http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-3D-SIM-data-tp6251420p6280528.html
Thanks for the plug, Mark! What 2-photon does is give you the psf squared – so the resolution is improved by root 2. The formula becomes 0.61 lambda / NA x 1.414. In principle that’s what confocal does, but this requires an infinitely small pinhole and so cannot be achieved. In 2P it is automatic. What Colin and I did in that paper is calculate what resolution you could actually get with pinholes of different finite size. We showed (experimentally and theoretically) that the root 2 resolution improvement makes 2-photon nearly as good as real-world confocal in spite of the longer wavelength. Presumably in 3-photon you would get a root 3 (1.732) resolution improvement.
GUY COX AND COLIN J.R. SHEPPARD
microscopy (Pawley) as well. The importance of RI mismatch cannot be
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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
>
>
>>
>> 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
>>>
>>> Building location:
>>> Marchioninistr. 27, München-Großhadern
>>