Posted by
Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Leica-TCS-SP2-LCS-and-Win7-tp7582853p7582946.html
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David,
that is a good idea. So with the one-photon PSF data from PSFLab, image
multiplication in Fiji and normalization in Excel I should be getting
somewhere.
However, when I create a psf intensity distribution in the focal plane
with PSFlab for NA 1.4, lambda 0.5 µm (all Ris set to 1.518, calculation
in x from -1 µm to +1 µm in 10 nm steps, in y 30 nm wide), I get the
first minimum at 0.27 µm away from the maximum intensity. But according
to r=0.61*lambda/NA it should be at 0.22 µm.
Also, when I superimpose two intensity profiles with the max of the
second in the first min of the first (Rayleigh-criterion), the maximal
intensity drop between the two profiles is about 55% while it should be
only 26%. As Arne mentioned, PSFlab describes the excitation PSF only,
not the theoretical confocal 1/sqrt(2) case.
So apparently PSFlab does not calculate the simple theoretical Airy
pattern but some more complicated (and supposedly more realistic) case,
resulting in different results.
I am wondering, does that mean we should throw the Rayleigh formula out
the window, including the 26%-intensity dip if the max of one is on the
first min of the other pattern? Else, I am still looking for the right
numbers. I didn't yet get around to try Cosmos, as was suggested by Sergey.
Steffen
Am 17.11.2014 15:29, schrieb David Baddeley:
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> Dear Steffen,
> just to add to the suggestions already made, confocal and two-photon PSFs are really easy to derive from an emission or excitation case, if you have a psf generation program for widefield.
> To a 1st appoximation (i.e. infinitely small pinhole):
> confocal PSF = widefield PSF at exctitation wavelength multiplied by widefield PSF at detection wavelength
> Or for a large pinhole in 2-photon
> 2-photon PSF = widefield PSF at excitation wavelength squared.
> These multiplications should be easy to do in ImageJ (convert the data type to floating point first)
> If you want to model a finite pinhole size you can convolve the detection PSF with a flat disc with the pinhole diameter before doing the multiplication (doable in ImageJ, but somewhat harder, as the disc needs to be centred to avoid shifting the excitation and detection PSFS wrt each other).
> cheers,David
>
--
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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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