Saturated patterned excitation microscopy—a concept for optical resolution improvement

TLDR

Optical microscopy resolution is limited by numerical aperture and wavelength, and existing methods such as 4Pi, I5M, STED, and GSD improve resolution by increasing NA or exploiting nonlinear excitation–emission relationships but require complex optical setups. This work develops a theory of nonlinear patterned excitation microscopy that achieves substantial resolution improvement by deliberately saturating the fluorophore excited state. Simulations compare saturated patterned excitation microscopy to linear patterned excitation (structured illumination) and widefield imaging, incorporating photon noise to assess performance. Although post‑acquisition data manipulation is computationally more demanding than in STED or GSD, the experimental requirements remain simple.

Abstract

The resolution of optical microscopy is limited by the numerical aperture and the wavelength of light. Many strategies for improving resolution such as 4Pi and I5M have focused on an increase of the numerical aperture. Other approaches have based resolution improvement in fluorescence microscopy on the establishment of a nonlinear relationship between local excitation light intensity in the sample and in the emitted light. However, despite their innovative character, current techniques such as stimulated emission depletion (STED) and ground-state depletion (GSD) microscopy require complex optical configurations and instrumentation to narrow the point-spread function. We develop the theory of nonlinear patterned excitation microscopy for achieving a substantial improvement in resolution by deliberate saturation of the fluorophore excited state. The postacquisition manipulation of the acquired data is computationally more complex than in STED or GSD, but the experimental requirements are simple. Simulations comparing saturated patterned excitation microscopy with linear patterned excitation microscopy (also referred to in the literature as structured illumination or harmonic excitation light microscopy) and ordinary widefield microscopy are presented and discussed. The effects of photon noise are included in the simulations.