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Wide field, Epi illumination, overfilling the aperture?

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Lu Yan

Wide field, Epi illumination, overfilling the aperture?

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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

Re: Wide field, Epi illumination, overfilling the aperture?

*****
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 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%3D

at

How to use shading correction and background subtraction

http://mdc.custhelp.com/app/answers/detail/a_id/19319/~/metamorph%C2%AE-software-technical-notes

The 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/georgemcnamara
https://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/44962650
http://confocal.jhu.edu

July 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract
supporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023
figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures

mmodel

Re: Wide field, Epi illumination, overfilling the aperture?

In reply to this post by Lu Yan

*****
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*****

Dear Lu,

In practice, we focus the collector lens at whatever plane gives us the best combination of uniformity and brightness. But it is never perfectly flat across the field unless, as George McNamara said in his post, you use a scrambler or something. I suppose that by simply projecting something as variable as mercury arc you cannot expect to get perfect illumination.

Mike

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Yan, Lu <[hidden email]>
Sent: Friday, August 4, 2017 12:45 AM
To: [hidden email]
Subject: Wide field, Epi illumination, overfilling the aperture?

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cmmodel%40KENT.EDU%7Ca9ba5fb033a34939f9d708d4daf3d3ad%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=B4%2BxjBLa9gf30TE7Uy4np07eadfth81QXu0vG9qbOqk%3D&reserved=0 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: https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fconfocal-microscopy-list.588098.n2.nabble.com%2FObjective-back-focal-plane-vs-exit-pupil-plane-locations-td7584559.html%23a7584565&data=01%7C01%7Cmmodel%40KENT.EDU%7Ca9ba5fb033a34939f9d708d4daf3d3ad%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=N9twDfsIfwM4sXG6QVDH2%2FbyDzlxxjN%2BzQgEcrr4trA%3D&reserved=0, 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://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Ffigshare.com%2Farticles%2FZemax_optical_design_files_of_microscope_objectives_tube_lenses_and_Fourier_imaging_setups%2F1481270&data=01%7C01%7Cmmodel%40KENT.EDU%7Ca9ba5fb033a34939f9d708d4daf3d3ad%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=cGMeYj3U8Twi0kiG2dOcGXeZN1TVADipZ%2F1zZKm33QA%3D&reserved=0 ). 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

Lu Yan

Re: Wide field, Epi illumination, overfilling the aperture?

In reply to this post by George McNamara

*****
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 George,

Thanks for your detailed input. I read about KD's nature photonics paper. Actually, I was planning to implement that with fly-eyes lenses. But before that I would like to try several other ways of getting uniform illumination, including launching laser light into a multimode fiber at an angle to have flat-top beam output (something like this: https://www.thorlabs.com/images/TabImages/MM_Fiber_Lab.pdf, which seems quite appealing but I have not heard anyone actually tried it), or using a square core multimode fiber to get square flat-top beam output etc.

I am currently using a Optotune speckle reducer (6 degrees), and it work well so far with regular multimode fiber (200 um core; normal incident angle launching condition).

I will definitely read through all papers you referenced. It felt good to always be able to get help/ideas/comments from the list. I really enjoyed it.

Regarding the shading correction, I will look into it. I also found a nice article from Kurt Thorne's blog regarding shading correction for stitching images (http://nic.ucsf.edu/blog/2014/04/shading-correction-for-different-objectives-and-channels/ ). Pretty useful information to generated large FOV, which kind of brings me back to one of my earlier question: what is limiting the FOV of a microscopy system (supposed that one can choose different tube lens size, and camera size). Is then the FOV limit a property of the objective lens?

Thanks,
Lu

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Friday, August 04, 2017 4:09 AM
To: [hidden email]
Subject: Re: Wide field, Epi illumination, overfilling the aperture?

*****
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 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%3D

at

How to use shading correction and background subtraction

http://mdc.custhelp.com/app/answers/detail/a_id/19319/~/metamorph%C2%AE-software-technical-notes

The 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:

Lu Yan

Re: Wide field, Epi illumination, overfilling the aperture?

In reply to this post by mmodel

*****
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*****

Agreed, Mike.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
Sent: Friday, August 04, 2017 4:31 AM
To: [hidden email]
Subject: Re: Wide field, Epi illumination, overfilling the aperture?

*****
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.
*****

Dear Lu,

In practice, we focus the collector lens at whatever plane gives us the best combination of uniformity and brightness. But it is never perfectly flat across the field unless, as George McNamara said in his post, you use a scrambler or something. I suppose that by simply projecting something as variable as mercury arc you cannot expect to get perfect illumination.

