Reverberation multiphoton microscopy for volumetric imaging in scattering media

Abstract

Multiphoton microscopy has become an extremely valuable tool for peering deeply into thick, scattering media such as biological tissue. However, the traditional multiphoton beam-scanning approach is held back because only one thin plane is observed at a time. The reverberation loop elegantly overcomes this limitation by generating an infinite series of foci at depths spanning the sample, all sampled individually but near-simultaneously. With the inclusion of some additional interleave steps, it is possible to quickly scan a sample at video rates – allowing volumetric imaging at or near the rate one would traditionally image planes. In neural imaging, this enables a reverberation multiphoton microscope to simultaneously monitor relationships in neuronal activity not only horizontally across samples, but vertically across many layers of the brain. In imaging of engineered cardiac tissues, this enables high resolution observation of three-dimensional structures in a live sample, even as it actively beats and moves.