CAREER: Imaging vesicular neurotransmitter dynamics with high-sensitivity stimulated Raman scattering microscopy

在化学系化学测量和成像项目的支持下，华盛顿大学西雅图分校的Dan Fu教授和他的团队正在设计研究神经递质的新方法——小分子（如多巴胺和乙酰胆碱），它们在大脑神经元之间的化学信使中起着关键作用。具体来说，Fu小组正在开发超灵敏成像工具来探测神经递质的储存、释放和回收。更好地理解这些动态对于理解大脑中的神经元功能至关重要。傅教授的方法使用强大的成像技术，依靠独特的神经递质光谱来定量绘制它们在模型神经元中的分布。该项目通过提供神经传递中缺失的化学信息，与BRAIN计划保持一致。最终目标是深入了解各种神经递质在调节大脑活动的基本过程（如学习和记忆）中的作用。傅小组利用他们在光谱学和显微镜方面的专业知识，针对所有年龄段的学生，特别是当地高中和社区大学中代表性不足的少数民族学生，开发演示和教学实验室实验，目的是提高他们的物理科学技能和兴趣，为未来的科学和工程职业生涯做好准备。这些活动旨在通过使用3D打印和现成的消费产品来构建教室使用的廉价光谱仪，从而揭开“黑匣子”研究光谱仪的神秘面纱。这些新仪器和实验室的影响将通过一个专门的网站和YouTube教学视频在资源有限的大学和高中进行在线传播，从而得到放大。现有的小分子神经递质研究工具（如安培法和循环伏安法）通常局限于细胞外分子的检测，缺乏定量空间分解囊泡神经递质含量的能力。受激拉曼散射（SRS）显微镜是一种新兴的脉冲激光化学成像技术，它能有效地激发分子的固有振动。为了使用SRS对各种神经递质的分布和动态进行无创监测，Fu小组正在开发新的SRS成像技术，该技术基于超快激光工程、脉冲整形、傅里叶变换检测和新型显微镜仪器的结合，以将SRS显微镜的灵敏度提高50倍。这一改进将使测量单个囊泡的神经递质含量和多重检测神经元细胞中不同的神经递质成为可能。通过电化学测量验证后，该方法将首次提供神经递质分布、释放百分比和模型神经元细胞循环时间的非扰动测量。本CAREER提案的教育计划侧重于开发低成本、模块化的仪器和设计科学实验室，以满足以下方面的需求：(1)教育本科生仪器科学和技术基础知识，(2)加强少数族裔（URM）高中学生接受STEM教育的学术准备。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。

With support from the Chemical Measurement and Imaging Program in the Division of Chemistry, Professor Dan Fu and his group at the University of Washington - Seattle are devising new ways of studying neurotransmitters - the small molecules (such as dopamine and acetylcholine) that play key roles as chemical messengers between neurons in the brain. Specifically, the Fu group is developing ultrasensitive imaging tools to probe the storage, release, and recycling of neurotransmitters. Better understanding of these dynamics is essential to understanding neuronal function in the brain. Professor Fu's approach uses powerful imaging techniques that rely on the unique spectra of neurotransmitters to quantitatively map their distribution in model neurons. The project aligns well with the BRAIN initiative by providing missing chemical information on neurotransmission. The ultimate goal is to gain insight into the roles of various neurotransmitters in modulating brain activities in fundamental processes such as learning and memory. The Fu group is leveraging their expertise in spectroscopy and microscopy to develop demonstrations and teaching laboratory experiments targeting students across all ages, especially underrepresented minority students in local high schools and community colleges, with the goal of enhancing their physical science skills and interests for future careers in science and engineering. These activities are designed to demystify "blackbox" research spectrometers by using 3D printing and off-the-shelf consumer products to build cheap spectrometers for classroom use. The impact of these new instruments and labs will be amplified by online dissemination to resource-limited colleges and high schools through a dedicated website and YouTube instructional videos.Existing tools for studying small molecule neurotransmitters (e.g. amperometry and cyclic voltammetry) are often limited to detection of extracellular molecules and lack the capability of quantifying spatially resolved vesicular neurotransmitter content. Stimulated Raman scattering (SRS) microscopy is an emerging pulsed-laser chemical imaging technique that efficiently excites intrinsic molecular vibrations. To enable use of SRS for noninvasive monitoring of the distribution and dynamics of various neurotransmitters, the Fu group is developing new SRS imaging techniques based on a combination of ultrafast laser engineering, pulse shaping, Fourier-transform detection, and new microscopy instrumentation to improve the sensitivity of SRS microscopy by 50-fold. This improvement will enable measurement of neurotransmitter content of single vesicles and multiplex detection of different neurotransmitters in neuronal cells. Following validation by electrochemical measurements, the approach will provide the first nonperturbative measurements of neurotransmitter distribution, release percentage, and recycling time in model neuronal cells. The educational plan of this CAREER proposal focuses on developing low-cost, modular instruments and designing science labs to fill unmet needs in (1) educating undergraduate students on fundamentals of instrument science and technology, and (2) enhancing academic preparation of underrepresented minority (URM) high school students for STEM education.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.