Mike

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Yan, Lu <[hidden email]>
Sent: Friday, August 4, 2017 12:45 AM
To: [hidden email]
Subject: Wide field, Epi illumination, overfilling the aperture?

*****
To join, leave or search the confocal microscopy listserv, go to:
https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=01%7C01%7Cmmodel%40KENT.EDU%7Ca9ba5fb033a34939f9d708d4daf3d3ad%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=wbx4UwbUCyfd7kwJdrd5n%2FnGXlDKROgBspzdg4o0igM%3D&reserved=0
Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=01%7C01%7Cmmodel%40KENT.EDU%7Ca9ba5fb033a34939f9d708d4daf3d3ad%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=B4%2BxjBLa9gf30TE7Uy4np07eadfth81QXu0vG9qbOqk%3D&reserved=0 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: https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fconfocal-microscopy-list.588098.n2.nabble.com%2FObjective-back-focal-plane-vs-exit-pupil-plane-locations-td7584559.html%23a7584565&data=01%7C01%7Cmmodel%40KENT.EDU%7Ca9ba5fb033a34939f9d708d4daf3d3ad%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=N9twDfsIfwM4sXG6QVDH2%2FbyDzlxxjN%2BzQgEcrr4trA%3D&reserved=0, 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://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Ffigshare.com%2Farticles%2FZemax_optical_design_files_of_microscope_objectives_tube_lenses_and_Fourier_imaging_setups%2F1481270&data=01%7C01%7Cmmodel%40KENT.EDU%7Ca9ba5fb033a34939f9d708d4daf3d3ad%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata=cGMeYj3U8Twi0kiG2dOcGXeZN1TVADipZ%2F1zZKm33QA%3D&reserved=0 ). 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

Eric Girard

Re: Wide field, Epi illumination, overfilling the aperture?

*****
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Hi Lu,

We have means of removing laser speckle within fiber, up to 600 um core.
Optical power loss is greatly reduced compared to diffusers and less
complicated than getting out of the fiber.
Typical integration time with >1/5 spatial noise is in the 5 ms range.

Using a non circular fiber, it is possible to also produce a flat top ,
although laser coupling options in standard fiber may be less expensive.

Eric Girard

-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De
la part de Yan, Lu
Envoyé : August-14-17 11:24 PM
À : [hidden email]
Objet : Re: Wide field, Epi illumination, overfilling the aperture?

*****
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.
*****

Agreed, Mike.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of MODEL, MICHAEL
Sent: Friday, August 04, 2017 4:31 AM
To: [hidden email]
Subject: Re: Wide field, Epi illumination, overfilling the aperture?

*****
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.
*****

Dear Lu,

In practice, we focus the collector lens at whatever plane gives us the best
combination of uniformity and brightness. But it is never perfectly flat
across the field unless, as George McNamara said in his post, you use a
scrambler or something. I suppose that by simply projecting something as
variable as mercury arc you cannot expect to get perfect illumination.

Mike

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf
of Yan, Lu <[hidden email]>
Sent: Friday, August 4, 2017 12:45 AM
To: [hidden email]
Subject: Wide field, Epi illumination, overfilling the aperture?

*****
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ata=wbx4UwbUCyfd7kwJdrd5n%2FnGXlDKROgBspzdg4o0igM%3D&reserved=0
Post images on
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m&data=01%7C01%7Cmmodel%40KENT.EDU%7Ca9ba5fb033a34939f9d708d4daf3d3ad%7Ce5a0
6f4a1ec44d018f73e7dd15f26134%7C1&sdata=B4%2BxjBLa9gf30TE7Uy4np07eadfth81QXu0
vG9qbOqk%3D&reserved=0 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:
https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fconfocal-mic
roscopy-list.588098.n2.nabble.com%2FObjective-back-focal-plane-vs-exit-pupil
-plane-locations-td7584559.html%23a7584565&data=01%7C01%7Cmmodel%40KENT.EDU%
7Ca9ba5fb033a34939f9d708d4daf3d3ad%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sd
ata=N9twDfsIfwM4sXG6QVDH2%2FbyDzlxxjN%2BzQgEcrr4trA%3D&reserved=0, 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://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Ffigshare.co
m%2Farticles%2FZemax_optical_design_files_of_microscope_objectives_tube_lens
es_and_Fourier_imaging_setups%2F1481270&data=01%7C01%7Cmmodel%40KENT.EDU%7Ca
9ba5fb033a34939f9d708d4daf3d3ad%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1&sdata
=cGMeYj3U8Twi0kiG2dOcGXeZN1TVADipZ%2F1zZKm33QA%3D&reserved=0 ). 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

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