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

IDBR：开发阴极发光近场能量转移显微镜，用于水生物动力学的高帧率、纳米级、非侵入性观察

• 批准号: 1152656
• 负责人: Naomi Ginsberg
• 金额: $ 52.19万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1152656&HistoricalAwards=false
• 关键词: IDBR; Development; cathodoluminescent; near; field

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.

IDBR：开发高框架速率，纳米级，非侵入性观察水性生物动力学的无侵蚀性观察的开发，该项目的目的是在生理条件下开发适合研究纳米级的水性生物样品的高亮度，可快速扫描，可快速扫描的纳米光源。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。为了实现这些努力，需要一种结合电子和光学光学元件最兑现的元素的方法：在扫描电子显微镜内部，将使用聚焦的电子束在薄膜闪光灯（一种被电子撞击时产生光线的材料）中形成纳米光点。此点又将通过高度局部的近场谐振能量转移来激发封装水性样品中的相邻荧光团。通过利用纳米级的分辨率和对电子显微镜的快速扫描，同时可以对水性动力学进行频谱选择性的生物兼容测量，这项创新将为近场扫描光学显微镜提供全新的方式，以执行传统上限制的诸如低光的互动效果，并有效地互动，并构成良好的效果。横向和轴向上的10 nm都将彻底改变许多生物物理荧光技术。在生理浓度下测量单分子荧光波动的能力比传统的荧光相关光谱（FCS）高一百万倍（FCS），将产生一种急需的方法来研究在复合物稳定的情况下，研究结合和酶催化。这在理解阿尔茨海默氏病等疾病以及核糖体等分子机械的形成方面具有重要意义。光漂白后的荧光恢复（FRAP）在膜上的扩散测量本身小于衍射极限，例如，在植物叶绿体的令人印象深刻的拥挤的植物grana中，将揭示组织异质性和局部变化的迁移率和其他超级鉴定式摩擦式的局部变化。 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将电子束转换为高度定位的光场以进行样品照明的窗口。这项工作将在两个不同的SEMS上进行，都属于共享设施。在一种情况下，PI小组将培训所有新的SEM用户，并将为他们提供教学的帮助，以使用开发的纳米光功能。 PI的小组将指导该设施的志愿者本科生，以开发用于显微镜的不同纳米阶层。第二个SEM是在没有相关用户费用的国家设施中，并且可以为开发技术提供广泛的用户群。除了这些设施之外，该技术还可以轻松地重现，作为其他机构希望将其纳入其现有的SEM设施中的可访问扩展，扩大了其用户群，以将更多用户包括在生命科学中。在本科课程材料中，PI将使用该研究项目的示例来强调如何将物理原理纳入现实世界的生物学研究中。 PI还促进了妇女和其他少数民族在科学方面的参与，作为自然科学妇女学生群体的常客演讲嘉宾，PI的研究小组成员通过课堂课程中的科学社区社区资源参加了课堂访问。