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Perhaps Alan is really asking "What happens to the light from all the 
molecules excited by the original Airy disk but residing in the 
"donut" area of the STED beam?"

These emissions are "swept away" (entrained?) by the STED beam. They 
become sort of a mini-laser and the emitted photons leave the area 
totally coherent with the STED beam (i.e., in phase, at the same 
wavelength as, and going in the same directions as the STED beam).

It is sometimes forgotten that the STED beam is at a wavelength that 
is considerably longer than the peak of the emission spectrum. So, 
even if you detect your signal fluorescence in the the transition 
mode, it is relatively easy to use filters to separate the 
fluorescence swept up by the STED beam (not to mention the STED beam 
itself) from the fluorescence emitted by molecules near the center of 
the donut hole because these latter photons are mostly at wavelenghts 
near the emission peak.

Cheers,

Jim Pawley


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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
>6-145 Jackson Hall, 321 Church St. SE    Dept Fax: (612) 626-5009
>Minneapolis, MN  55455                    e-mail: [log in to unmask]


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