Two-photon excitation structured illumination super-resolution microscopy

Three dimensional (3D) fluorescence microscopy has proven essential in biological studies. It allows interrogation of structure and function at spatial scales spanning the macromolecular, cellular, and tissue levels. Two-photon excitation fluorescence microscopy (TPM) is especially well suited to 3D imaging in samples tens to hundreds of microns in thickness, enabling better background rejection than alternatives due to the long wavelength, nonlinear excitation of fluorescence. However, the spatial resolution of conventional TPM is limited by diffraction to similar to 0.3 um laterally and similar to 0.8 um axially. In this report, we will introduce our new developed two-photon excitation structured illumination microscopy which combines the two-photon microscopy capability and the super-resolution microscopy capability in the same system. In addition, optical aberrations caused by optical system and biological samples are determined using direct wavefront sensing with a nonlinear guide star (two-photon-excited fluorescence emitted either from the labeled sample or an exogenous marker) and subsequently corrected using a deformable mirror, restoring super-resolution information that is otherwise lost. We demonstrate that both resolution and fluorescence intensity of our super-resolution microscope is improved on a variety of samples, including bead phantoms, cultured cells in collagen gels, and Drosophila brain tissue slides.