Fundamental properties of light unavoidably impose features on images collected using fluorescence microscopes. Accounting for these features is often critical in quantitatively interpreting microscopy images, especially those gathering information at scales on par with or smaller than light's emission wavelength. Here the optics responsible for generating fluorescent images, fluorophore properties, and microscopy modalities leveraging properties of both light and fluorophores, in addition to the necessarily probabilistic modeling tools imposed by the stochastic nature of light and measurement, are reviewed.

This feature issue commemorating 25 years of STED microscopy and 20 years of SIM is intended to highlight the incredible progress and growth in the field of superresolution microscopy since Stefan Hell and Jan Wichmann published the article Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy in Optics Letters in 1994.