment, and the highest NA condenser I could find on hand was 0.9NA, so
> I started thinking about how we could get a comparably high resolution with
> our setup.
>
> For a 1.4NA objective and a 1.4NA condenser, with white light BF
> illumination, one would calculate the lateral resolution to be
> approximately:
>
> (0.6 * 575nm) / ((1.4 + 1.4) / 2) = 246nm
>
> For a 1.4NA objective and a 0.9NA condenser, with white light BF
> illumination, one would calculate the lateral resolution to be
> approximately:
>
>
> (0.6 * 575nm) / ((1.4 + 0.9) / 2) = 300nm
>
> However, if you then simply put a blue emission filter (such as a DAPI
> filter cube) into the light path, then one would calculate the lateral
> resolution to be:
>
>
> (0.6 * 445nm) / ((1.4 + 0.9) / 2) = 232nm
>
> Which is now a slightly better lateral resolution then even the 1.4NA
> condenser setup.
>
> I tested this out on a diatom slide, and the results perfectly matched
> the theory, with the white BF image maxing out at 300nm resolution, and the
> blue BF image maxing out at 230nm resolution. You can also clearly see
> additional detail in the blue BF image:
>
> White BF Image -
>
https://drive.google.com/file/d/0B7pDqE0lTjQXT3VKc2Y0ckFEU2s/view> Blue BF Image -
>
https://drive.google.com/file/d/0B7pDqE0lTjQXVUhBODJ4NUZMS3c/view> FFT of White BF -
>
https://drive.google.com/file/d/0B7pDqE0lTjQXb2lBR2dwRXEzVVE/view> FFT of Blue BF -
>
https://drive.google.com/file/d/0B7pDqE0lTjQXZU5GQWNaTE5aUGM/view>
> Upon further investigation, I found this great write-up by René van
> Wezel discussing the same and other ideas for boosting resolution:
>
>
http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artapr09/rvw-contrast.html>
>
> However, in my hands, annular illumination generated a ringing
> artifact, although this is likely because the NA of the condenser is much
> lower than the NA of the objective. All in all, I'm sure for experienced
> microscopists this is likely an obvious solution, but for newer
> microscopists, it may be surprising just how much higher the resolution
> becomes simply by putting a short wavelength dichroic filter into the light
> path (especially when comparing the FFTs), and serves as a reminder that
> transmitted light resolution isn't primarily about NA alone. I know for
> myself, I qualitatively knew that blue light would boost resolution, but it
> wasn't until I did out the math, and verified it experimentally, that I
> realized that blue light with a conventional dry condenser can even
> out-perform white light with a 1.4NA oil immersion condenser.
>
> Have a great Friday,
> Ben Smith
>
> Benjamin E. Smith, Ph.D.
> Samuel Roberts Noble Microscopy Laboratory
> Research Scientist, Confocal Facility Manager
> University of Oklahoma
> Norman, OK 73019
> E-mail:
[hidden email]
> Voice 405-325-4391
> FAX 405-325-7619
>
http://www.microscopy.ou.edu/>