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It's multiphoton excitation so the effective PSF is much smaller than  
the 1 photon PSF. This has been described in numerous texts. Not  
wanting to rain on your parade but for THG the resolution should be  
better than you measured.

Cheers



On 15/04/2011, at 5:52 AM, Steffen Dietzel wrote:

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>
> Hi everybody,
>
> somewhat related to the ongoing discussion on resolution, I came  
> across a puzzle today concerning the resolution of multi-photon  
> microscopy.
>
> I measured the resolution of our third harmonic generation (THG)  
> microscope and surprisingly I came up with a full width half maximum  
> (FWHM) slightly better than theory allows. Seems the most likely  
> explanation is I applied the wrong theory. But which one is the  
> correct one?
>
> The experiment:
> THG with 1275 nm, Objective 0.95 NA (water, 20x), beads 60 nm in 2%  
> agarose, voxel size 0.136 x 0.136 x 0.5 µm. Result: FWHM ~0.7 µm  
> (for both, forward and backward THG)
>
> Assuming that for multi-photon point-scanners only the excitation  
> wavelength is relevant, I used 1275 nm for the theory (Rayleigh):  
> r=0.61λ/NA = 0.82 µm
>
> So, the measured resolution is one pixel better than the theoretical  
> limit. You don't get that lucky every day ;-)
>
> Possibilities I have considered:
> - I messed up the experiment. I wouldn't know, however, how I could  
> get a better result by messing up.
> - Microscope settings are wrong (wrong pixel size). Possible of  
> course but not very likely.
> - Rayleigh does not apply to multi-photon, I overlooked something.  
> If so, please help out.
> - THG requires 3 photons to take place. Maybe the photon density is  
> low enough in the outer areas of the PSF so that signal generation  
> is limited to inner areas of the PSF? (Now that would be really  
> interesting from an academic point of view, since it would mean you  
> could squeeze the size of the excitation spot relative to the  
> wavelength with 4, 5, etc. photon effects. Although it probably  
> wouldn't do much good for practical purposes since you would have to  
> start with long wavelengths to end up with a visible (=easy  
> detectable) signal.)
>
>
> Any ideas? Could people share measured FWHMs from their multi-photon  
> setup? Maybe even from a 3 photon process?
>
> Steffen
>
>
> -- 
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
> Head of light microscopy
>
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