CAREER: Tip-Enhanced Fluorescence Microscope for Resolving Single Molecules Within Dense Biomolecular Networks

该奖项是根据2009年《美国复苏和再投资法案》(公法111-5)资助的。该项目的主要目标是开发一种光学显微镜，能够在分子长度尺度上破译完整的生物分子网络的组织，特别强调与膜相关的网络。这种网络的功能与其结构密切相关，这种结构可以被遗传或环境触发的分子成分的构象变化破坏，有时会导致疾病或其他功能障碍。大多数用于破译网络结构的高分辨率结构技术的一个关键弱点是它们无法确定分子组成，因为它们通常缺乏识别单个分子的敏感性。只有光学光谱学才能常规地实现单分子检测，但传统的光学技术不够灵敏，无法将样品的分子特征分辨出近两个数量级。该奖项的技术部分涉及对一种名为尖端增强荧光显微镜(TEFM)的新兴高分辨率结构分析技术的许多创新改进。TEFM是一种近场光学方法，通过这种方法，激光束的光学强度集中在类似于避雷针的尖端。增强的强度区域增加了尖端附近的荧光率，这使得空间分辨率仅受其锐度的限制。TEFM已经被用来以~10纳米的空间分辨率对单个分子进行成像，并且在研究生物膜等平面样品方面特别有前途。该项目的几项创新，包括偏振调制方案和定制的单光子分析算法，提高了TEFM对比度，并改善了与软生物样品的兼容性。为了增加其多功能性，TEFM在单个集成平台上与其他成像手段相结合，如广场荧光显微镜、共焦显微镜、相差显微镜、全内荧光显微镜、荧光相关显微镜和荧光寿命显微镜。还实现了几个独立的光谱通道，以支持同时进行多色成像。最后，将开发温度调节和闭环聚焦控制来成像活细胞膜。根据该奖项开发的仪器预计将对生物成像研究产生广泛的影响。结果将发表在科学文献上，也将发表在公众可访问的网页上。详细信息将根据要求共享。该奖项的教育和推广部分涉及发展本科综合科学专业；为高中生和大学新生提供各种户外活动(如滑雪、帆船、潜水、冲浪)物理的混合媒体模块；以及为小学生提供的科学模块。这些项目正在进行中，结果将在可公开访问的网页上说明。根据本奖项开发的教育材料将根据要求提供。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).The main objective of this project is to develop an optical microscope that is capable of deciphering the organization of intact biomolecular networks at the molecular length scale, with a particular emphasis on membrane-associated networks. The function of such a network is closely related to its structure, which can be disrupted by genetically or environmentally triggered conformational changes to the molecular components, sometimes resulting in disease or other malfunction. A key weakness of most high-resolution structural techniques for deciphering the network architecture is their inability to determine molecular composition, since they generally lack the sensitivity to identify individual molecules. Only optical spectroscopy can routinely achieve single-molecule detection, but traditional optical techniques are not sensitive enough to resolve the molecular features of a sample by almost two orders of magnitude. The technical component of this award involves a number of innovative improvements to an emerging high-resolution structural analysis technique called tip-enhanced fluorescence microscopy (TEFM). TEFM is a near-field optical method whereby the optical intensity of a laser beam is concentrated at the apex (tip) of a sharp needle analogous to a lightning rod. The enhanced-intensity region increases the fluorescence rate in the vicinity of the tip, which enables spatial resolution limited only by its sharpness. TEFM has been used to image individual molecules with ~10 nanometer spatial resolution, and is particularly promising for studying planar samples such as biological membranes. Several innovations in this project, including a polarization modulation scheme and customized single-photon analysis algorithms, increase TEFM contrast and improve compatibility with soft biological samples. To increase its versatility, TEFM combines on a single integrated platform with other imaging modalities such as wide-field fluorescence microscopy, confocal microscopy, phase contrast microscopy, total internal fluorescence microscopy, fluorescence correlation microscopy, and fluorescence lifetime microscopy. Several independent spectral channels are also implemented to enable simultaneous multi-color imaging. Finally, temperature regulation and closed-loop focus control will be developed for imaging live cell membranes. The instrumentation developed under this award is expected to have broad implications for biological imaging research. Results will be published in the scientific literature and also on a publicly accessible webpage. Detailed information will be shared upon request. The education and outreach component of this award involves the development of an undergraduate integrated science major; mixed-media modules for high-school students and college freshmen on the Physics of various outdoor activities (e.g. skiing, sailing, SCUBA, surfing); and science modules for elementary school students. These projects are ongoing and the results will be described on a publicly accessible webpage. Education materials developed under this award will be provided upon request.