Contrast, resolution, pixelation, dynamic range and signal‐to‐noise ratio: fundamental limits to resolution in fluorescence light microscopy

In a perfect optical system numerical aperture and wavelength determine resolution. In a real optical system, however, the number of photons collected from a specimen determines the contrast and this limits the resolution. Contrast is affected by the number of picture elements per unit area, the number of photons and the aberrations present in every optical system. The concept of contrast vs. distance functions is used to compare the resolution achievable in confocal and wide‐field fluorescence microscopes and the effect of a further reduction of the observable volume. In conclusio: (a) real optical systems will never be able to achieve the theoretical resolution, (b) wide‐field fluorescence microscopy will often provide a better resolution than confocal fluorescence microscopy, (c) decreasing the observed volume does not necessarily increase the resolution and (d) using multiple fluorophores can improve the accuracy with which distances are measured. Some numbers for typical situations are provided.