http://confocal-microscopy-list.275.s1.nabble.com/Boosting-bright-field-resolution-with-dichroic-filters-tp7583983p7583993.html
Just a comment on the George's discussion. These
get a double bang for the buck. It only takes a
>*****
>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.
>*****
>
>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.html>
>
>enjoy,
>
>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