Multi-color two-photon scanning structured illumination microscopy imaging of live cells

Multi-color two-photon microscopy imaging of live cells is essential in biology. However, the limited diffraction resolution of conventional two-photon microscopy restricts its application to subcellular organelle imaging. Recently, we developed a laser scanning two-photon non-linear structured illumination microscope (2P-NLSIM), whose resolution improved three-fold. However, its ability to image polychromatic live cells under low excitation power has not been verified. Here, to improve the reconstruction super-resolution image quality under low excitation power, we increased the image modulation depth by multiplying the raw images with the reference fringe patterns in the reconstruction process. Simultaneously, we optimized the 2P-NLSIM system to image live cells, including the excitation power, imaging speed, and field of view. The proposed system could provide a new imaging tool for live cells.