Super-resolution Imaging by Two-photon Structured Illumination Microscopy

Abstract

The application of two-photon excitation to confocal laser scanning microscope (CLSM) leads two-photon laser scanning microscopy (TPLSM) to become an important tool in biology. The simultaneous absorption of two photons can reduce the phototoxicity and scattering, which can realize better imaging depth and high quality image. However, the spatial resolution of TPLSM is limited by optical diffraction, which is a barrier for its application. Structured illumination microscopy (SIM) offers rapid imaging speed for living cells and resolution enhancement by a factor of two. In this paper, we propose a nonlinear two-photon structured illumination microscopy (NTPSIM) that combines SIM with two-photon excitation, enabling resolution in thick specimens imaging twice of the normal resolution. Our method has the ability of less scattering and better depth penetration. Through theoretical simulation, we demonstrated that NTPSIM can improve imaging resolution significantly and provide strong penetrability with rapid imaging speed.