Improved and fast biotissue imaging by temporal focusing widefield multiphoton microscopy with spatially modulated illumination

Abstract

A developed temporal focusing-based multiphoton microscope (TFMPM) has a digital micromirror device (DMD) which is adopted not only as a blazed grating for light spatial dispersion but also for patterned illumination simultaneously. Herein, the TFMPM has been extended to implement spatially modulated illumination at structured frequency and orientation to increase the beam coverage at the back-focal aperture of the objective lens. The axial excitation confinement (AEC) of TFMPM can be condensed from 3.0 mu m to 1.5 mu m for a 50% improvement. Furthermore, a multiline scanning mechanism based on the DMD can push its AEC nearly equivalent to line scanning-based TFMPM to 1.5 mu m from optimal 3.0 mu m of the conventional TFMPM. By using the TFMPM with structured illumination and multiline scanning, reconstructed biotissue images according to the condensed AEC structured illumination are shown obviously superior in contrast and better scattering suppression.