Fast Functional Imaging of Single Neurons Using Random-Access Multiphoton (RAMP) Microscopy

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Abstract
The successful study of dendritic signaling and computation requires the ability to simultaneously monitor neuronal activity at multiple cellular sites. While the difficulties of accessing dendritic submicron structures with conventional micropipette approaches are generally overcome by optical recording techniques, their spatio-temporal resolution has limited such studies to few sites or slow signals. Here we present a novel approach to functional imaging, termed random-access multiphoton (RAMP) microscopy, which combines multiphoton excitation with an inertia-free scanning mechanism. RAMP microscopy employs two-dimensional acousto-optic deflection to rapidly position a focused near-infrared ultrafast laser beam between dwell periods at multiple user-selected sites. Because neuronal structures are generally sparse, activity located throughout various compartments, including thin dendritic branches and spines, can be mapped at high frame rates while maintaining the signal-to-noise ratio of conventional scanning microscopy. Moreover, RAMP microscopy maintains the excellent structural imaging capability of multiphoton excitation, i.e., intrinsic optical sectioning and high lateral resolution from within highly light-scattering brain tissue. RAMP microscopy thus comprises a versatile tool for investigating correlations of dendritic structure and function with significantly enhanced experimental throughput.