New Photostable Nanoprobes for Real-time Imaging of Single Live Cells

用于单个活细胞实时成像的新型光稳定纳米探针

• 批准号: 9468747
• 负责人: X. Nancy Xu
• 金额: $ 8.96万
• 依托单位: OLD DOMINION UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9468747
• 关键词: Address; Binding; Biochemical; Biological Markers; Biomedical Research; Biosensor; CD44 gene; Cancer Biology; Cell physiology; Cells; Color; Complex; Darkness; Detection; Disease; Early Diagnosis; Early treatment; Educational Activities; Event; Fluorescence; Fluorescence Microscopy; Halogens; Health; Hour; Image; Imaging Device; In Situ; Individual; Institution; Lasers; Ligands; Light; Malignant Neoplasms; Malignant neoplasm of ovary; Microscope; Molecular; Molecular Probes; Nanoscopy; Noise; Optics; Peroxisome Proliferator-Activated Receptors; Photobleaching; Population; Proteins; Research Activity; Research Infrastructure; Resolution; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Source; Students; Time; Time Study; Tumor stage; Work; cancer stem cell; cancer therapy; cell type; design; educational atmosphere; fluorescence imaging; fluorophore; imaging detector; imaging modality; imaging platform; imaging probe; instrument; instrumentation; liquid crystal; live cell imaging; molecular imaging; nanoparticle; nanoprobe; neoplastic cell; novel; optical spectra; outreach; programs; public health relevance; quantitative imaging; receptor; single molecule; tool

 DESCRIPTION: New Photostable Nanoprobes for Real-time Imaging of Single Live Cells. Different types of cells express trace amounts of distinctive sets of receptors, enabling them serve as biomarkers for cell identification. Binding of a few ligand molecules with its receptors on single live cells can initiate dynamic cascades of cellular signaling pathways, alter cellular functions and cause diseases. Multiple types of ligand-receptor (L-R) interactions typically work together to regulate and alter the cellular functions. Such signaling pathways can take minutes, hours or days. Thus, it is crucial to simultaneously visualize different types of receptors on single live cells with single-molecule sensitivity in order to quantify various types of receptor molecules, to directly capture how L-R interactions work together to achieve given cellular functions, and to effectively detect rare subsets of single live cells. Currently, fluorescence microscopy using fluorescence imaging probes is the primary workhorse for live cell imaging. Unfortunately, fluorescence probes (fluorophor, fluorescence protein, QD) suffer intrinsic photobleaching, making them unable to continuously capture the dynamic events of single live cells over hours. Photobleaching also makes quantitative analysis over time difficult. Separation of different excitation and emission of various fluorophores leads to complex and expensive instrument and restricts their capacity of multiplexing study of multiple types of molecules simultaneously. All fluorescence imaging methods including conventional, confocal and super-resolution fluorescence microscopy suffer these same fundamental limitations. In this proposal, we aim to develop a novel molecular imaging platform including photostable multicolored and multifunctional single molecule nanoparticle optical biosensors (SMNOBS) and far-field photostable optical nanoscopy (PHOTON) for real-time study of functions of single live cells. To demonstrate the-proof-of-concept, we will use them to quantitatively image and molecular characterize roles and functions of multiple types of the receptors on rare subsets of ovarian cancer stem cells (oCSCs) in highly heterogeneous tumor cell populations and to study their differentiation mechanisms. These powerful new tools are expected to address a wide range of pressing biochemical and biomedical questions about molecular and real-time characterization of functions of single receptor molecules on single live cells and their related signaling pathways in real time. The proposed study will enable us to create an interdisciplinary educational environment for students; expose them to leading-edge biomedical research program; involve more students in biomedical research activities; enhance and strengthen institutional biomedical research program and infrastructure.

 描述：用于单个活细胞实时成像的新型光稳定纳米探针。 不同类型的细胞表达微量的独特受体组，使它们能够作为细胞识别的生物标志物。一些配体分子与其在单个活细胞上的受体的结合可以启动细胞信号传导途径的动态级联，改变细胞功能并引起疾病。 多种类型的配体-受体（L-R）相互作用通常共同作用来调节和改变细胞功能。这种信号传导途径可能需要几分钟、几小时或几天。因此，以单分子敏感性同时可视化单个活细胞上不同类型的受体至关重要，以便量化各种类型的受体分子，直接捕获L-R相互作用如何协同工作以实现给定的细胞功能，并有效检测单个活细胞的稀有子集。目前，使用荧光成像探针的荧光显微镜是活细胞成像的主要工具。不幸的是，荧光探针（荧光团、荧光蛋白、QD）会遭受内在的光漂白，使得它们无法在数小时内连续捕获单个活细胞的动态事件。随着时间的推移，光漂白也使得定量分析变得困难。各种荧光团的不同激发和发射的分离导致仪器复杂且昂贵，并限制了它们同时对多种类型分子进行多重研究的能力。所有荧光成像方法，包括传统荧光显微镜、共焦荧光显微镜和超分辨率荧光显微镜，都受到这些相同的基本限制。在本提案中，我们的目标是开发一种新型分子成像平台，包括光稳定的彩色多功能单分子纳米颗粒光学生物传感器（SMNOBS）和远场光稳定光学纳米镜（PHOTON），用于实时研究单个活细胞的功能。为了证明概念验证，我们将使用它们对高度异质性肿瘤细胞群中卵巢癌干细胞 (oCSC) 稀有亚群上多种受体的作用和功能进行定量成像和分子表征，并研究它们的分化机制。这些强大的新工具有望解决有关单个活细胞上单个受体分子功能及其相关信号通路的分子和实时表征的广泛紧迫的生化和生物医学问题 实时。拟议的研究将使我们能够为学生创造一个跨学科的教育环境；让他们接触前沿的生物医学研究项目；让更多的学生参与生物医学研究活动；加强和加强机构生物医学研究计划和基础设施。