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于研究细胞中特定蛋白质集合的行为和功能。另一方面，基于单分子的活细胞蛋白质功能分析是出了名的困难，因为自体荧光的时空变化会影响图像对比度。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，因此开发新的光学探头、成像显微镜和相关分析将导致极大地改善从少数分子到单分子水平的图像对比度，这将是至关重要的。这项拟议的研究的目的是提高对培养细胞和活生物体中极少到单一蛋白质分子的检测能力，使其提高到&gt；100倍。为了实现这一目标，我们将开发新的合成和遗传编码的光学开关探针，并优化一种快速标签方法，将光学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细胞内马达蛋白的单分子成像研究。调制光学开关探头发射的能力是开发一种用于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。来自OLID显微镜的改进的图像对比度是通过从包括自体荧光的大背景噪声环境中提取其强度根据定义的扰动波形进行调制的光开关探头的荧光来实现的；执行锁定检测和关联图像分析以通过在逐个像素的基础上显示时生成相关系数图或相关图像来提取嵌入在背景中的信号调制。在这项提案中，我们还将描述提高OLID成像效率的新方法，以及适用于区分活细胞中特定的单一蛋白质偶联及其复合体的新光学探针的设计。这项以多研究者为基础的研究建议有四个目标，重点在于：(A)改进我们先前用于群体和单分子成像的光学开关类型；(B)开发用于控制OLID显微镜和实时图像分析的自动化分析；(C)利用OLID成像显微镜研究发动机蛋白Myosin V和Myosin VI在神经细胞中的作用以及确定这些马达的突变如何影响神经细胞内的囊泡运输特性。与公共健康相关：这项研究描述了新型光学探针和相关成像技术，它们在图像对比度方面取得了令人印象深刻的改进，可以对活细胞中的特定蛋白质进行少量到单分子的成像。这些技术将用于单分子成像研究，以研究肌球蛋白马达蛋白在正常小鼠和阿尔茨海默病小鼠模型小鼠神经元内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.