High-contrast imaging of single molecules in live cells

描述（由申请人提供）：现有的成像技术非常适合研究细胞中特定蛋白质系综的行为和功能。另一方面，活细胞中蛋白质功能的基于单分子的分析是非常困难的，因为自发荧光的时空变化损害了图像对比度。由于蛋白质的功能往往是在活细胞或生物体的背景下揭示，这将是必不可少的，以开发新的光学探针，成像显微镜和相关的分析，这将导致显着改善图像对比度的水平上的几个到单个分子。拟议研究的目的是将培养细胞和活生物体中少数至单个蛋白质分子的检测提高100倍以上。为了实现这一目标，我们将开发新的合成和遗传编码的光开关探针，并优化Snap标签方法，将光开关探针靶向活细胞中的特定蛋白质。特别是这些探针将用于神经元细胞内的运动蛋白的单分子成像研究。调制光开关探针发射的能力是发展高对比度荧光成像的范式转换方法的关键，我们称之为光学锁定检测（OLID）成像显微镜。OLID显微镜的改进的图像对比度是通过从包括自发荧光的大背景噪声环境中提取光开关探针的荧光来实现的，所述光开关探针的强度根据定义的扰动波形来调制;执行锁定检测和相关联的图像分析，以通过生成相关系数图，或相关图像，当在逐像素的基础上显示时。在该提案中，我们还将描述新的方法，以提高效率的OLID成像和新的光学探针的设计，是适合于区分检测特定的单蛋白缀合物从它们的复合物在活细胞中。这项基于多研究者的研究计划有四个目标，重点是：（a）改进我们早期的用于系综和单分子成像的光学开关;（B）开发用于OLID显微镜控制和实时图像分析的自动分析;（c），使用OLID成像显微镜研究神经细胞中马达蛋白肌球蛋白V和肌球蛋白VI的作用，并确定这些马达中的突变如何影响神经元细胞内囊泡运输的性质。公共卫生相关性：该研究描述了新的光学探针和相关的成像技术，这些技术为活细胞中特定蛋白质的少数分子到单分子成像提供了令人印象深刻的图像对比度改进。这些技术将被用于单分子成像研究，以研究肌球蛋白马达蛋白在轴突运输的特定类型的囊泡内的神经元来自正常小鼠和阿尔茨海默病的小鼠模型的作用。

DESCRIPTION (provided by applicant): Existing imaging technologies are well-suited to study the behavior and functions of a specific protein ensemble in cells. On the other hand, single molecule based analysis of protein function in living cells is notoriously difficult, as spatio-temporal variations in auto-fluorescence compromise image contrast. Since protein function is often revealed within the context of a living cell or organism, it will be essential to develop new classes of optical probe, imaging microscopy and associated analyses that will lead to dramatic improvements in image contrast at the level of few to single molecules. The objective of the proposed research is to improve the detection of few to single protein molecules in cells in culture and in living organisms by a factor of >100. To meet the objective we will develop new classes of synthetic and genetically-encoded optical switch probes and optimize a Snap-tag approach to target optical switch probes to specific proteins in live cells. In particular these probes will be used for single molecule imaging studies of motor proteins within neuronal cells. The ability to modulate the emission from optical switch probes is key to the development of a paradigm-shifting approach for high-contrast fluorescence imaging that we term optical lock-in detection (OLID) imaging microscopy. The improved image contrast from OLID microscopy is achieved through extracting the fluorescence of an optical switch probe, whose intensity is modulated according to a defined perturbation waveform, from a large background noise environment including autofluorescence; lock-in detection and associated image analysis are performed to extract the signal modulation embedded in the background by generating a map of correlation coefficients, or a correlation image, when displayed on a pixel-by-pixel basis. In this proposal we will also describe new approaches to increase the efficiency of OLID-imaging and the design of new optical probes that are suitable for differentiate detection of specific single protein conjugates from their complexes in living cells. This multi-investigator based research proposal has four aims that focus on (a), improving our earlier classes of optical switch for ensemble and single molecule imaging; (b), developing automated analysis for the control of OLID microscopy and real-time image analysis; (c), using OLID imaging microscopy to study the roles of motor proteins myosin V and myosin VI in nerve cells and to establish how mutations in these motors affect the properties of vesicle transport within neuronal cells. Public Health Relevance: The research describes new classes of optical probes and associated imaging techniques that provide impressive improvements in image contrast for few to single molecule imaging of specific proteins in living cells. These technologies will be used within single molecule imaging studies to investigate the roles of myosin motor proteins in axonal transport of specific types of vesicles within neurons derived from normal mice and those from a mouse model of Alzheimers disease.