Adaptive three-photon microscopy for fine structure imaging deep in the mouse brain

In order to study dynamic biological processes in-vivo in mammalian organisms techniques are required which enable non-invasive imaging at large tissue depth with sub-cellular resolution. However, optical aberrations and scattering in biological tissue lead to signal loss and a degradation of both spatial resolution and penetration depth. Here, we combine two powerful optical techniques, multi-photon microscopy and adaptive optics, to push the depth limit further while retaining diffraction limited resolution. We apply these techniques to open questions in the field of neuron and glia biology. By utilizing three-photon excitation at the 1300 nm spectral excitation window we achieve highresolution imaging of GFP-labeled neurons up to a depth of 1.2 mm in the in-vivo mouse brain. Furthermore, we have combined our approach with indirect modal-based wavefront correction and synchronization of our microscope to the animal’s heart pulsation. This allowed us to improve the resolution up to ~6-fold and achieve synaptic resolution throughout an entire cortical column. With adaptive optics correction, small structures such a dendritic branches thus become clearly visible at over 1mm depth in the hippocampus. Furthermore, our adaptive three-photon microscope system enabled, for the first time, to investigate calcium dynamics of fibrous astrocytes which reside in the corpus callosum and whose dynamics have previously not been possible to image.