Does the PSF need to be re-measured when one changes the zoom?
In principle, it is sufficient to record bead images for a PSF calculation at the system Nyquist rate, say 50 nm × 50 nm × 150 nm for a confocal setup (or doubled figures for widefield).
If your sample of interest has been imaged at a different sampling rate, then the PSF can be adjusted by bandlimited interpolation. This is automatically done by the Huygens CMLE and QMLE deconvolution methods.
You can use our Nyquist Calculator for determining the ideal sampling values for your experimental conditions.
There are two problems, though:
- In some microscopes the magnification at high zoom is unreliable, with errors up to 30%.
- In case of widefield data, the Nyquist sampled PSF might be too small (in terms of microns) to be used in deconvolving physically large data. Make sure you specify, during the PSF distillation, a large enough required size for the final PSF. However, in extreme cases memory limits might get in the way.
Keywords: scaling psf bandlimited interpolation<br>
Categories: Faq Deconvolution, Faq Microscopy, Huygens Faq, Imported Faqs<br>
Platforms: Irix Linux Windows Mac AIX<br>
Related products: Hu Ess Hu Pro<br>
