What should be the total distance from the top to the bottom of the PSF?
It all greatly depends on the Numerical Aperture (NA), to a lesser degree on WaveLengths. For a 'typical' Confocal Microscope (NA = 1.3) 5 micron is fine, 3 will do. For a Wide Field Microscope the situation is different because the PSF does not stop in Z. For restoration the software needs a PSF which is a bit larger in Z than the image to be restored. Now the problem is that this needed extent can't be known to the software during the measurement, i.e. bead averaging/cleaning + PSF reconstruction.
It turns out that in practice recorded PSFs are often too shallow, so we have fitted out the reconstruct tool with a powerful extrapolator and a manual Z-size setting. Provided the extrapolator has a big enough foothold it can extrapolate to whatever size you need. Rule of thumb for the foothold is 5 micron. Ideally the extrapolator is called as part of the preprocessing phase of the restoration tools, but because it is rather lengthy we put it in the reconstructor. We did put a light-weight extrapolator in the restoration tools to be able to extend a PSFs a little bit.
See also RecordingBeads.
Keywords: psf size sampling
Categories: Faq Deconvolution, Huygens Faq, Imported Faqs
Platforms: Irix Linux Windows Mac AIX
Related products: Hu Ess Hu Pro Hu Script
