Plasmon-Enhanced Imaging of Single Membrane Proteins

光学天线是光学物理领域的一个新概念，本课题旨在利用光学天线的特性，从根本上提高光学成像的分辨率。目的是开发一种仪器，用于天然细胞膜中单个蛋白质的成像，鉴定和表征。具有这种能力的仪器将对生物学研究的各个领域产生重大影响，因为膜蛋白经历许多特定的任务，从识别和信号传导到跨细胞膜的分子运输。尽管目前的方法，如x射线晶体学或核磁共振，提供了高分辨率的结构细节，但它们受到静态观察和集合平均的限制。因此，膜蛋白的空间分布和共定位以及与膜拓扑结构的相关性在很大程度上是未知的。在这个项目中开发的仪器使用光学天线来定位和增强入射激光辐射，从而产生纳米级光源。这种光源将在生物膜表面用荧光标记的蛋白质进行光栅扫描。样品表面上的每个点都将以荧光光谱的形式具有其独特的指纹特征，使我们能够定位，识别和研究单个蛋白质。初步结果支持该技术的可行性，但还需要解决各种问题。我们将使用熔融金纳米粒子和金纳米棒作为光学半波天线，并将它们连接到尖锐的介电毛细血管上。不同的方法抑制背景荧光源自细胞内部将进行研究。该仪器将用于研究内皮细胞膜中粘附蛋白的分布，粘附蛋白在白细胞-内皮细胞相互作用中起关键作用，因此在炎症反应中起关键作用。单层内皮细胞将直接在功能化的玻璃底物上生长，p -选择素蛋白将被荧光抗体标记。目的是在生理条件下对单个蛋白质进行空间解析，并研究其与膜拓扑结构的相关性。

0930074NovotnyThis proposal seeks to fundamentally improve the resolution of optical imaging by exploiting the properties of optical antennas, a new concept in the field of optical physics. The objective is to develop an instrument for the imaging, identification and characterization of single proteins in native cell membranes. An instrument with such capability will have a high impact on various fields of biological research because membrane proteins undergo a multitude of specific tasks ranging from recognition and signaling to molecular transport across the cell membrane. Although current methods, such as X-ray crystallography or NMR, provide high-resolution structural details, they are limited by their static observation and ensemble averaging. As a consequence, the spatial distribution and co-localization of membrane proteins as well as the correlations with membrane topology are largely unknown. The instrument to be developed in this project uses an optical antenna to localize and enhance incident laser radiation thereby creating a nanoscale light source. This light source will be rasterscanned over the surface of a biological membrane with fluorescently labeled proteins. Each point on the sample surface will be characterized by its unique fingerprint in form of a fluorescence spectrum allowing us to localize, identify and study single proteins. Preliminary results support the feasibility of this technique but various issues need yet to be addressed. We will use fused gold nanoparticles and gold nanorods as optical half-wave antennas and attach them to pointed dielectric capillaries. Different approaches for suppression of background fluorescence originating from the cell interior will be investigated. The instrument will be used to study distributions of adhesion proteins in endothelial cell membranes which play a key role in leukocyte-endothelial cell interactions and thus in the inflammatory response. Single layers of endothelial cells will be grown directly on functionalized glass substrates and P-selectin proteins will labeled with fluorescent antibodies. The goal is to spatially resolve individual proteins under physiological conditions and to study the correlation with membrane topology.