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：阴极发光近场能量转移显微镜的发展，用于高帧率，纳米级，非侵入性的水生物动力学观察本项目的目标是开发一种高亮度，快速扫描，纳米级光源，适用于研究生理条件下的水生物样品在纳米级。预期的应用将调查复杂的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，需要一种方法来联合收割机结合电子光学和光光学的最可取的元素：在扫描电子显微镜内，聚焦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）中制造纳米光点。该点将进而通过高度局部化的近场共振能量转移激发封装的水性样品中的相邻荧光团。通过利用电子显微镜的纳米级分辨率和快速扫描，同时实现水动力学的光谱选择性生物相容性测量，这项创新将提供一种全新的方式来执行近场扫描光学显微镜，不受传统引用的挑战，如低光学通量，不必要的尖端相互作用，实现横向和轴向都在10 nm量级上的更紧密的焦点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）高出一百万倍，这将产生一种急需的方法来研究复合物稳定的体系中的结合和酶催化。这对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。荧光恢复后，光漂白（FRAP）扩散测量膜本身小于衍射极限，例如在植物叶绿体的令人印象深刻的拥挤的基粒的情况下，将揭示组织的异质性和流动性的局部变化，尚未被发现，因为其他超分辨率显微镜是不兼容的FRAP。这些在纳米尺度上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级起源联系起来。开发的平台将包括标准扫描电子显微镜（SEM）中的光学检测，以及纳米制造的液体池，其将在真空中安全地容纳含水样品，并且包括薄膜蒸发窗口以将电子束转换成高度-用于样品照明的局部光场。该工作将在两个不同的SEM进行，两者都位于共享设施中。在一种情况下，PI团队将培训所有新的SEM用户，并帮助教他们使用开发的纳米光学功能。PI的小组将指导该设施的志愿大学生开发用于显微镜的不同纳米闪烁器。第二个扫描电镜是在一个国家设施，没有相关的用户费，并定位为提供广泛的用户基础，接触到开发的技术。除了这些设施之外，该技术将成为一种易于复制的扩展，其他机构将希望将其纳入其现有的SEM设施，扩大其用户群，以包括生命科学领域的更多用户。在本科课程材料中，PI将使用本研究项目的例子来强调物理原理如何融入真实的世界生物研究。PI还促进妇女和其他少数群体参与科学，经常作为自然科学领域女学生团体的特邀演讲人，PI的研究小组成员通过“课堂科学社区社区资源”方案，参加对邻近小学的课堂访问。