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.

项目摘要（摘要） 单个活细胞成像的多重定量光稳定纳米镜检查 单细胞正在建立所有生物体的块。不同类型的单元表达痕量的痕量 独特的受体集，可以用作细胞鉴定和疾病的生物标志物 诊断。值得注意的是，细胞种群通常表现出高异质性。单个活细胞的罕见子集可以 行动明显，它们可以随着时间的推移影响和改变整个细胞种群的功能。那是 开发可以在单细胞分辨率下绘制细胞群的异质性的新工具至关重要 随着时间的流逝，原位和时空分辨率随着时间的流逝，以了解 单细胞及其与邻近细胞的通信，以及它们如何从健康状态发展到 随着时间的流逝，病理状态。当前方法（流式细胞术，遗传分析方法和 基于荧光的成像和测定法，包括用于识别的高素质筛选，HCS） 特定的单个活细胞无法提供足够的光稳定性，多路复用能力，选择性，单个 分子敏感性，临时和空间分辨率，以专门研究单个受体分子 在本机环境中的单个活细胞上 时间（天）。通常需要隔离或预先审查单个单元的罕见子集 进一步的分析，这将失去机会在其本地环境中不断研究它们 时间。我们建议开发多色的多功能光稳定单分子纳米粒子 光学生物传感器（M2-SMNOB）和远场多路复用定量​​光稳定光纳米镜检查 （MQ-PHOTON）用于定量对单个活细胞上多个接收器的成像，旨在识别 高度异质细胞中的单个活细胞，尤其是单个活细胞的稀有子集（≤5％） 人群并研究了它们在平时颞的原位中的功能 决议。为了证明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脑肿瘤中单脑癌干细胞（BCSC） 细胞并研究其在天然环境中的分化（48小时）与时空的分化 决议。拟议研究的结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中稀有和重要子集功能的功能 高度异质的细胞群，具有空间时间分辨率的实时在单一实时原位 分子分辨率。这些强大的新工具将变得非常有价值，以解决广泛的范围 关于关于分子和实时表征的生化和生物医学问题 随着时间的推移，单个活细胞上的单个接收器和生物标志物的功能。