Illumination homogenizer for wide-field

[Csúcs Gábor-3]

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Dear All,

I have just today learned about a new sCMOS camera with 29mm Field of View. With some of the new large FOV microscope bodies this really makes sense, but I was thinking about the vignetting effect that can be quite significant with such a large FOV. Of course, there are numerous computational methods how you can correct for this, but still the best way would be to have a more homogenous illumination. I am fully aware of some developments for laser-based systems (homogenizer for various spinning disk systems by various providers) but I was wondering whether anyone knows about a commercial homogenizer solution for wide-field systems (using e.g. LED light sources)?

Thanks                                  Gabor

[Sylvie Le Guyader]

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If I remember well the Nikon Ti2 has 32mm FOV with some vignetting and 25mm without. I wonder how it performs at 29.

Med vänlig hälsning / Best regards
Sylvie
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
LCI microscopy blog

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Csucs Gabor
Sent: Thursday, 3 September, 2020 12:52
To: [hidden email]
Subject: Illumination homogenizer for wide-field

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Dear All,

I have just today learned about a new sCMOS camera with 29mm Field of View. With some of the new large FOV microscope bodies this really makes sense, but I was thinking about the vignetting effect that can be quite significant with such a large FOV. Of course, there are numerous computational methods how you can correct for this, but still the best way would be to have a more homogenous illumination. I am fully aware of some developments for laser-based systems (homogenizer for various spinning disk systems by various providers) but I was wondering whether anyone knows about a commercial homogenizer solution for wide-field systems (using e.g. LED light sources)?

Thanks                                  Gabor


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[Benjamin Smith]

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The key is to understand why there is the vignetting issue.  Some of the
vignetting is because as you increase the angle you approach the aperture
of the objective, the narrower the narrower aperture gets.  You can see
this effect by simply looking straight down on the back of an objective,
and then tilt it to one side and see how the effective back aperture gets
smaller.  With this in mind, the easiest way I know of to flatten
illumination vignetting would be to have your illumination source underfill
the back aperture of the objective.  Of course, this also requires ensuring
that the back focal plane is focused onto the back aperture of the
objective.  You could try this by closing the aperture stop in the
reflected light path.  That said, this would also decrease the illumination
intensity.

One other consideration is that underfilling the back aperture only solves
one half of the vignetting equation, because the fluorescence collected by
the objective will also be vingetted.  This is for the exact same reason
that the illumination gets vingetted, where when you increase the tilt of
the back aperture (i.e. towards the edge of the FOV), the effective
diameter of the back aperture is decreasing.

For me, the optimal solution is the computational approach you hinted at
where you simply take a high dynamic range reference image on a fluorescent
test slide, and just divide your sample images by the reference image to
get a perfect correction (this will even correct for specs of dust on the
optics, camera sensor non-uniformities, etc.)  The nice thing is you only
need to do this once, and then you are good to go.  If you are measuring
objects near the optical resolution limit of the microscope, then you will
also want to carefully measure the resolution at the corners of the FOV and
apply a Gaussian blur to the whole image to ensure your resolution is
isotropic across the FOV.  This is because vignetting simultaneously
reduces intensity and resolution, both of which should be corrected against
for analysis.

-Ben Smith

On Thu, Sep 3, 2020 at 3:52 AM Csucs Gabor <[hidden email]>
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.
> *****
>
> Dear All,
>
> I have just today learned about a new sCMOS camera with 29mm Field of
> View. With some of the new large FOV microscope bodies this really makes
> sense, but I was thinking about the vignetting effect that can be quite
> significant with such a large FOV. Of course, there are numerous
> computational methods how you can correct for this, but still the best way
> would be to have a more homogenous illumination. I am fully aware of some
> developments for laser-based systems (homogenizer for various spinning disk
> systems by various providers) but I was wondering whether anyone knows
> about a commercial homogenizer solution for wide-field systems (using e.g.
> LED light sources)?
>
> Thanks                                  Gabor
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
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Fax [phone]
e-mail: [hidden email]
https://vision.berkeley.edu/faculty/core-grants-nei/core-grant-microscopic-imaging/