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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 <[log in to unmask]>
Komu: [log in to unmask]
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?

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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: +44 (0)20 7679 7806"