Multiphoton In Vivo Microscopy (Core 2)

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Discovering the basic molecular and cellular mechanisms of post-traumatic epileptogenesis in a biophysically realistic model of traumatic brain injury will require having longitudinal, high-resolution access to the gyrencephalic, large animal brain. Fluorescence microscopy enhances such capabilities, enabling multimodal data collection through the use of genetically targeted and ion-selective fluorophores. In the tool development Phase I of this project, we developed an instrument and supporting technologies that afford us the unique ability to perform such measurements. These technologies include a custom two-photon microscope with a gantry design that can accommodate imaging in vivo, with subcellular resolution, brains of animals weighing >80kg. Microscopic in vivo imaging in animals of this size (to our knowledge, the largest by a factor of 2-4x) required substantial development and optimization of motion artifact mitigation tools, including heartbeat-triggered, high speed image stack acquisition; flexible, penetrable, transparent sub-dural windows; and post-hoc image registration algorithms. Finally, the microscope can perform digital fluorescence lifetime imaging, enabling quantification of extracellular chloride using novel single-wavelength dyes. The Microscopy Core will implement the imaging tools developed in Phase I, as well as optimize for calcium imaging and long-term repeated access. As these tools require substantial cost and technical expertise, the Microscopy Core represents a cost-effective consolidation and consistent implementation for the proposed program.