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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
>
> Mail room:
> Marchioninistr. 15, D-81377 München
>
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