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

该研究项目旨在开发一种革命性的显微镜，用于研究大脑的神经活动。通过结合工程、物理和神经科学的专业知识，多学科团队正在努力克服现有成像技术的局限性，这是一项高风险、高回报的工作。研究人员将重点放在基因编码电压指示器（GEVIs）上，以捕捉快速的神经元放电和亚阈值电压变化。这种创新的方法结合了双光子显微镜和电润湿自适应光学微棱镜技术，能够以高速率选择性地激发和成像神经元群。这一进展有可能开启对大脑功能的关键见解，并在治疗神经系统疾病方面取得突破。通过让研究生参与进来，参与有效的外展和促进跨学科合作，该项目旨在塑造认知科学、神经科学和教育的未来。在清醒行为动物的细胞内钙的双光子成像已经在认知神经过程的理解的前所未有的进步的催化剂。然而，钙时间过程是神经元尖峰与钙外排动力学的噪声卷积，隐藏了阈下电压变化和快速动作电位放电的潜在关键信息。在过去的三十年中，一些研究小组已经开发出了有前途的新方法，通过使用遗传编码电压指示器（GEVIs）成像神经元膜电位来克服这一固有的缺点。由广泛的神经科学团体对gevi进行成像，将提供钙成像中丢失的信息，可能在理解行为的神经基础方面产生关键突破。然而，GEVI成像由于信噪比低、对大神经元群的成像能力有限以及难以被科学界广泛采用而未被广泛采用。在这里，研究人员将开发一种新的大集合GEVI成像技术，并将其应用于了解小脑参与快速决策的复杂过程。提出了一种双光子显微与自适应光学微棱镜技术相结合的大集合GEVI成像新方法。结果是高成像率（500赫兹）的神经元子集的波束激发。该小组将继续利用该项目开发的技术进行传播工作。最后，研究人员将努力通过丹佛自然与科学博物馆和科罗拉多大学科学发现计划等场所向更广泛的受众传播科学。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.