Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution

TLDR

The fluorescence microscope can, in principle, achieve unlimited resolution, contrary to the diffraction limit. The study experimentally demonstrates saturated structured‑illumination microscopy, using excitation saturation to generate nonlinear fluorescence. The method employs spatially structured illumination and fluorescence saturation in a simple wide‑field microscope with a single inexpensive laser, and its resolution is limited by signal‑to‑noise and photobleaching. Experimental results show a two‑dimensional point resolution below 50 nm on bright, photostable samples.

Abstract

Contrary to the well known diffraction limit, the fluorescence microscope is in principle capable of unlimited resolution. The necessary elements are spatially structured illumination light and a nonlinear dependence of the fluorescence emission rate on the illumination intensity. As an example of this concept, this article experimentally demonstrates saturated structured-illumination microscopy, a recently proposed method in which the nonlinearity arises from saturation of the excited state. This method can be used in a simple, wide-field (nonscanning) microscope, uses only a single, inexpensive laser, and requires no unusual photophysical properties of the fluorophore. The practical resolving power is determined by the signal-to-noise ratio, which in turn is limited by photobleaching. Experimental results show that a 2D point resolution of <50 nm is possible on sufficiently bright and photostable samples.