Posted by
James D. Manton on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Re-Tetraspeck-beads-for-PSFs-vendor-reply-tp7588497p7588499.html
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> Aren’t 100 nm beads too big for PSF measurements on the Elyra? 40 nm should be better.
There are a couple of methods that allow you to use larger beads, and
hence benefit from the increased signal, which I like but which seem to
be infrequently used. Both these approaches are detailed in an excellent
article from Hanser et al. in the Journal of Microscopy
(
https://doi.org/10.1111/j.0022-2720.2004.01393.x).
The first method relies on knowing the Fourier transform of the bead
shape and dividing the measured optical transfer function by this
distribution (as what you have actually measured is the true
instrumental point spread function convolved with the intensity
distribution of the bead). For a uniformly filled spherical bead, the
Fourier domain distribution function, b(k), is 3h(πkd), where d is the
bead diameter and h(x) = sin(x) / x^3 - cos(x) / x^2.
The second method, and the main focus of the Hanser et al. article, is
generating a pupil function via phase retrieval and using this to
produce a simulated point spread function. As the final PSF is
simulated, no noise corrupts the result, which can aid in deconvolution
if the only available experimental PSFs have a low SNR. In addition,
Zernike polynomials can be fitted to this pupil function to provide a
quantitative measure of aberrations present within the microscope. Once
again, the final OTF is produced by dividing the inferred OTF by the
bead distribution.
While the first method is relatively simple to implement, the second is
quite complex, especially for high numerical aperture objectives in
which a vectorial theory of diffraction must be used. The second also
relies on an empirical scaling function to better match experimental
results to those generated by the theory, as high spatial frequencies
are over-emphasised in the phase retrieval process. For this reason,
unless I want a measure of the aberrations present in a system, I
generally stick with just dividing the experimentally-measured OTF by
the Fourier transform of the bead.
Best wishes,
James