Rayleigh criterion

The Rayleigh criterion establishes a standard to characterize the Spatial Resolution of an optical device, that is: the minimum resolvable detail, or how much can two points be close to each other before they become indistinguishable due to the Diffraction limit.

The criterion is satisfied when the central maximum of the Airy Disk of one imaged point falls below the first minimum of the other. This determines a resolution r

$$r\ =\ 0.61\ \frac{\lambda}{\text{NA}}$$

where λ is the radiation WaveLength and NA the objective Numerical Aperture. This is approximately the Half Intensity Width of the Airy Disk. For a Fluorescence Microscope of NA = 1.3 and λ = 500 nm, this stablishes a resolution limit of 235 nm. Two distinct objects closer than that will be imaged as a single object. But with Image Restoration one can obtain noticeable Resolution Improvements.

See this interactive tutorial for more details.