Scanning Probes to Image Cellular Signaling Processes

扫描探针对细胞信号传导过程进行成像

• 批准号: 7120500
• 负责人: BORIS MIZAIKOFF
• 金额: $ 55.83万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7120500
• 关键词: adenosine triphosphate; atomic force microscopy; bioimaging /biomedical imaging; biological models; biological signal transduction; biosensor device; cell communication molecule; computer simulation; cystic fibrosis; electrochemistry; electrodes; exocytosis; image processing; interdisciplinary collaboration; method development; microscopy; molecular dynamics; molecular film; nanotechnology; neurotransmitter transport; optics; three dimensional imaging /topography; tissue /cell culture

DESCRIPTION (provided by applicant): The present project proposes an innovative, multidisciplinary approach to the investigation of cell communication processes at the molecular level. In this project, novel, innovative and interdisciplinary research is emphasized with a focus on the application of microfabricated integrated scanning nanoprobe and nanobiosensing systems. Scanning probe microscopy (SPM) techniques provide powerful means for obtaining chemical, topographical and optical information with high spatial resolution. Each technique - atomic force microscopy (AFM), scanning nearfield optical microscopy (SNOM) and scanning electrochemical microscopy (SECM) - is designed to provide specific information based on interaction with the sample surface in the nearfield regime. SECM is especially interesting for biochemical/biological investigation, since this technique provides information on electrochemical, chemical and biochemical (re)activity at sample surfaces. The proposed multifunctional tool based on combination of scanning probe techniques will provide simultaneous information with a high selectivity for individual transmitters, high temporal resolution of changes in transmitter concentration, and high spatial resolution to distinguish which cells are releasing transmitter molecules, information required for in-situ investigations of complex biological systems and heterogeneous matrices. Many pathological events involve disruption of chemical communication between cells, most notably in the central nervous system, lungs, and kidneys. An important component of cell communication is the exocytotic release of transmitters from a variety of cells (e.g., the presynaptic terminal), their diffusion across between cells (e.g., a cross the synaptic cleft to the postsynaptic membrane) and the selective binding to suitable receptors, which lead to the initiation of cellular signalling events specific to the transmitter and the receptor. Little is known about the formation of secretory granules and the characteristics of transmitter release, which is frequently altered during physiological or pathophysiological conditions. Hence, detailed knowledge and multiparametric analytical assessment of exocytotic events will help understand the underlying mechanisms of cellular communication. Multifunctional microfabricated SECM-AFM, SECM-SNOM and SECM-AFM-SNOM with integrated nanobiosensors will provide simultaneous topographical, optical/fluorescence and electrochemical information at nanometer resolution for the first time. We will apply these combined multifunctional techniques in combination with tip-integrated nanobiosensors to a relatively simple model biological system to demonstrate the feasibility of the approach for measuring biologically relevant transmitter molecules. The model system will involve the exocytotic release of ATP from a monolayer of epithelial cells in culture. This system is a good model for a variety of epithelial tissues in mammals and the question of the amount and regulation of ATP release is a contemporary issue following the hypothesis that abnormalities in ATP release could play a significant role in the pathophysiology of cystic fibrosis (CF). Quantitative theoretical models and simulations of multifunctional scanning probes in order to facilitate interpretation of electrochemical results and imaging data will complement the proposed tasks.

描述（由申请人提供）： 本项目提出了一种创新的、多学科的方法来研究分子水平上的细胞通讯过程。该项目强调新颖、创新和跨学科研究，重点关注微加工集成扫描纳米探针和纳米生物传感系统的应用。扫描探针显微镜 (SPM) 技术为获取高空间分辨率的化学、形貌和光学信息提供了强大的手段。每种技术 - 原子力显微镜 (AFM)、扫描近场光学显微镜 (SNOM) 和扫描电化学显微镜 (SECM) - 旨在根据近场区域与样品表面的相互作用提供特定信息。 SECM 对于生化/生物学研究特别有趣，因为该技术提供了有关样品表面电化学、化学和生化（再）活性的信息。所提出的基于扫描探针技术组合的多功能工具将为各个发射器提供高选择性的同步信息、发射器浓度变化的高时间分辨率以及区分哪些细胞正在释放发射器分子的高空间分辨率、信息所需的信息。复杂生物系统和异质基质的原位研究。 许多病理事件涉及细胞之间化学通讯的破坏，尤其是在中枢神经系统、肺和肾脏中。细胞通讯的一个重要组成部分是来自各种细胞（例如，突触前末梢）的递质的胞吐释放、它们在细胞之间的扩散（例如，穿过突触间隙到突触后膜）以及与合适的受体的选择性结合，这导致特定于发射器和受体的细胞信号传导事件的启动。关于分泌颗粒的形成和递质释放的特征知之甚少，这些特征在生理或病理生理条件下经常发生变化。因此，胞吐事件的详细知识和多参数分析评估将有助于理解细胞通讯的潜在机制。 具有集成纳米生物传感器的多功能微加工SECM-AFM、SECM-SNOM和SECM-AFM-SNOM将首次以纳米分辨率同时提供形貌、光学/荧光和电化学信息。我们将把这些组合的多功能技术与尖端集成的纳米生物传感器结合应用到一个相对简单的模型生物系统中，以证明该方法测量生物相关递质分子的可行性。该模型系统将涉及培养物中单层上皮细胞中 ATP 的胞吐释放。该系统是哺乳动物多种上皮组织的良好模型，并且 ATP 释放的量和调节问题是一个当代问题，因为假设 ATP 释放异常可能在囊性纤维化 (CF) 的病理生理学中发挥重要作用。 ）。为了促进电化学结果和成像数据的解释，多功能扫描探针的定量理论模型和模拟将补充所提出的任务。