Resolution enhancement of two-photon microscopy via intensity-modulated laser scanning structured illumination.

Conventional structured illumination microscopy (SIM) with wide-field illumination is an applicable tool to provide resolution enhancement. And yet its applications in thick specimens are still full of challenges. By combing the structured illumination concept with two-photon excitation, a laser scanning two-photon structured illumination microscope (LSTP-SIM) was constructed to gain ∼1.42-fold lateral resolution enhancement in contrast to two-photon fluorescence microscopy. With a point-scanning geometry, an acoustic-optical modulator was used to modulate temporally the excitation intensity in order to produce the structured illumination pattern. The theoretical models of image formation and image reconstruction were clearly established. Simulation and experiments were both performed to show the capability of this system to enhance the lateral resolution. Combined with the inherent optical sectioning power of the two-photon excitation, LSTP-SIM would have the potential for applications in optically-thick specimens.