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不同受体，使它们能够作为细胞识别的生物标志物。少数配体分子与单个活细胞上的其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）相互作用通常共同调节和改变细胞功能。这种信号通路可能需要几分钟、几小时或几天。因此，至关重要的是，以单分子灵敏度同时可视化单个活细胞上的不同类型的受体，以定量各种类型的受体分子，直接捕获L-R相互作用如何共同作用以实现给定的细胞功能，并有效检测单个活细胞的罕见子集。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主力。不幸的是，荧光探针（荧光体，荧光蛋白，QD）遭受内在的光漂白，使它们无法连续捕捉单个活细胞的动态事件超过数小时。光漂白也使得随时间的定量分析变得困难。不同荧光团的不同激发和发射的分离导致复杂和昂贵的仪器，限制了它们同时多重研究多种类型分子的能力。所有的荧光成像方法，包括传统的，共聚焦和超分辨率荧光显微镜遭受这些相同的基本限制。在这个提议中，我们的目标是开发一种新的分子成像平台，包括光稳定的多色和多功能的单分子纳米颗粒光学生物传感器（SMNOBS）和远场光稳定的光学纳米显微镜（PHOTON），用于实时研究单个活细胞的功能。为了证明概念验证，我们将使用它们来定量成像和分子表征高度异质性肿瘤细胞群中卵巢癌干细胞（oCSCs）罕见子集上多种类型受体的作用和功能，并研究其分化机制。这些功能强大的新工具有望解决一系列紧迫的生物化学和生物医学问题，这些问题涉及单个活细胞上单个受体分子的功能及其相关信号通路的分子和实时表征 在真实的时间里。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;让他们接触到前沿的生物医学研究计划;让更多的学生参与生物医学研究活动;提高和加强机构生物医学研究计划和基础设施。