Nanophotonic Approach to Imaging Exocytosis

胞吐作用成像的纳米光子方法

基本信息

批准号：
8035518
负责人：
ZYGMUNT GRYCZYNSKI
金额：
$ 14.2万
依托单位：
UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-01 至 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8035518
关键词：
Address BODIPY Biochemistry Biological Biological Process Biology Biophysics Buffers Calcium Calcium Signaling Caliber Cancer Detection Cancerous Cell Proliferation Cell membrane Cell physiology Cell surface Cells Cellular biology Chemicals Clinical Medicine Colloids Communication Complex Deposition Development Diffusion Discipline Dyes Endocytosis Environment Epithelial Cells Event Exocytosis Extracellular Space Fluorescence Fluorescence Microscopy Fluorescence Spectroscopy Generations Goals Gold Grant Growth Hormones Image Imaging Techniques Immunochemistry In Vitro Ionic Strengths Kinetics Label Life Lung Malignant Neoplasms Malignant neoplasm of lung Membrane Membrane Proteins Methodology Methods Molecular Molecular Profiling Monitor Muscle Fibers Nanostructures Nanotechnology Neoplasm Metastasis Neurotransmitters Nutrient P2X-receptor Pathology Pathway interactions Physiological Play Positioning Attribute Process Proteins Pump Purinergic P1 Receptors Quinacrine Recording of previous events Recycling Regulation Research Personnel Resolution Role Secretory Vesicles Signal Transduction Signaling Molecule Silicon Dioxide Silver Solutions Spectrum Analysis Stimulus Structure Surface System Systems Biology Technology Testing Transmembrane Transport Vesicle Waste Products Work autocrine base cancer cell cell growth cell motility directional cell experience fluorophore imaging modality improved malignant breast neoplasm molecular imaging neurotransmitter uptake novel novel strategies photonics plasmonics public health relevance receptor function response tool transmission process tumor uptake

项目摘要

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.

描述（由申请人提供）：基于荧光的成像本身就发展成为一门学科，并在生物学，临床医学和癌症检测中进行了许多应用。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，营养素的摄取，化学和电信号的产生和传播。囊泡胞吐作用是一种重要的机制，信号分子（例如激素和神经递质）越过表面膜。胞吐病 - 细胞增多症的过程也参与了质膜和细胞内室之间的膜蛋白的回收。选择性地可视化此类过程和活细胞中的亚膜结构的能力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。但是，如今，除了TIRF的某些例外，没有成像方法能够直接在活细胞中可视化胞外事件，这些事件涉及在质膜上运输和分泌囊泡货物。细胞膜的大小仅为约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应用的长期目标是通过利用荧光团与表面沉积金属纳米结构的近场相互作用引起的巨大荧光信号增强来开发和验证一种新的方法来监测膜过程。该应用是基于我们最近发现的，即自组装的胶体结构（SAC）可为紧密定位的荧光团产生高达1000倍的荧光信号增强。我们的近期目标是将这种纳米光子现象与TIRF显微镜结合使用，以用前所未有的灵敏度监测胞吐过程。这将允许关闭囊泡募集到质膜和货物动力学释放的动力学，即使对于通常构成分泌囊泡池最大部分的最小囊泡也是如此。我们要解决的生物学问题是胞吐ATP从非抗癌性肺上皮细胞中释放出来。 TIRF显微镜将可视化囊泡，该囊泡以荧光场激发的距离，距离为200 nm。在细胞外空间中的胞吐释放后，标记的货物将位于金属纳米结构的增强场内，从而产生巨大，易于检测的信号，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：建议在迁移细胞的前沿释放ATP释放，从而有助于与定向细胞运动有关的许多过程的协调。拟议的纳米光学方法将允许以未经表达的灵敏度研究这些复杂的过程，并有助于更好地了解肿瘤转移（肿瘤扩散）和侵袭性的调节。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Hybrid optical materials of plasmon-coupled CdSe/ZnS coreshells for photonic applications.

DOI：
10.1364/ome.2.001026
发表时间：
2012-08-01
期刊：
Optical materials express
影响因子：
2.8
作者：
Seo J;Fudala R;Kim WJ;Rich R;Tabibi B;Cho H;Gryczynski Z;Gryczynski I;Yu W
通讯作者：
Yu W

Fluorescence instrument response standards in two-photon time-resolved spectroscopy.

双光子时间分辨光谱中的荧光仪器响应标准。

DOI：
10.1366/000370210792081000
发表时间：
2010
期刊：
Applied spectroscopy
影响因子：
3.5
作者：
Luchowski,Rafal;Szabelski,Mariusz;Sarkar,Pabak;Apicella,Elisa;Midde,Krishna;Raut,Sangram;Borejdo,Julian;Gryczynski,Zygmunt;Gryczynski,Ignacy
通讯作者：
Gryczynski,Ignacy