Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Boosting-bright-field-resolution-with-dichroic-filters-tp7583983p7583990.html
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Hi Ben,
thanks for initiating a great thread (ex. Barbara Foster's reply).
Be sure the microscope is optimally focused for each wavelength!
You wrote:
(0.6 * 445nm) / ((1.4 + 0.9) / 2) = 232nm
but for $97 you could buy a 400 nm filter
https://marketplace.idexop.com/store/IdexCustom/PartDetails?pvId=35105(or buy a filter from Semrock or Chroma).
(0.6 * 400nm) / ((1.4 + 0.9) / 2) = 209nm
Could go shorter than this with appropriate light source, such as the
~370 nm peak of the XLED1, in transmitted mode
http://www.excelitas.com/Pages/Product/X-Cite-XLED1.aspx (0.6 * 370nm) / ((1.4 + 0.9) / 2) = 193nm
or in epi mode, 1.4 NA objective lens (and assuming clean light path):
(0.6 * 370nm) / ((1.4 + 0.9) / 2) = 158nm
//
A bit more challenging: diameter of the pores of /Pleurosigma
angulatum/, instead of just the spacing of the pores - see bottom of
http://www.microscopy-uk.org.uk/mag/artjan13/fs-diatom-micro.htmlenjoy,
George
p.s. reflection confocal microscopy of diatoms is also a lot of fun ...
especially <<1.0 Airy diameter pinhole. Hopefully someone will publish
results of this for the Zeiss AiryScan. STED with the right fluorophore
in the mounting medium (or thin coating the diatom) would also be
interesting.
On 7/10/2015 8:59 AM, Smith, Benjamin E. wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
http://www.imgur.com and include the link in your posting.
> *****
>
> 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/>
>
--
George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales
http://works.bepress.com/gmcnamara/42