What is an optimal sampling density in Z?
What is an optimal sampling density in Z?
The Confocal Microscope provides more information than the widefield system; in theory the confocal Sampling Density should be twice the widefield density. This holds for in all 3 dimensions, so you'd get 8 times more voxels.
A 'typical' widefield setup (1.3 effective NA lens, 500nm emission) is sampled well at 100 nm laterally, 300 nm axially. However, due to good SNR ratio's deconvolution can often gain a lot in Z, so you might as well go for 200 nm. With a 100x lens and a CCD with 6.7 micron cells you get 67 nm laterally. (This is assuming that there is no extra magnification; otherwise the total magnification must be used when calculating the pixel size).
If this bloats your data too much you can try binning to increase the lateral size to 134 nm, but you will already start seeing some 'staircasing' effects on thin filaments in the deconvolved image (Aliasing Artifacts).
In the same typical confocal case a nice sampling rate would be 50 nm in Z, 150 nm axially. In case of bleaching problems you can stretch this up to 75 nm lateral, and after that increase the Z-sampling.
If Essential starts coloring the sampling fields orange, then you start to undersample; red means severely undersampled.
See Nyquist Rate.
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Keywords: sampling density Z<br>
Categories: Faq Deconvolution, Faq Microscopy, Huygens Faq, Imported Faqs<br>
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