In STED the depletion 'doughnut' (controversial term) limits the fluorescence emission to a spot ~50-80nm radius.  So we know that that is where the signal has come from, even though the detection optics cannot actually resolve it.  

The parallel with MP is very appropriate - in MP we typically use widefield detection so the detection optics cannot resolve anything at all.  But again the scanning beam provides the resolution.

It is just the same in scanning electron microscopy - the resolution all comes from the scanning beam, the detection system has no resolution capability.

                                                                    Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Alan Smith
Sent: Friday, 31 August 2012 10:22 PM
To: [log in to unmask]
Subject: STED detection

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Dear list,

Firstly, sorry if this is not confocally enough for this forum, but I hope someone would know. I was wondering if anyone could help with some a confusion I have about STED detection.

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.

I know that for MP excitation the psf is solely determined by the illumination psf, but again, I am a little unsure as to the reason for this.
Plus STED can obviously be CW also.

Any help or suggestions would be brilliant!
Much appreciated.
Alan Smith