Posted by
Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-3D-SIM-data-tp6251420p6273791.html
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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
--
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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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Marchioninistr. 15, D-81377 München
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