Nanoscale Imaging of Subcellular Processes

描述（由申请人提供）： 在过去的一年半中，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）。该技术利用位于反射表面上方几个波长距离处的荧光团发射的光谱振荡。这些光谱振荡是由于直接发射光和反射发射光的自干扰造成的，对其进行分析可以得出荧光团的垂直位置在几纳米之内。该提案旨在证明 SFM 作为人工和生物模型系统中的纳米分辨率荧光显微镜技术，将其独特的分辨率能力应用于体内新的生物问题，最后将该功能扩展到任意切片和最终的 3D 实时纳米级荧光成像。总体长期目标是开发、演示和应用最终分辨率为 10 纳米的体内亚细胞显微镜。 原核细胞内蛋白质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、运动和粘附。迄今为止，介导原核生物中蛋白质三维靶向的基本机制仍然很大程度上未知。 MB Goldberg 博士的实验室表明，志贺氏菌肌动蛋白组装蛋白 IcsA 向细菌老极的靶向发生在细菌细胞质中，并涉及多肽的两个特定区域。我们将应用本提案中开发的 SFM 以亚细胞分辨率分析活细菌中 IcsA 与参与其分泌的蛋白质的特异性相互作用。

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.