Nanophotonic Approach to Imaging Exocytosis

描述（由申请人提供）：荧光成像已经发展成为一门独立的学科，在生物学、临床医学和癌症检测中有着广泛的应用。活细胞的许多基本过程都发生在细胞表面的质膜上，包括激素和神经递质的分泌、营养物质的吸收、化学和电信号的产生和传递。水疱胞吐是信号分子（如激素和神经递质）穿过细胞膜表面的重要机制。胞吐-内吞过程也参与了膜蛋白在质膜和细胞内隔室之间的再循环。有选择地可视化这些过程和活细胞中的亚膜结构的能力将极大地有助于提高我们对细胞生理学和病理学基本原理的理解。然而，除了TIRF之外，目前还没有一种成像方法能够在活细胞中直接观察到涉及囊泡货物跨质膜运输和分泌的胞外事件。细胞膜的大小只有~ 10nm，许多分泌囊泡的直径通常远低于50nm，因此所有的成像方式都缺乏足够的分辨率。该R21应用程序的长期目标是开发和验证一种监测膜过程的新方法，该方法利用荧光团与表面沉积的金属纳米结构的近场相互作用产生的巨大荧光信号增强。这个应用建立在我们最近的发现，自组装胶体结构（SACS）产生高达1000倍的荧光信号增强紧密定位的荧光团。我们的直接目标是将这种纳米光子现象与TIRF显微镜相结合，以前所未有的灵敏度监测胞外过程。这将允许密切跟踪囊泡招募到质膜和货物释放动力学，即使是通常构成分泌囊泡池的最大部分的最小囊泡。我们想要解决的生物学问题是从不可兴奋的、癌变的肺上皮细胞中释放胞外ATP。TIRF显微镜将可视化囊泡装载荧光标记的ATP通过消失场激发距离高达200nm。随着胞外释放到细胞外空间，被标记的货物将被定位在金属纳米结构的增强场内，产生一个巨大的，易于检测的信号，由于荧光团自由扩散到细胞外空间而消失。

DESCRIPTION (provided by applicant): Fluorescence based imaging has evolved into a discipline in its own right with numerous applications in biology, clinical medicine and cancer detection. Many fundamental processes of a living cell take place at the cell surface plasma membrane, including secretion of hormones and neurotransmitters, uptake of nutrients, generation and transmission of chemical and electrical signals. Vesicular exocytosis is an important mechanism by which signaling molecules, such as hormones and neurotransmitters cross the surface membrane. The process of exocytosis-endocytosis is also involved in the recycling of membrane proteins between the plasma membrane and intracellular compartments. The ability to selectively visualize such processes and the sub-membrane structures in living cells would be tremendously helpful in improving our understanding of the fundamental principles of cell physiology and pathology. However, today no imaging method, with some exception for TIRF, is capable of directly visualizing in live cells the exocytotic events that involve transport and secretion of vesicular cargo across the plasma membrane. The size of the cell membrane is only ~10 nm and the diameter of many secretory vesicles is often well below 50 nm, thus all imaging modalities lack sufficient resolution. The long-term goal of this R21 application is to develop and validate a novel approach for monitoring membrane processes by utilizing enormous fluorescence signal enhancement resulting from near field interactions of fluorophores with surface deposited metallic nanostructures. This application builds on our recent discovery that self assembled colloidal structures (SACS) produce up to 1000- fold fluorescence signal enhancement for closely positioned fluorophores. Our immediate goal is to apply this nanophotonic phenomenon in combination with TIRF microscopy for monitoring exocytotic process with unprecedented sensitivity. This will allow close following of vesicle recruitment to the plasma membrane and kinetics of cargo release even for the smallest vesicles that typically constitute the largest portion of the secretory vesicular pool. The biological problem we want to address is the exocytotic ATP release from non-excitable, cancerous lung epithelial cells. TIRF microscopy will visualize vesicles loaded with fluorescently-tagged ATP by evanescence field excitation in distances up to 200 nm. Following the exocytotic release into the extracellular space the labelled cargo will be positioned within the enhancement field of the metallic nanostructure, producing a huge, easy to detect signal that disappears due to free diffusion of the fluorophore into the extracellular space. PUBLIC HEALTH RELEVANCE: ATP release is suggested to take place at the leading edge of a migrating cell, contributing to a coordination of many processes that are involved in a directional cell movement. Proposed nanophotonic approach will allow studying these complex processes with unprescedented sensitivity and help to better understand regulation of tumour metastasis (tumour spread) and invasiveness.