Nyquist-Shannon Sampling in Optical Microscopy
The Nyquist-Shannon theorem requires sampling at ≥2× the highest spatial frequency to avoid aliasing. In microscopy the diffraction limit sets the highest frequency, so the required pixel size depends on the optical resolution.
Practical rule: divide the resolution by 2.3 (rather than exactly 2) to provide a small safety margin. This applies independently in X/Y (lateral) and Z (axial) dimensions.
Under-sampling causes aliasing artifacts and lost information. Over-sampling wastes acquisition time, disk space, and signal-to-noise without improving real resolution.