Multiplexing Quantitative Photostable Nanoscopy for Single Live Cell Imaging

用于单活细胞成像的多重定量光稳定纳米显微镜

• 批准号: 10453061
• 负责人: X. Nancy Xu
• 金额: $ 2万
• 依托单位: OLD DOMINION UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2023-07-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10453061
• 关键词: Address; Affect; BMP4; Biochemical; Biological Assay; Biological Markers; Biomedical Research; Biosensor; Brain Neoplasms; CD44 gene; Cell physiology; Cells; Color; Communication; Complex; Detection; Development; Environment; Event; Exhibits; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Halogens; Health; Heterogeneity; Hour; Image; Imaging Device; Immunohistochemistry; In Situ; In Situ Hybridization; Individual; Kinetics; Lasers; Light; Magnetic Resonance Imaging; Malignant neoplasm of brain; Maps; Methods; Microscope; Molecular; Nanoscopy; Noise; Optics; Organism; Outcome; Pathologic; Photobleaching; Photons; Population; Positron-Emission Tomography; Research; Resolution; Signal Transduction; Source; Stains; Time; U251; Work; base; biomedical imaging; cancer stem cell; cell fixing; cell population study; cell type; cellular imaging; disease diagnosis; early detection biomarkers; fluorescence imaging; fluorophore; genetic analysis; imaging detector; imaging probe; innovation; instrumentation; live cell imaging; multiplex detection; nanoparticle; neoplastic cell; optical nanoscopy; optical spectra; photoacoustic imaging; quantitative imaging; receptor; screening; single molecule; spatiotemporal; tool; ultrasound

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.

项目概要（摘要） 用于单活细胞成像的多重定量光稳定纳米显微镜 单细胞是所有生物体的组成部分，不同类型的细胞表达微量的。 独特的受体组，可以作为细胞识别和疾病的生物标志物 值得注意的是，细胞群通常表现出高度异质性。 它们的作用独特，随着时间的推移，它们可以影响和改变整个细胞群的功能。 开发能够以单细胞分辨率绘制细胞群异质性的新工具至关重要 原位并随着时间的推移具有时空分辨率，以便了解稀有子集的功能 单细胞及其与邻近细胞的通讯，以及它们如何从健康状态演变为 当前的方法（流式细胞术、遗传分析方法和 基于荧光的成像和测定，包括用于识别的高内涵筛选 (HCS) 特定的单个活细胞无法提供足够的光稳定性、多重能力、选择性、单一 分子灵敏度、时间和空间分辨率，可专门研究单个受体分子 在其原生环境中对单个活细胞进行研究，并在原位和长期表征其功能 稀有的单细胞亚群通常需要分离或预浓缩一段时间（天）。 进一步的分析，从而失去了在其本地环境中持续研究它们的机会 我们建议开发多色多功能光稳定单分子纳米粒子。 光学生物传感器（m2-SMNOBS）和远场复用定量光稳定光学纳米显微镜 (mq-PHOTON) 用于对单个活细胞上的多个受体进行定量成像，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%） 细胞群并随着时间的推移利用时空研究它们在原位细胞群中的功能 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 这些新工具可检测高度异质性脑肿瘤中的单个脑癌干细胞 (bCSC) 细胞并通过时空研究它们在原生环境中随时间（48 小时）的分化 拟议研究的成果包括开发高度创新的工具。 用于分子鉴定和表征单个活细胞的稀有和重要亚群的功能 单次实时原位分析具有时空分辨率的高度异质性细胞群 这些强大的新工具对于解决广泛的问题将变得极其有价值。 关于分子和实时表征的紧迫生化和生物医学问题 随着时间的推移，原位单个活细胞上的单个受体和生物标志物的功能。