Re: Resolution/PSF/FWHM in multi-photon microscopy

Posted by Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-3D-SIM-data-tp6251420p6275467.html

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On 14.04.2011 22:38, Mark Cannell wrote:
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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.

Mark, I am glad to hear that. I didn't come across those texts, though.
Could you give me some references? The text books I tried doesn't seem
to cover this, or not in a way that is intelligible without a physics
degree.


Not wanting
> to rain on your parade but for THG the resolution should be better than
> you measured.

Oh, I am not short of reasons why the measured PSF could be worse than
theoretically achievable, starting with Ri mismatch. But again, if you
could be more specific, that would be helpful.

Thanks,
Steffen

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> 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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>> Building location:
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