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

MRI：开发先进的双光子显微镜，用于活细胞大分子系统的五维成像

• 批准号: 1126386
• 负责人: Valerica Raicu
• 金额: $ 29.99万
• 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: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1126386&HistoricalAwards=false
• 关键词: MRI; Development; Advanced; Two; Photon

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.

任何活着的有机体的正常功能和生存都取决于其蛋白质相互作用形成短期或长期大分子复合体的能力。首席研究员和他的合作者最近的研究表明，有可能确定活细胞中大分子复合体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包括一种新型的双光子显微镜，它可以在单个图像像素的水平上提供完整的发射光谱。这项技术允许像素级的Foerster共振能量转移(FRET)测量，即能量从被激发的荧光分子--称为“供体”(D)--到附近的非激发“受体”(A)的非辐射转移。特别感兴趣的量是FRET效率，它是从通过光谱分解获得的D和A的组成光谱中的每个像素确定的。这项拟议的技术将把现有技术带到一个全新的水平，允许无与伦比地跟踪含有荧光团的蛋白质，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，新仪器获取图像的速度将比之前的仪器快几个数量级--它本身在该领域仍然是独一无二的，而且是在三个空间维度而不是两个空间维度上做到这一点。这一前沿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种群的动态特征，包括共定位和运输、蛋白质复合体运输以及配体诱导的构象和寡聚体状态的变化。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，拟议中的仪器开发还将为密尔沃基大学密尔沃基分校、密尔沃基大学麦迪逊分校和国内外其他研究机构的40多名本科生、研究生和研究生提供极好的培训机会。