What is the maximal voxel size at which Huygens can still do a good job.
This greatly depends on the optical paramaters, in particular on the NA and microscope type. Together with the wavelenghts and refractive indexes these determine the so called Nyquist sample distance, the maximum sampling distance at which all image information is captured. For a plot of the dependancy of the Nyquist rate on the NA and microscope type see the User guides, or go to Nyquist Rate. To calculate this figures online see the Nyquist Calculator.
Confocal microscopes
While sampling at the Nyquist rate is a very good idea, it is in many practical situations hard to attain. In these cases larger sampling distances may be used. For confocal images with a Airy disk sized pinhole the lateral sampling distance may be up to about 1.6 of the Nyquist distance. When much larger pinholes are used, up to 2x.When very small pinholes are used to optimize resolution, or any other case where high resolution is required, we do not not recommend undersampling since this would defeat the purpose, and since it might limit the deconvolution result.
Two photon microscopes
Two photon microscopes without a detection pinhole (e.g. non descanned systems) follow the rules of a small pinhole confocal microscope relative to the longer excitation wavelength. The addition of a detection pinhole increases the theoretical bandwidth, but since usually large pinholes are used the practical bandwidth increase is small.Spinning (Nipkow) disk microscopes
Regarding sampling criteria spinning disk microscopes behave like confocal microscopes with, depending on the disk, a fairly large pinhole. For a typical system we do not recommend more than 1.6 times undersampling.Widefield
Widefield data are more sensitive to undersampling; stay below 1.3. In case of low numerical apertures like 0.4 we recommend not to undersample in the axial direction.STED
The remarkable property of STED microscopes is that they do not have a band limit in the strict sense. That means that any sampling rate is a compromise between practical considerations and the to be reached resolution. Since lateral STED HIW resolution can achieve 50nm in good conditions we recommend sampling around 25nm. In difficult condition this can be increased, but that will limit deconvolution.Multi channel data
The sampling rate should be derived from the highest resolution channel.Tips
The actual and the ideal sampling distances of an image can be seen in Huygens Essential right-clicking on the image thumbnail and choosing 'Show parameters'.To compute the ideal Nyquist rate of an image in Huygens Professional, select the image and choose Edit -> Nyquist rate.
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Keywords: sampling NA Nyquist numerical aperture<br>
Categories: Faq Deconvolution, Faq Microscopy, Huygens Faq, Imported Faqs<br>
Platforms: Irix Linux Windows Mac<br>
Related products: Hu Ess Hu Pro Hu Script<br>
