IDBR: Development of cathodoluminescent near-field energy transfer microscopy for high frame-rate, nanoscale, non-invasive observation of aqueous biodynamics

IDBR：用于高帧率、纳米尺度、无创观察水生物动力学的阴极发光近场能量转移显微镜的开发本项目的目标是开发一种高亮度、可快速扫描的纳米级光源，适合在生理条件下研究纳米尺度的水生物样品。预期的应用将研究络合可溶性生物分子和异质脂质膜的动力学。要实现这些努力，需要一种结合电子光学和光光学最重要的元素的方法：在扫描电子显微镜内，聚焦的电子束将被用来在薄膜闪烁体（一种被电子撞击时产生光的材料）中制造纳米光学光斑。这个点将反过来激发相邻的荧光团封装的水样品通过高度局域的近场共振能量转移。通过利用纳米级分辨率和电子显微镜的快速扫描，同时实现水动力学的光谱选择性生物相容性测量，这一创新将为执行近场扫描光学显微镜提供一种全新的方式，不受传统引用的挑战（如低光学吞吐量，不必要的尖端相互作用和主动机械稳定）的限制。在10纳米左右的横向和轴向上实现更紧密的焦点将彻底改变许多生物物理荧光技术。测量生理浓度下单分子荧光波动的能力比传统荧光相关光谱（FCS）高100万倍，将为研究复合物稳定状态下的结合和酶催化提供一种急需的方法。这对于理解阿尔茨海默氏症等疾病以及核糖体等分子机制的形成具有重要意义。在本身小于衍射极限的膜上进行光漂白后荧光恢复（FRAP）扩散测量，例如在植物叶绿体令人印象深刻的密集颗粒中，将揭示尚未发现的组织异质性和迁移率的局部变化，因为其他超分辨率显微镜与FRAP不兼容。这些纳米尺度的光谱选择性研究将为生物分子相互作用的内部工作提供一个窗口，并将复杂生物材料的高级功能与其纳米起源联系起来。开发的平台将包括标准扫描电子显微镜（SEM）中的光学检测，以及一个纳米制造的液体池，该液体池将在真空中安全地容纳水样品，并包括一个薄膜闪烁窗口，将电子束转换为用于样品照明的高度局部光学场。这项工作将在两个不同的中小企业进行，这两个企业都位于共用的设施中。在一种情况下，PI的小组将培训所有新的SEM用户，并将提供帮助，教他们使用已开发的纳米光学功能。PI的小组将指导该设施的志愿者本科生开发用于显微镜的不同纳米闪烁体。第二个SEM位于国家设施中，没有相关的用户费用，并且定位于为广泛的用户群提供接触发达技术的机会。除了这些设施之外，该技术将很容易复制，作为一种可访问的扩展，其他机构将希望将其纳入其现有的SEM设施，扩大其用户基础，以包括更多生命科学领域的用户。在本科课程材料中，PI将使用该研究项目中的例子来强调如何将物理原理纳入现实世界的生物学研究。PI还促进妇女和其他少数民族参与科学，经常作为自然科学女性学生团体的客座演讲者，PI的研究小组成员通过社区科学资源社区课堂计划参加对邻近小学的课堂访问。

IDBR: Development of cathodoluminescent near-field energy transfer microscopy for high frame-rate, nanoscale, non-invasive observation of aqueous biodynamicsThe objective of this project is to develop a high-brightness, rapidly scannable, nanoscale light source appropriate for studying aqueous biological samples at the nanoscale under physiological conditions. Anticipated applications will investigate the dynamics of complexing soluble biomolecules and of heterogeneous lipid membranes. To realize these efforts requires a way to combine the most redeeming elements of electron and light optics: Inside a scanning electron microscope, a focused electron beam will be used to make a nano-optical spot in a thin film scintillator (a material that produces light when struck by electrons). This spot will in turn excite adjacent fluorophores in an encapsulated aqueous sample via highly localized near-field resonant energy transfer. By leveraging the nanoscale resolution and fast scanning of electron microscopy while enabling spectrally selective bio-compatible measurements of aqueous dynamics, this innovation will provide an entirely new way to perform near-field scanning optical microscopy, uninhibited by traditionally cited challenges such as low optical throughput, unwanted tip interactions, and active mechanical stabilization.Achieving tighter focal spots on the order of 10 nm both laterally and axially will revolutionize a host of biophysical fluorescence techniques. The ability to measure single-molecule fluorescence fluctuations at physiological concentrations up to a million times greater than traditional fluorescence correlation spectroscopy (FCS) will yield a much needed way to study binding and enzyme catalysis in the regime where complexes are stable. This has important implications in the understanding of diseases such as Alzheimer's and in the formation of molecular machinery such as the ribosome. The enabling of fluorescence recovery after photobleaching (FRAP) diffusion measurements on membranes that are themselves smaller than the diffraction limit, as is the case for example in the impressively crowded grana of plant chloroplasts, will reveal organizational heterogeneity and local variations in mobility that have yet to be uncovered, as other super-resolution microscopies are not compatible with FRAP. These spectrally selective studies at the nanoscale will literally provide a window into the inner workings of biomolecular interactions and will connect the high-level function of complex biomaterials with their nanoscopic origins.The developed platform will comprise optical detection in a standard scanning electron microscope (SEM), and a nanofabricated liquid cell that will both safely house the aqueous sample in vacuum and include a thin film scintillating window to convert an electron beam into a highly-localized optical field for sample illumination. The work will be performed at two different SEMs, both residing in shared facilities. In one case, the PI's group will train all new SEM users, and will offer assistance in teaching them to use the developed nano-optical capabilities. The PI's group will mentor the facility's volunteer undergraduates in the development of different nano-scintillators for use in the microscopy. The second SEM is in a national facility that has no associated user fees, and is positioned to provide a broad user base with exposure to the developed technology. Beyond these facilities, the technology will become easily reproducible as an accessible extension that others institutions will wish to incorporate into their pre-existing SEM facilities, broadening their user base to include more users in the life sciences. In undergraduate course material, the PI will use examples from this research project to highlight how physical principles are incorporated into real world biological research. The PI also promotes the participation of women and other minorities in science, as a frequent guest speaker for women's students groups in the natural sciences, and the PI's research group members participate in classroom visits to neighboring elementary schools through the Community Resources for Science Community in the Classroom program.