Should I oversample since the beads I'm using are only 175nm diameter?
Using the Nyquist calculation I calculate that the minimum step in Z should be 170nm (x40water, NA 1.15, ex 364nm). I see from the example in the manual that you sampled a 230nm bead at 25nm steps.
If you like you can oversample the beads at say 2x the Nyquist rate in the lateral direction. <br>
In general it is best to really match the Nyquist criterion (or better) in z since highest resolution gain is in Z. It also depends on the capabilities of the z-stepper. If you use finer 170nm beads instead of 230nm beads (and get sufficient signal), so much the better. It has no impact on the sampling density. Of course there is a relation between the Nyquist rate and the bead size, but it is a weak one. Literature: van der Voort HTM and Strasters KC (1995) Restoration of confocal images for quantitative image analysis. J.Micr. Vol 178, pp 43-54.
The deconvolution tools are capable of adapting a PSF derived from such bead images to the sampling density of the specimen. Still, it is best to sample at the same density as at which you are going to collect the biological images later, or with densities which differ by a factor of 2 or 3.
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Keywords: bead Nyquist sampling<br>
Categories: Faq Deconvolution, Huygens Faq, Imported Faqs<br>
Platforms: Irix Linux Windows Mac AIX<br>
Related products: Hu Pro<br>
