Should the sampling density used in PSF measurement be equal to the of the specimen?
Do we have to deal with the same PSF when we record fluorescent beads with the zooming function from our confocal microscope while we record the specimen without the zoom function?
Measuring both the PSF and the sample with the Ideal Sampling is always nice, but not strictly necessary. If required, the deconvolution functions in the Huygens Software will automatically scale the measured PSF to adapt it to the image sampling.
However, the PSF should always be ideally sampled or better. In order to do deconvolution, a slight undersampling can occur in the sample image but only to a certain extent.
Why is the Nyquist Rate sampling so relevant for deconvolution? The (degrading) imaging process acts at the scale of the PSF, and therefore this must be precisely acquired in order to restore the image properly. See Ideal Sampling for more details.
Therefore, in any case, the beads for PSF acquisition should be imaged with a Sampling Density at least according to the Nyquist Rate, or even better. Like that the PSF would contain all the information about the imaging properties of the microscope, and can be adapted to other imaging conditions that are → slightly undersampled.
PSF measurement
The Huygens Software will reject beads that are severely Under Sampled if you try to distill a PSF from them, because in that case they do not contain the necessary information to do it!!!
The Nyquist Rate is the minimum sampling required for a proper PSF measurement. OverSampling the bead image can be a good idea (it increases the Signal To Noise Ratio of this fundamental image), but in practice this is not possible in the widefield case, because the image would be too large. Because other microscope's PSF are smaller, you can afford some oversampling there. If possible limit the differences in sampling density to factors 2 or 3, thus making the later scaling of the PSF easier and more precise.
In practice (and with good signal) it is not necessary to sample finer than 25 nm lateral and 100 nm axial for confocal systems or 50 nm lateral and 100 nm axial for widefield systems. Fair numbers in a typical confocal case are 50 nm lateral and 150 nm axial.
Caveat: at high zoom factors the magnification as reported by the microscope is not always reliable.
In the widefield case best record with no Pixel Binning. This usually results in a lateral sampling density in the 67-100 nm range. Axial sampling should match the sampling of the specimen if it is below 250 nm.
For more information see Recording Beads and Parameter Variation.
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Keywords: zoom scale PSF sampling density<br>
Categories: Faq Deconvolution, Faq Microscopy, Huygens Faq, Imported Faqs<br>
Platforms: Irix Linux Windows Mac AIX<br>
Related products: Hu Pro<br>
