Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Wide-field-Epi-illumination-overfilling-the-aperture-tp7587113p7587114.html
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Hi Lu,
Best wishes getting uniform illumination by lenses and focal points
only. See:
Super-resolution imaging of multiple cells by optimised flat-field
epi-*illumination*. <
https://www.ncbi.nlm.nih.gov/pubmed/27818707>
Douglass KM, Sieben C, Archetti A, Lambert A,*Manley S*.
Nat Photonics. 2016 Nov;10(11):705-708. Epub 2016 Oct 17. No abstract
available.
PMID:
27818707
Efficient homogeneous illumination and optical sectioning for
quantitative single-molecule localization microscopy.
<
https://www.ncbi.nlm.nih.gov/pubmed/27906373>
Deschamps J, Rowald A, Ries J.
Opt Express. 2016 Nov 28;24(24):28080-28090. doi: 10.1364/OE.24.028080.
PMID:
27906373
At the QBI 2017 conference at Texas A&M, Prof. Manley briefly mentioned
a new generation of their device, so please keep a look out for that.
To the best of my knowledge, the original fiber optic light scrambler
was developed by Gordon Ellis for Shinya Inoue at MBL. See Shinya's
Video Microscopy book (i.e. edition with Ken Springs) and papers.
final step can be digital shading correction. Probably very thin,
uniformly fluorescent, target best for the reference image. See
SIPcharts, such as these two:
Quantitative image correction and calibration for confocal fluorescence
microscopy using thin reference layers and SIPchart-based calibration
procedures. <
https://www.ncbi.nlm.nih.gov/pubmed/18638190>
Zwier JM, Oomen L, Brocks L, Jalink K, Brakenhoff GJ.
J Microsc. 2008 Jul;231(Pt 1):59-69. doi: 10.1111/j.1365-2818.2008.02017.x.
PMID:
18638190
Characterization of sectioning fluorescence microscopy with thin uniform
fluorescent layers: Sectioned Imaging Property or*SIPcharts*.
<
https://www.ncbi.nlm.nih.gov/pubmed/16176252>
Brakenhoff GJ, Wurpel GW, Jalink K, Oomen L, Brocks L, Zwier JM.
J Microsc. 2005 Sep;219(Pt 3):122-32.
PMID:
16176252
PubMed related articles of Zweir (sorted for newest) should also be useful.
Can also see
http://mdc.custhelp.com/app/answers/detail/a_id/18800/session/L2F2LzEvdGltZS8xNTAxODQ0NzE5L3NpZC9nVFFZbGlwbg%3D%3Dat
How to use shading correction and background subtraction
http://mdc.custhelp.com/app/answers/detail/a_id/19319/~/metamorph%C2%AE-software-technical-notesThe latter page is a list of a lot of MetaMorph technical and
application notes. Disclosure: I wrote early (1992-1997) versions of
many of these (fig 1 of the shading note was probably drawn by Ted Inoue
- definitely not by me).
enjoy,
George
On 8/4/2017 12:45 AM, Yan, Lu 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.
> *****
>
> Hi,
>
> I was searching for back aperture (or exit aperture plane) and the back focal plane of high NA objective lenses, and found this thread that Kyle posted in end of 2015:
http://confocal-microscopy-list.588098.n2.nabble.com/Objective-back-focal-plane-vs-exit-pupil-plane-locations-td7584559.html#a7584565, in which all agreed most of cases, these two planes are not coplanar.
>
> I understood that, for confocal scope, one would want to 'overfill' the back aperture of an objective, as described in several textbooks, to utilize the full NA of the lenes, and achieve smallest spot size possible. There, since the laser beam comes in collimated, it of course also overfills the back focal plane which often lays inside of the objective lens (I found this website providing Zemax models of several Nikon lenses based on publicly released documentations:
https://figshare.com/articles/Zemax_optical_design_files_of_microscope_objectives_tube_lenses_and_Fourier_imaging_setups/1481270 ). I have simulated several of them, and found that the 'back focal plane' is indeed residing inside of the lens. Some of them are actually almost in midway of the lens housing.
>
> Based on these information, I was wondering, for wide field scope, epi-illumination (non-critical illumination), what would be the best practice to choose the relay lenses to focus onto this back focal plane to get most uniform, and possibly large illuminated FOV, considering there is a hard stop up stream, i.e. the exit aperture of the lens. It seems to me that depending on the distance between these two plane, and the size of the exit aperture, there is a limited NA for lens that focuses the collimated laser beam onto the back focal plane. Is there an 'overfilling'-equivalent concept for epi-illumination? Does it matter if one comes with quite large cone angle to focus onto the back focal plane, and the beam is clipped by the back aperture of the objective lens? Would the amount of clipping affect the illumination quality in general? I guess this kind of clipping will play more important role in TIRF or SIM case where one wants to focus beam(s) onto the edge of the back focal plane of the objective. Smaller cone angel will give you small beam size (collimated) on the sample plane), but large cone angle will be clipped by the back aperture of the objective. I am trying to collect all relevant thoughts from experts here on the list, and any inputs will be welcome. I have to get several concept clear in my head.
>
>
> Thanks,
> Lu
--
George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamarahttps://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650http://confocal.jhu.eduJuly 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstractsupporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures