Observing the IgE and FceRI signaling pathway via dielectric-filled nanoapertures

描述(申请人提供)：这项研究旨在了解过敏原引起免疫系统反应的分子过程。特别是，我们打算促进对抗体诱导的膜受体交联性并引发过敏和炎症反应的细胞内过程的理解。该项目将通过开发一系列小孔来提高超分辨率显微镜的实验能力，每个小孔利用近场光学来照明小样本体积。这项研究的长期目标是了解用于改变质膜局部组成的物理和化学过程。这个项目的目标是用传统的荧光显微镜技术测量具有80 nm和1ps分辨率的纳米图案化光学表面的膜重组和信号传递过程。该项目将产生一种新技术，以克服目前的实验障碍，并应用于各种生物学假说。这项研究的短期目标是(1)开发一种能够在集中的生物环境中进行单分子检测的技术和(2)进一步了解IgE-FccRI聚集、蛋白质募集和局部膜异质性的诱导。这项研究的中心假设是，介质填充的零模波导将在细胞膜上提供足够的时间和空间分辨率，以测量受体刺激诱导的蛋白质-蛋白质联系。这一假设建立在相关的纳米结构和理论预测的基础上，如下所述。这样的设备将提供一种测量膜(重新)组织的手段，检验以前无法回答的假说，并推进基于膜受体的食物的治疗。这个项目提供了一个独特的培训机会，在纳米制造和膜生物学的指导下，在我的生物物理化学背景的基础上再接再厉。这项研究将创造一项新技术，并将其应用于回答抗原刺激和Fc？RI膜受体交联后细胞内信号级联中的紧迫问题。这项研究的结果将直接适用于诊断和治疗过敏反应。具体目标1：研制用于近场光学显微镜(ANOM)的孔阵列特定目标2：表征用于定量超分辨显微镜的ANOM的光学性质

DESCRIPTION (provided by applicant): This research aims to understand the molecular processes by which an allergen elicits an immune system response. In particular, we intend to advance understandings of the intracellular processes that result from antibody-induced cross-linking of the membrane receptors and induce an allergic and inflammation response. This project will advance the experimental capabilities for super-resolution microscopy through the development of an array of apertures, each illuminating a small sample volume utilizing near-field optics. The long-term goal of this research is to understand the physical and chemical processes used to modify local composition of the plasma membrane. The objective of this project is to measure the membrane reorganization and signaling processes with nano-patterened optical surfaces capable of 80 nm and 1 ps resolution with otherwise conventional fluorescent microscopy techniques. This project will yield a new technology to overcome current experimental barriers and have applications to a variety of biological hypotheses. The short-term goals of this study are (1) to develop a technique capable of single-molecule detection in concentrated biological environments and (2) to further understand IgE-FccRI clustering, protein recruitment, and the induction of local membrane heterogeneity. The central hypothesis of this research is that dielectric-filled zero-mode waveguides will provide sufficient time and space resolution on cell membranes to measure protein-protein associations induced by receptor stimulation. This hypothesis is built upon both related nanoscale structures and theoretical predictions, as described below. Such devices will provide a means for measuring membrane (re)organization, testing previously unanswerable hypotheses, and advancing the treatment of membrane-receptor based aliments. This project provides a unique training opportunity to build upon my background in biophysical chemistry, guided by mentoring in nanofabrication and membrane biology. This research will create a new technology and apply it to answer pressing questions in the intracellular signaling cascade following antigen stimulation and cross-linking of FC?RI membrane receptors. Results from this research will be directly applicable to diagnosing and treating allergic responses. Specific Aim 1: Develop an array of apertures for near-field optical microscopy (ANOMs) Specific Aim 2: Characterize the optical properties of ANOMs for quantitative super-resolution microscopy Specific Aim 3: Measure local membrane heterogeneity induced by FC?RI clustering