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集合的行为和功能。另一方面，基于活细胞中蛋白质功能的单分子分析是出了名的困难，因为自动荧光的时空变化会损害图像对比度。由于蛋白质的功能通常是在活细胞或生物体的背景下揭示的，因此开发新型光学探针、成像显微镜和相关分析将是必不可少的，这将导致在少数到单分子水平上的图像对比度的显着提高。本研究的目的是将培养细胞和活生物体中少量到单个蛋白质分子的检测提高100倍。为了实现这一目标，我们将开发新型的合成和遗传编码光开关探针，并优化Snap-tag方法，将光开关探针靶向于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开发高对比度荧光成像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。通过从包括自身荧光在内的大背景噪声环境中提取光开关探针的荧光来提高OLID显微镜的图像对比度，该探针的强度根据定义的扰动波形进行调制；在逐像素显示时，通过生成相关系数图或相关图像，执行锁定检测和相关图像分析以提取嵌入背景中的信号调制。在本提案中，我们还将描述提高oled成像效率的新方法，以及适用于活细胞中特定单蛋白偶联物及其复合物的区分检测的新型光学探针的设计。这项基于多研究者的研究计划有四个目标，重点是：(a)改进我们早期用于集成和单分子成像的光开关；(b)开发用于OLID显微镜控制和实时图像分析的自动化分析；(c)利用OLID成像显微镜研究运动蛋白myosin V和myosin VI在神经细胞中的作用，并确定这些运动蛋白的突变如何影响神经元细胞内囊泡运输的特性。公共卫生相关性：该研究描述了新型光学探针和相关成像技术，为活细胞中特定蛋白质的少数到单分子成像提供了令人印象深刻的图像对比度改进。这些技术将用于单分子成像研究，以研究肌球蛋白运动蛋白在来自正常小鼠和阿尔茨海默病小鼠模型的神经元中特定类型囊泡的轴突运输中的作用。

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.