What happens if I dont meet Abbe's sine condition by underfilling a tube lens with a laser?

[Pedro Almada]

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Dearest mailing list,

I am a cell biologist currently assembling a super-resolution microscope.
Currently I am trying to get my head around the consequences of not
fulfilling etendue. Our plan is to underfill the tube lens since we would
like to restrict the laser illumination to only the areas which are covered
by the camera sensor, as to make efficient use of our laser power (this is
to be a dSTORM system).

Reading through the handbook, there is only a few passing mentions of
etendue/Abbe sine condition and one of them is in the coma section, which
is worrying since we would like to do 3D dSTORM. My question is: what
aberrations should I expect from not filling the aperture of the tube lens
to the field number of the microscope objective? It's not going to be a
massive underfill as we are planning on using an sCMOS camera (18.8mm
diagonal) with a Nikon objective (25mm FN).

Any input would be appreciated!

Thank you,
Pedro

PhD Student

MRC Laboratory for Molecular Cell Biology
University College London
Gower Street
London
WC1E 6BT
Telephone: [phone]

[Andrew York]

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If I understand you correctly, you mean you'll restrict the diameter of
your excitation laser so it doesn't overfill your tube lens? This is a fine
plan. The shape of your excitation has no effect on your emission PSF. Pay
careful attention to your emission path, but the excitation path for
palm/storm doesn't matter much.

On Fri, Jan 16, 2015 at 11:17 AM, Pedro Almada <[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.
> *****
>
> Dearest mailing list,
>
> I am a cell biologist currently assembling a super-resolution microscope.
> Currently I am trying to get my head around the consequences of not
> fulfilling etendue. Our plan is to underfill the tube lens since we would
> like to restrict the laser illumination to only the areas which are covered
> by the camera sensor, as to make efficient use of our laser power (this is
> to be a dSTORM system).
>
> Reading through the handbook, there is only a few passing mentions of
> etendue/Abbe sine condition and one of them is in the coma section, which
> is worrying since we would like to do 3D dSTORM. My question is: what
> aberrations should I expect from not filling the aperture of the tube lens
> to the field number of the microscope objective? It's not going to be a
> massive underfill as we are planning on using an sCMOS camera (18.8mm
> diagonal) with a Nikon objective (25mm FN).
>
> Any input would be appreciated!
>
> Thank you,
> Pedro
>
> PhD Student
>
> MRC Laboratory for Molecular Cell Biology
> University College London
> Gower Street
> London
> WC1E 6BT
> Telephone: [phone]
>

[Zdenek Svindrych-2]

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Dear Pedro,
this has nothing to do with Abbe sine condition. And the term 'underfilling
tube lens' is somewhat exotic...
I believe the illumination is important in STORM. Although it won't affect
your widefield resolution, it may have great impact on background
fluorescence (and high background kills superresolution very efficiently :-)
.
I got good results following these steps:
1) strong illumination - you need quite strong solid-state lasers, the
bigger the are of the iluminated FOV the higher power is needed. Although I'
ve observed blinking with LEDs, Hg lamps and even a spinning disc confocal,
it's impractical. Bear in mind that by blocking some light you won't get
more light anywhere...
2) homogeneous illumination - surprisingly, in STORM areas that receive less
illumination may produce much more fluorescence, possibly saturating your
detector. Even minor variations in intensity may bring about artefacts in
the reconstructed images. Sharply limited illumination is a good idea. It's
simple, you just put an iris into a position that is conjugated with the
sample plane (i.e. in focus at the same time). Try to get your hands on some
microscope with an adjustable field stop in the fluorecence illumination
path, e.g. Olympus IX with an 'L-shaped illuminator'.
3) good cooled camera - don't forget we're photon-limited!
4) good localisation and rendering software
5) perfect sample - that's the tricky part - to get reproducible blinkink
with more than 1 in 100 samples. There are number of recipes to try out.
Note: It's hard to achieve uniform illumination with a laser. The speckle is
everywhere. Generally I got good results when following 'telecentric' (also
called '4-f') principles with either 'Abbe' (for single mode free beam or
vibrating diffusers) or 'critical' (for multimode, vibrating fiber)
illumination. It always helps if there is some sort of mechanical vibration
in your setup to destroy the laser coherence...
Best, zdenek
P.S.: there are a couple of state-of-the-art STORM labs in UK, I'm sure it's
going to be very useful (and fun) to visit one of those...
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, USA
http://www.kcci.virginia.edu/workshop/index.php



---------- Původní zpráva ----------
Od: Pedro Almada <[hidden email]>
Komu: [hidden email]
Datum: 16. 1. 2015 11:19:21
Předmět: What happens if I dont meet Abbe's sine condition by underfilling a
tube lens with a laser?

"*****
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.
*****

Dearest mailing list,

I am a cell biologist currently assembling a super-resolution microscope.
Currently I am trying to get my head around the consequences of not
fulfilling etendue. Our plan is to underfill the tube lens since we would
like to restrict the laser illumination to only the areas which are covered
by the camera sensor, as to make efficient use of our laser power (this is
to be a dSTORM system).

Reading through the handbook, there is only a few passing mentions of
etendue/Abbe sine condition and one of them is in the coma section, which
is worrying since we would like to do 3D dSTORM. My question is: what
aberrations should I expect from not filling the aperture of the tube lens
to the field number of the microscope objective? It's not going to be a
massive underfill as we are planning on using an sCMOS camera (18.8mm
diagonal) with a Nikon objective (25mm FN).

Any input would be appreciated!

Thank you,
Pedro

PhD Student

MRC Laboratory for Molecular Cell Biology
University College London
Gower Street
London
WC1E 6BT
Telephone: [phone]"