Scientific Volume Imaging b.v.
Please make sure you understand the descriptive → Microscopic Parameters necessary to properly do the calculations. Only the first five parameters in the form are used when calculating the Nyquist rate, the rest are considered when also calculating the PSF. The pinhole size doesn't alter the bandwidth of the detection system!
To calculate the PSF keep in mind that Pinhole Radius and Pinhole Distances are not the physical ones, but Back Projected (i.e. divided by the total magnification of the system). You may find the Backprojected Pinhole Calculator useful. See further details below.
The ideal → Sampling Density (or inversely, Voxel Size) during image acquisition depends on the optics of the microscope. It is recommended to sample the image at a rate close to the ideal Nyquist Rate. Images obtained with sampling distances (voxel dimensions) larger than those established by this rate suffer from UnderSampling. See the examples on Anti Aliasing and Aliasing Artifacts, and some consequences in Quality Vs Sampling.
With the form at the end of this page you can calculate the Ideal Sampling that corresponds to your optical conditions in order to acquire a Well Sampled image. (To see what equations are used in this calculator and some theory behind the scenes read the Nyquist Rate). The data will be returned in nanometers (nm). You can optionally also generate an image of the Point Spread Function (PSF) calculated at that rate, that takes only a few seconds more. The size of the PSF image will be given in µm. The images are returned as Maximum Intensity Projections along Z and Y, and they are upscaled to allow a better view. The pixelation corresponding to the Nyquist rate will be clearly seen.
A common rule of thumb defines the Ideal Sampling in terms of spatial resolution ("sample with half of the resolution") but this is not exactly correct, and in some cases will lead to UnderSampling. The correct Nyquist rate is defined in terms of the system Band Width (in the frequency domain) which is determined by the Point Spread Function.
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, and still a good job can be done when Doing Deconvolution. For Confocal Microscope images sampling distances may be up to 1.7 times the Nyquist ones. When large pinholes are used, up to 2 times larger. Widefield microscopy data is more sensitive to undersampling so it is better to stay below a factor of 1.5. In case of low Numerical Apertures like 0.4 we recommend not to undersample in the axial direction.
Hint: If you have a data stack that is dramatically undersampled in Z (not fulfilling the Nyquist Criterion by a large factor) you better interpret the different planes as independent (i.e. as 2D images) and do 2D deconvolution in the Huygens Software planewise. See Convert The Data Set*.
You can use the PSF calculator option in the form below for example to see how large is the PSF expected to be in your setup, and accordingly image beads for an Experimental Psf distillation.
See also Bleaching Vs Sampling.
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