Nyquist rate and PSF calculator
Please make sure you have the correct values for the MicroscopicParameters necessary for the calculations. Only the first five parameters in the form are used when calculating the Nyquist rate, the others are considered when calculating the PSF itself. Note that the pinhole size doesn't alter the bandwidth of the detection system!!!
The PinholeRadius and Pinhole Distances parameters are not physical sizes, but rather BackProjected sizes, i.e. divided by the total magnification of the system. To compute these backprojected values, see BackprojectedPinholeCalculator. See below for further details.
Explanation: proper images
The ideal SamplingDensity (or inversely, VoxelSize) during image acquisition depends on the optics of the microscope. It is recommended to sample the image at a rate close to the ideal NyquistRate. Images obtained with sampling distances (voxel dimensions) larger than those established by this rate suffer from UnderSampling. See the examples on AntiAliasing and AliasingArtifacts, and some consequences in QualityVsSampling.
With the form at the end of this page you can calculate the IdealSampling that corresponds to your optical conditions in order to acquire a WellSampled image. (To see what equations are used in this calculator and some theory behind the scenes read the NyquistRate). The data will be returned in nanometers (nm). You have the option to generate an image of the PointSpreadFunction (PSF) also, 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 IdealSampling 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 BandWidth (in the frequency domain) which is determined by the PointSpreadFunction.
Exceptions in practice
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 DoingDeconvolution. For ConfocalMicroscope 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 NyquistCriterion by a large factor) you better interpret the different planes as independent (i.e. as 2D images) and do 2D deconvolution in the HuygensSoftware planewise. See ConvertTheDataSet.
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 ExperimentalPsf distillation.
See also BleachingVsSampling.
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