MRI: Development of an Advanced Two-Photon Microscope for Five-Dimensional Imaging of Macromolecular Systems in Living Cells

任何生物体的正常功能和生存关键取决于其蛋白质相互作用形成短寿命或长寿命大分子复合物的能力。首席研究员及其合作者最近的研究表明，有可能确定活细胞中大分子复合物中蛋白质单体的数量和相对分布，这是当代科学的主要挑战之一。为进行这些实验而开发的实验装置由一种新型的双光子显微镜组成，该显微镜在单个图像像素的水平上提供整个发射光谱。该技术允许对Foerster共振能量转移（FRET）进行像素级测量，即能量从被称为“供体”（D）的激发荧光分子到位于附近的非激发“受体”（A）的非辐射转移。特别感兴趣的量是FRET效率，其是从通过光谱解混获得的D和A的组成光谱中针对每个像素确定的。这项技术将把现有技术提升到一个全新的水平，允许对携带荧光团的蛋白质进行无与伦比的跟踪，并动态监测活细胞中的蛋白质-蛋白质相互作用。更具体地说，新仪器将比其前身更快地获得图像数量级-其本身在该领域仍然是独一无二的，并且在三维空间而不是二维空间中这样做。这种尖端技术的发展将使细胞和分子生物学家，生物化学家和其他生命科学家能够研究多个蛋白质群体的动态特征，包括共定位和运输，蛋白质复合物运输以及配体诱导的构象和寡聚状态变化。仪器的设计将提供给其他研究人员和显微镜制造公司，而仪器本身将提供给感兴趣的研究小组使用。除了影响众多主要研究人员的研究计划外，拟议的仪器开发还将为UW-Milwaukee，UW-Madison和全国各地和国外的其他研究机构的40多名本科生，研究生和研究生学员提供精致的培训机会。

The proper function and survival of any living organism depends critically upon the ability of its proteins to interact with one another to form short- or long-lived macromolecular complexes. Recent investigations by the Principal Investigator and his collaborators have demonstrated that it is possible to determine the number and relative disposition of protein monomers in macromolecular complexes in living cells, which is one of the major challenges of contemporary science. The experimental setup developed for conducting those experiments consisted of a novel two-photon microscope which provided entire emission spectra at the level of single image pixels . This technology permits pixel-level measurements of Foerster resonance energy transfer (FRET), that is, the non-radiative transfer of energy from an excited fluorescent molecule - called a 'donor' (D) to a non-excited 'acceptor' (A) that resides nearby. The quantity of particular interest is the FRET efficiency, which is determined for every pixel from the constituent spectra of D and A as obtained by spectral un-mixing. The proposed technology will take the existing technology to a whole new level, by allowing unmatched tracking of fluorophore-bearing proteins and dynamic monitoring of the protein-protein interactions in living cells. More specifically, the new instrument will acquire images orders of magnitude faster than its predecessor - which itself remains unique in the field and do so in three spatial dimensions rather than two. The development of this cutting edge technology will allow cellular and molecular biologists, biochemists, and other life-scientists to investigate dynamic features of multiple protein populations, including co-localization and trafficking, protein-complex trafficking, and ligand-induced changes in conformation and oligomeric status. The design of the instrument will be made available to other researchers and to microscope manufacturing companies, while the instrument itself will be made available for use by interested research groups. In addition to impacting the research programs of numerous principal investigators, the proposed instrument development will provide exquisite training opportunities for over forty undergraduate, graduate, and postgraduate trainees at UW-Milwaukee, UW-Madison and other research institutions around the nation and abroad.