As the light is diffracted on the apperture of the objective of an optical microscope, a point-shaped object will not be imaged as a point but
as a intensity distribution that can be described by the Point Spread Function (PSF). This has the consequence that two objects whose distance is
smaller than a certain value cannot be distinguished any more as their PSF will merge.
This distance or resolution limit can be estimated by the Rayleigh criterion:
where λ is the excaitation wavelength, n the refraction index of the medium and α
half of the angle of the aperture of the objective.
Different ways have been found to circumvent this barrier, either by stochastically allowing only one fluorescent emitter being active in a
diffraction limited area (STORM/PALM) or by actively modelling the shape of the PSF
(STED).