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Dear Alan--

On 8/31/2012 7:22 AM, Alan Smith wrote:

> I understand how the illumination psf is formed in STED using a depletion
> beam. However, if the emission is detected is collected in epi-detection,
> why is the resolution not determined by the diffraction limit for the objective.
>
> As an example, if a single molecule was producing fluorescence, the image
> will be an airy disk determined by the NA of the detecting objective and the
> wavelength of light. Despite the fluorescence solely coming from a single
> molecule.

You're right: with both a single molecule and with STED, you see a 
diffraction-limited Airy disk on the emission side.  However, with STED, 
you know to a high degree of precision the position of the excitation 
beam eliciting that Airy disk.  Thus, as you scan that small excitation 
beam over a small structure, you can obtain resolution that's many times 
better than confocal.  In that way, it's similar to near-field 
super-resolution methods.

Hope that helps!

Martin Wessendorf
-- 
Martin Wessendorf, Ph.D.                   office: (612) 626-0145
Assoc Prof, Dept Neuroscience                 lab: (612) 624-2991
University of Minnesota             Preferred FAX: (612) 624-8118
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