Nanophotonic Approach to Imaging Exocytosis

描述（由申请人提供）： 基于荧光的成像已经发展成为一门学科，在生物学，临床医学和癌症检测中有许多应用。活细胞的许多基本过程发生在细胞表面质膜，包括激素和神经递质的分泌，营养物质的摄取，化学和电信号的产生和传递。囊泡胞吐作用是信号分子如激素和神经递质穿过表面膜的重要机制。胞吐-胞吞过程还涉及质膜和细胞内区室之间的膜蛋白的再循环。选择性地可视化活细胞中的这些过程和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，今天没有成像方法，TIRF的一些例外，是能够直接可视化在活细胞中的胞吐事件，涉及运输和分泌的囊泡货物跨质膜。细胞膜的大小仅为~10 nm，许多分泌囊泡的直径通常远低于50 nm，因此所有成像方式都缺乏足够的分辨率。该R21应用的长期目标是开发和验证一种新的方法，用于通过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来监测膜过程。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。我们的近期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吐过程。这将允许密切关注囊泡向质膜的募集和货物释放的动力学，即使是通常构成分泌囊泡池的最大部分的最小囊泡。我们要解决的生物学问题是从非兴奋性癌性肺上皮细胞中释放的胞吐ATP。TIRF显微镜将可视化囊泡加载荧光标记的ATP的倏逝场激发的距离高达200 nm。在胞吐释放到细胞外空间中之后，标记的货物将位于金属纳米结构的增强场内，产生巨大的、易于检测的信号，该信号由于荧光团自由扩散到细胞外空间中而消失。 公共卫生相关性： ATP的释放被认为发生在迁移细胞的前缘，有助于协调参与定向细胞运动的许多过程。提出的纳米光子方法将允许以前所未有的灵敏度研究这些复杂的过程，并有助于更好地理解肿瘤转移（肿瘤扩散）和侵袭性的调节。

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.