Boosting bright field resolution with dichroic filters

Posted by Smith, Benjamin E. on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Boosting-bright-field-resolution-with-dichroic-filters-tp7583983.html

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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]
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