Fast dynamic wavefront correction for multi-photon microscopy with a high resolution MEMS phase-only modulator

Abstract: Multi-photon microscopy is a powerful technique for deep-tissue imaging, providing high spatial resolution at increased penetration depth. Nevertheless, imaging remains largely restricted to superficial tissue layers well below 1 mm. Adaptive optics based on indirect wavefront sensing can significantly extend the accessible imaging depth, but their iterative operation is typically time-consuming and therefore limits the correction of strong, spatially complex aberrations. Until recently, this limitation was partly due to the lack of high-speed phase modulators offering sufficiently large numbers of actuators. Recent technological advances have addressed this bottleneck with the emergence of megapixel phase modulators operating at kilohertz rates. Here, we demonstrate wavefront correction in multi-photon microscopy using a high-speed phase-only MEMS modulator combined with a rapidly converging scatter-compensation algorithm. We experimentally correct complex aberrations comprising 144 spatial modes in less than one second, resulting in signal enhancements exceeding a factor of two. Benchmarking against a fast liquid-crystal spatial light modulator reveals a sixfold increase in correction speed. We further demonstrate adaptive optics imaging in mouse brain tissue using both two-photon and three-photon excitation fluorescence microscopy. These results indicate that high-resolution MEMS-based spatial light modulators enable efficient indirect wavefront sensing and represent a promising platform for high-speed wavefront shaping in non-linear microscopy.