Improving optical access, sampling speed, and resolution for in vivo multiphoton microscopy

Multiphoton microscopy is a powerful optical imaging modality renowned for its non-invasive nature and relatively affordable characteristics. In particular, it has found its niche in neuroimaging due to its ability to probe in vivo biological processes in scattering brain tissue approaching millimeter depths with cellular resolution. However, the brain is a large and complex organ, and in order to fully understand its heterogeneous architecture and associated functional roles, several distal regions must be imaged simultaneously. Moreover, due to the critical implications of organelle features in various macroscale processes, whole-brain imaging at subcellular resolution scales presents itself as one of the outstanding challenges faced by the neuroscientific community today. Primarily, this research aims to expand the depth, field-of-view, and temporal throughput of multiphoton microscopy to enable large volume imaging of microvasculature at greater acquisition speeds. To accomplish this, we combine multi-faceted efforts focused on the engineering and development of advanced multiphoton microscopy techniques and technologies. This includes the characterization of novel contrast agents, the optimization of scan system optics, and the integration of high-repetition rate lasers with a resonant galvanometer. In addition, we develop a two-color imaging system capable of enhancing excitation efficiency, improving signal-to- background ratio, and further extending imaging depth. Finally, we present a novel application for two-color non-degenerate mode mixing to effectively circumvent the diffraction-limited nature of optical resolution and enable subcellular imaging. Collectively, these efforts advance the state-of-the art of multiphoton microscopy for routine cerebrovascular and neuroimaging.Biomedical Engineerin