In Vivo Three-Photon Microscopy of Mouse Brain Excited at the 2200 nm Window

Three-photon microscopy (3PM) excited at both the 1300 and 1700 nm window are established deep-brain imaging technologies. Beyond 1700 nm lies the last unexplored optical window potential for deepbrain 3PM: the 2200 nm window. However, there has been no experimental demonstration of brain 3PM at this window, and hence no quantitative comparison with other excitation windows. Here we demonstrate brain 3PM excited at 2200 nm in vivo, by developing an energetic femtosecond laser and a laser scanning microscope with high transmittance and minimum pulse broadening. We also perform 3PM excited at 1700 nm for direct comparison. Our results show that the effective attenuation length, a measure of the maximum achievable imaging depth, at 2200 nm is smaller than that at 1700. Besides, the spatial resolution of 2200 nm 3PM is lower than that of 1700 nm 3PM. We thus fill this last missing gap in deep-brain MPM, in terms of excitation wavelength selection.