Theoretical assessment of two-dimensional nonlinear structured illumination microscopy based on structured excitation and structured stimulated emission depletion

Abstract

The performance of nonlinear structured illumination microscopy (NSIM), which has theoretically infinite optical resolution, largely depends on nonlinear phenomena. Unfortunately, nonlinear effects of excitation saturation and photoswitchable fluorophores suffer from photobleaching and slow switching time, respectively. In contrast, stimulated emission depletion (STED) is also a nonlinear phenomenon, having fast switching time and being potentially harmless. We propose NSIM based on STED, which has structured excitation light and structured STED light, and both structured illuminations have the same pitch and orientation in the sample plane. Theoretical analysis shows that two-structured illumination having the same grating vector can efficiently increase nonlinearity caused by the STED effect and improve optical resolution. We also found that nonlinearity depends on the phase difference between two-structured illumination made by excitation and STED light and that opposite phase is the most efficient to increase harmonic strength. The feasibility study shows that our method can theoretically achieve optical resolution of 1/7 of wavelength with commercially available laser over large field of view.