Multiplexing Quantitative Photostable Nanoscopy for Single Live Cell Imaging

项目摘要（摘要） 用于单个活细胞成像的多路定量光稳定纳米显微术 单细胞是所有生物体的基本组成部分。不同类型的细胞表达微量的 独特的受体集，可以作为细胞识别和疾病的生物标志物 诊断.值得注意的是，细胞群体通常表现出高度异质性。单个活细胞的罕见亚群可以 作用独特，并且它们可以随着时间的推移影响和改变整个细胞群的功能。照经上所 至关重要的是开发新的工具，可以映射在单细胞分辨率的细胞群体的异质性， 随着时间的推移，以了解罕见的子集的功能， 单个细胞及其与邻近细胞的通信，以及它们如何从健康状态演变为 病理状态。目前的方法（流式细胞术、遗传分析方法和 基于荧光的成像和分析，包括高含量筛选，HCS），用于识别 特定的单个活细胞不能提供足够的光稳定性、多路复用能力、选择性、单个 分子灵敏度，时间和空间分辨率，以专门研究单个受体分子 在其天然环境中的单个活细胞上，并在原位和长时间内表征其功能 时间（天）。罕见的单细胞亚群通常需要分离或预浓缩， 进一步的分析，失去了在其原生环境中持续研究它们的机会， 时间我们提出发展多色多功能光稳定单分子纳米粒子 光学生物传感器（m2-SMNOBS）和远场多路复用定量光稳定光学纳米镜 （mq-PHOTON）用于单个活细胞上的多种受体的定量成像，旨在识别 单个活细胞，尤其是高度异质性细胞中罕见的单个活细胞亚群（≤ 5%） 群体，并研究它们在细胞群体中随时间的功能， 决议。为了展示这些新工具的独特功能，作为概念验证，我们将使用 这些新工具可以检测高度异质性脑肿瘤中的单个脑癌干细胞（bCSC）， 细胞，并研究它们在其天然环境中随时间（48小时）的分化， 决议。拟议研究的成果包括开发高度创新的工具 用于单个活细胞的稀有和重要子集的功能的分子鉴定和表征， 在真实的时间内，在单个细胞中具有时空分辨率的高度异质性细胞群体 分子分辨率这些功能强大的新工具将成为非常有价值的，以解决广泛的 迫切的生物化学和生物医学问题的分子和实时表征， 随着时间的推移，单个受体和生物标志物在单个活细胞上原位的功能。

Project Summary (Abstract) Multiplexing Quantitative Photostable Nanoscopy for Single Live Cell Imaging Single cells are building-block of all living organisms. Different types of cells express trace amounts of distinctive sets of receptors, which can serve as biomarkers for cell identification and for disease diagnosis. Notably, cell populations often exhibit high heterogeneity. Rare subsets of single live cells can act distinctively and they can affect and alter functions of an entire cell population over time. Thus, it is vital to develop new tools that can map the heterogeneity of a cell population at single cell resolution in situ and with spatiotemporal resolutions over time, in order to understand the functions of rare subsets of single cells and their communications with neighboring cells, and how they evolve from healthy states to pathological states over time. Current methods (flow cytometry, genetic analysis methods and fluorescence-based imaging and assays including high-content screening, HCS) that are used to identify specific single live cells cannot offer sufficient photostability, multiplexing capability, selectivity, single molecule sensitivity, temporal and spatial resolutions to specifically study individual receptor molecules on single live cells at its native environments and characterize their functions in situ and over a long period of time (days). Rare subsets of single cells often need to be isolated or pre-concentrated for further analysis, which loses the opportunity to study them continuously in its native environments over time. We propose to develop multicolored multifunctional photostable single-molecule nanoparticle optical biosensors (m2-SMNOBS) and far-field multiplexing quantitative photostable optical nanoscopy (mq-PHOTON) for quantitatively imaging of multiple receptors on single live cells, aiming to identify single live cells, especially rare subsets of single live cells (≤ 5%) in highly heterogeneous cell populations and study their functions in the cell population in situ over time with spatiotemporal resolutions. To demonstrate unique capabilities of these new tools, as a proof-of-concept, we will use these new tools to detect single brain cancer stem cells (bCSCs) in highly heterogeneous brain tumor cells and study their differentiation in their native environment over time (48 hours) with spatiotemporal resolutions. The outcomes of the proposed research include the development of highly innovative tools for molecular identification and characterization of functions of rare and vital subsets of single live cells in highly heterogeneous cell populations with spatiotemporal resolutions in situ in real time at single molecule resolution. These powerful new tools will become extremely valuable to address a wide range of pressing biochemical and biomedical questions about molecular and real-time characterization of functions of single receptors and biomarkers on single live cells in situ over time.