Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device

In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.