Adaptive optics two-photon microscopy enables near-diffraction-limited and functional retinal imaging in vivo.

In vivo fundus imaging offers non-invasive access to neuron structures and biochemical processes in the retina. However, optical aberrations of the eye degrade the imaging resolution and prevent visualization of subcellular retinal structures. We developed an adaptive optics two-photon excitation fluorescence microscopy (AO-TPEFM) system to correct ocular aberrations based on a nonlinear fluorescent guide star and achieved subcellular resolution for in vivo fluorescence imaging of the mouse retina. With accurate wavefront sensing and rapid aberration correction, AO-TPEFM permits structural and functional imaging of the mouse retina with submicron resolution. Specifically, simultaneous functional calcium imaging of neuronal somas and dendrites was demonstrated. Moreover, the time-lapse morphological alteration and dynamics of microglia were characterized in a mouse model of retinal disorder. In addition, precise laser axotomy was achieved, and degeneration of retinal nerve fibres was studied. This high-resolution AO-TPEFM is a promising tool for non-invasive retinal imaging and can facilitate the understanding of a variety of eye diseases as well as neurodegenerative disorders in the central nervous system.