MRI: Development of an Advanced Micro-Spectroscope for Imaging Quaternary Structure, Trafficking, and Dynamics of Macromolecular Systems in Live Cells

MRI：开发先进的显微光谱仪，用于对活细胞中大分子系统的四级结构、运输和动力学进行成像

• 批准号: 1626450
• 负责人: Valerica Raicu
• 金额: $ 45万
• 依托单位: University of Wisconsin-Milwaukee
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1626450&HistoricalAwards=false
• 关键词: MRI; Development; Advanced; Micro; Spectroscope

Recent investigations have shown that spectrally resolved fluorescence microscopy, in conjunction with Fluorescence Resonance Energy Transfer Spectrometry (or FRET Spectrometry), is an effective technique for uncovering the quaternary structure of membrane protein complexes in living cells. To realize the full potential of this approach and determine the supramolecular structures of protein complexes, as well as their relative concentrations, dynamics and spatial distributions Inside a cell or tissue, one must obtain three pieces of critical information at image pixel level: the concentrations of donor and acceptor molecules, and the FRET efficiency occurring between the two. Acquiring this information necessitates exciting the sample at two distinct wavelengths. Existing laser-scanning microscopes (including confocal, two-photon, and FLIM microscopes) perform two-wavelength excitation scans in a serial fashion. This leads to a long time delay (10-100 s) between the two successive scans on a pixel level. As membrane proteins can diffuse in and out of a pixel within 0.100 s, the two excitations are scanning different molecules, thereby compromising the molecular-level resolution. Furthermore, reducing the number of molecules per pixel requires increased spatial resolution, which is not available using current FLIM, confocal, or two-photon microscope technologies. Availability of this cutting-edge technology will open up a new avenue of research in cell signaling and provide an untapped source of pharmacological targets, which in turn may improve public health. The design of the instrument will be made available to other researchers, while the instrument itself will be made available for use by interested research groups. We will also partner with US companies to bring this instrument to the market. Besides to impacting the research programs of investigators around the nation and abroad, the proposed instrument will provide exquisite training opportunities for undergraduate, graduate, and postgraduate trainees in interdisciplinary research across the boundaries between biology, biochemistry, pharmacology and physics. This instrument also will be used for new education initiatives developed by the PI, which bring practical applications of physical and mathematical concepts to elementary and high school students as well as college freshmen, including underrepresented minorities.In this proposed instrument development project, we will design and construct a two-photon optical micro-spectroscope capable of quasi-parallel excitation of tens of focal spots spread in one or two dimensions, and rapid switching between two different excitation wavelengths. This instrument will present several advantages over existing ones: (1) The parallel excitation of multiple sample voxels will lead to dramatically increased signal-to-noise ratio of 20X to 100X compared with single point-scan microscopes. (2) Spectrally resolved fluorescence will be collected at two excitation wavelengths separated by 10 ms; this switching time is 100-1000X faster than existing technology and will enable the currently unattainable determination of the localization of differently sized oligomeric species together with their size distribution (e.g., monomers, dimers, tetramers) on a pixel level. (3) Finally, this instrument will present both reduced out-of-focus blur and increased axial resolution (by about 2X) compared to existing two-photon microscopes. The dramatic improvement in temporal and spatial resolution as well as data accuracy will provide biologists with a means to determine how protein oligomerization and function affect one another. The multidisciplinary team assembled by the PI is ideally suited to develop and validate this technology, since it combines exquisite technical expertise, facilities, experience with developing and running an imaging facility, and close collaborations between physicists, biologists and other life scientists.

最近的研究表明，光谱分辨荧光显微镜与荧光共振能量转移光谱（或FRET光谱）相结合，是揭示活细胞中膜蛋白复合物的四级结构的有效技术。为了充分发挥这种方法的潜力，确定蛋白质复合物的超分子结构，以及它们在细胞或组织内的相对浓度、动态和空间分布，必须在图像像素水平上获得三个关键信息：供体和受体分子的浓度，以及两者之间发生的FRET效率。要获得这一信息，必须在两个不同的波长激发样品。现有的激光扫描显微镜（包括共聚焦、双光子和FLIM显微镜）以串行方式执行双波长激发扫描。这导致两个连续扫描之间在像素级别上的长时间延迟（10-100秒）。由于膜蛋白可以在0.100秒内扩散进出一个像素，这两种激发扫描的是不同的分子，从而影响了分子水平的分辨率。此外，减少每像素的分子数量需要提高空间分辨率，这是目前使用FLIM，共聚焦或双光子显微镜技术无法实现的。这项尖端技术的可用性将开辟细胞信号研究的新途径，并提供尚未开发的药理学靶点来源，这反过来可能改善公众健康。仪器的设计将提供给其他研究人员，而仪器本身将提供给感兴趣的研究小组使用。我们还将与美国公司合作，将这种仪器推向市场。除了影响国内外研究者的研究计划外，该仪器还将为生物学、生物化学、药理学和物理学之间的跨学科研究的本科生、研究生和研究生提供良好的培训机会。该工具还将用于PI制定的新的教育倡议，这些倡议将物理和数学概念的实际应用带给小学和高中学生以及大学新生，包括代表性不足的少数民族。在本仪器开发项目中，我们将设计并构建一个双光子光学显微光谱仪，该光谱仪能够准平行激发分布在一维或二维上的数十个焦点，并在两种不同的激发波长之间快速切换。与现有的仪器相比，该仪器具有以下几个优点：(1)与单点扫描显微镜相比，多个样品体素的平行激发将导致信噪比显着提高20倍至100倍。(2)在两个激发波长间隔10 ms处采集光谱分辨荧光；这种切换时间比现有技术快100-1000倍，将使目前无法在像素水平上确定不同大小的寡聚物物种的定位及其尺寸分布（例如单体、二聚体、四聚体）。(3)最后，与现有的双光子显微镜相比，该仪器将减少失焦模糊并提高轴向分辨率（约2倍）。时间和空间分辨率以及数据准确性的显著提高将为生物学家提供一种方法来确定蛋白质寡聚化和功能如何相互影响。PI组建的多学科团队非常适合开发和验证这项技术，因为它结合了精湛的技术专长、设备、开发和运行成像设备的经验，以及物理学家、生物学家和其他生命科学家之间的密切合作。