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

最近的研究表明，光谱分辨荧光显微镜，结合荧光共振能量转移光谱（或FRET光谱），是一种有效的技术，用于揭示在活细胞中的膜蛋白复合物的四级结构。为了充分发挥这种方法的潜力并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、动力学和空间分布，必须在图像像素水平上获得三条关键信息：供体和受体分子的浓度，以及两者之间发生的FRET效率。获取这些信息需要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。这导致像素级上的两次连续扫描之间的长时间延迟（10-100 s）。由于膜蛋白可以在0.100 s内扩散进出像素，因此两种激发扫描不同的分子，从而影响分子水平的分辨率。此外，减少每个像素的分子数量需要增加空间分辨率，这是使用当前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传导研究的新途径，并提供尚未开发的药理学靶点来源，这反过来可能会改善公众健康。该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团体使用。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人员的研究计划外，该仪器还将为本科生，研究生和研究生学员提供跨生物学，生物化学，药理学和物理学之间跨学科研究的精美培训机会。该仪器还将用于PI开发的新教育举措，为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，带来物理和数学概念的实际应用。我们将设计并制作一台双光子光学显微光谱仪，数十个焦斑的平行激发在一维或二维上扩展，以及在两个不同激发波长之间的快速切换。与现有的仪器相比，该仪器将呈现以下几个优点：（1）与单点扫描显微镜相比，多个样品体素的并行激发将导致20倍至100倍的信噪比的显著增加。(2)光谱分辨荧光将在相隔10 ms的两个激发波长处收集;该切换时间比现有技术快100- 1000倍，并且将使得能够实现目前无法实现的对不同尺寸的寡聚物种类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，该仪器将同时减少失焦模糊和增加轴向分辨率（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确定蛋白质寡聚化和功能如何相互影响的方法。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业知识，设施，开发和运行成像设施的经验，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。

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.