Huygens excludes all the beads while trying to reconstruct PSFs from beads taken in widefield mode. What is wrong?
We tried to reconstruct PSFs from beads taken in wide-field mode. The problem is that Huygens excludes all the beads from the calculation because of different reasons: - too close to an edge or too close to each other. Even taking 100 slices did not improve the situation. We know that the image of the bead in widefield mode can be extremely large even if the cut-off is high.
Yes, this is the problem. Even worse: the thicker you make the stack the wider they become so the tops of the cones will tangle. That will really mess up the PSF. Best way to go is to set 'reduce PSF size', for instance to 2 (=high), and reduce the number of beads to a couple, even just one should be ok for widefield. Starting from Huygens version 2.16 it is less a problem if the PSF is somewhat truncated because of the build-in PSF extrapolation. This is also true for confocal PSFs which tend to be truncated always.
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Keywords: reconstruct PSF widefield<br>
Categories: Faq Microscopy, Huygens Faq, Imported Faqs<br>
Platforms: Irix Linux<br>
Related products: Hu Pro<br>
