Nanoscale Imaging of Subcellular Processes

描述(由申请人提供)： 在过去的一年半里，我们开发了一种新的荧光成像干涉技术，称为光谱自干涉荧光显微镜(SFM)。这项技术利用了位于反射面上方几个波长距离的荧光团发射的光谱振荡。这些光谱振荡是由于直接和反射发射的光的自干扰造成的，对光的分析得出荧光团的垂直位置在几纳米以内。这项提议旨在展示SFM作为一种纳米分辨率荧光显微镜技术在人工和生物模型系统中的应用，将其独特的分辨能力应用于体内新的生物学问题，最终将其能力扩展到任意切片和最终的3D实时纳米级荧光成像。总体上，长期目标是开发、演示和应用最终分辨率为10纳米的活体亚细胞显微镜。 蛋白质在原核细胞中的精确三维定位是许多细胞功能的关键，包括细胞周期、DNA复制、发育、运动和黏附。到目前为止，在原核生物中介导蛋白质三维靶向的基本机制仍然很大程度上是未知的。MB Goldberg博士的实验室表明，志贺氏菌肌动蛋白组装蛋白ICSA针对细菌旧极的靶向发生在细菌细胞质中，涉及多肽的两个特定区域。我们将应用在这项提议中开发的SFM来分析活细菌中ICSA与参与其分泌的蛋白质的特定相互作用。

DESCRIPTION (provided by applicant): Over the past year and a half, we have developed a new interferometric technique in fluorescent imaging called spectral self-interference fluorescent microscopy (SFM). The technique utilizes the spectral oscillations emitted by a fluorophore located a distance of several wavelengths above a reflecting surface. These spectral oscillations are due to the self-interference from the direct and reflected emitted light, analysis of which yields the vertical position of the fluorophore to within a few nanometers. This proposal is to demonstrate SFM as a nanometer resolution fluorescent microscopy technique in both artificial and biological model systems, apply its unique resolution capability to novel biological questions in vivo, and finally, extend the capabilities to arbitrary sectioning and eventual 3D, real-time nanoscale fluorescent imaging. The overall, long-term goal is to develop, demonstrate and apply in vivo subcellular microscopy at an ultimate resolution of 10 nanometers. The precise three-dimensional localization of proteins within prokaryotic cells is key to many cellular functions, including cell cycle, DNA replication, development, motility, and adhesion. As yet, the basic mechanisms that mediate three-dimensional targeting of proteins in prokaryotes remain largely unknown. Dr. MB Goldberg's laboratory has shown that the targeting of the Shigella actin assembly protein IcsA to the bacterial old pole occurs in the bacterial cytoplasm and involves two specific regions of the polypeptide. We will apply the SFM developed in this proposal to analyze with sub-cellular resolution in live bacteria specific interactions of IcsA with proteins involved in its secretion.