Collaborative Research: NCS-FO: Modified two-photon microscope with high-speed electrowetting array for imaging voltage transients in cerebellar molecular layer interneurons

The research project aims to develop a revolutionary microscope for studying the brain's neural activity. By combining expertise from engineering, physics, and neuroscience, the multidisciplinary team is working on a high-risk, high-payoff endeavor to overcome the limitations of existing imaging techniques. The investigators are focusing on genetically encoded voltage indicators (GEVIs) to capture fast neuronal firing and subthreshold voltage changes. The innovative approach combines two-photon microscopy with electrowetting adaptive optical microprism technology, enabling selective excitation and imaging of groups of neurons at high rates. This advancement has the potential to unlock crucial insights into brain function and lead to breakthroughs in treating neurological disorders. By involving graduate students, engaging in effective outreach and fostering interdisciplinary collaboration, the project aims to shape the future of cognitive science, neuroscience, and education. Two-photon imaging of intracellular calcium in awake behaving animals has been a catalyst for unprecedented advances in the understanding of cognitive neural processes. However, calcium time courses are a noisy convolution of neuronal spiking with calcium efflux dynamics that hide potentially crucial information on subthreshold voltage changes and fast action potential firing. In the last three decades several groups have developed promising new approaches to overcome this inherent disadvantage by imaging neuronal membrane potential using genetically encoded voltage indicators (GEVIs). Imaging GEVIs by a wide neuroscience community would provide information that is lost in calcium imaging potentially yielding crucial breakthroughs in understanding the neural basis of behavior. However, GEVI imaging has not been adopted widely because it suffers from low signal to noise ratio (SNR), limited ability to image large ensembles of neurons and difficult implementation by the scientific community. Here, the investigators will develop a novel large ensemble GEVI imaging technique and will apply it to understand the complex process of cerebellar involvement in rapid decision making. A new approach is proposed for large ensemble GEVI imaging in which two photon microscopy is coupled with adaptive optical microprism technology. The result is beamlet excitation of subsets of neurons, with high imaging rates (500 Hz). The team will continue dissemination efforts with the technology developed in this project. Finally, the investigators will endeavor to communicate science to the broader audience through venues such as the Denver Museum of Nature and Science and CU Science Discovery program. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.