Cellular-resolution multi-region optical recording of neural activity with wide-field two-photon microscopy

DESCRIPTION provided by applicant Objectives and optics for are rapidly evolving to allow wider and wider FOVs for two photon laser scanning microscopes PLSM This development introduces a new challenge point by point raster scanning over the entirety of the FOV requires the experimenter to sacrifice either single cell sensitivity because of faster scanning rates required to cover the larger field in the same amount of time or temporal resolution because of scanning at the same speed and thus taking longer to cover the FOV or both This tradeoff will be a major pain point for neuroscience labs moving to wide FOV PLSM and a major hindrance to harvesting the scientific benefits of wide FOV PLSM Current PLSM systems use two scanners in series either two galvanometer based scanners or an kHz resonant scanner paired with a galvanometer scanner Such scanner combinations allow addressable X Y point by point laser scanning at video rates for small FOV For larger FOV it is desirable to divide the FOV into smaller regions of interest to achieve higher scanning rates that retain cellular resolution and sensitivity The work described in this proposal will produce a nove PLSM three scanner hardware solution that will help address this challenge Combining a resonant R scanner with a galvanometer scanner G pair provides a means to perform faster scans in arbitrary rectangular regions of interest within a FOV Vidrio Technologiesandapos patent pending RGG scanning assembly will be a compact plug and play system that will easily integrate into existing PLSM systems The software required to drive this new scanner assembly will be added to Vidrioandapos s existing and popular PLSM imaging software ScanImage The multi region of interest imaging approach we describe is designed to scale up with the rapidly expanding reach of PLSM cellular resolution brain activity imaging thus providing a long term solution for large FOV imaging PUBLIC HEALTH RELEVANCE Two photon laser scanning microscopy has revolutionized neuroscience by allowing three dimensional imaging with cellular resolution within the brain an impact recently complemented by rapid developments in fluorescent biochemical reporter molecules Optical engineers and scientists have been recently developing new lenses to expand two photon imaging beyond the millimeter barrier thus enabling the imaging of multiple cortical columns that is required for studying integrative brain functions Traditional two photon laser scanning would become very slow if scanned over such new large fields of view we propose to develop a new scanning device and system to image rapidly in selectable smaller subregions of interest within these large new optical fields allowing scientists to continue looking at fast activity in single cells and synapses in the brain