EAGER: Developing a Live-cell, Multicolor Superresolution Imaging Method for Probing the Structural Dynamics of Bacterial Cytoskeletons

智力优势：本研究的目标是发展一种单分子超分辨率成像技术，能够直接探测单个活大肠杆菌中细菌细胞骨架的高分辨率结构和动态。coli细胞。 本研究基于新开发的光激活定位显微镜（PALM），通过开发新的策略以适应单活E. coli细胞。如果成功开发，这项技术将改变目前对细菌细胞骨架的研究，并使研究人员能够以目前方法无法获得的时空分辨率水平揭示细菌细胞骨架的结构和动态细节。这项工作的直接成果将是：（1）优化的成像策略，为单，两个和三个彩色超分辨率成像在生活E。coli细胞;（2）一套完整的PALM成像方案和重建算法，可以高速、高密度地绘制分子位置图;（3）在纳米分辨率水平上阐明单个活的E. coli细胞。更广泛的影响：活细胞超分辨率成像技术的发展将在许多方面产生深远的影响。首先，新的成像技术对细菌细胞生物学家来说将特别强大。 在过去，科学家们不得不依靠衍射限制的免疫荧光或传统的FP荧光成像方法来可视化小细菌细胞中的细胞结构，因为EM是其相关的高分辨率成像技术在识别细菌细胞结构方面没有成果。其次，虽然专门针对E.大肠杆菌细胞，新技术的基本成像概念和原理可以推广，使其应用到更高的真核细胞。第三，成像协议和成像后分析算法一旦制定，将传播和分发给广泛的研究界。最后，成功完成这项工作需要一支拥有生物学、化学、物理和工程专业知识的跨学科团队，为本科生、研究生和博士后提供良好的培训机会。该项目由分子和细胞生物科学部门的基因和基因组系统以及细胞系统集群共同资助。

Intellectual Merit: The goal of this research is to develop a single-molecule superresolution imaging technique that enables direct probing of high-resolution structures and dynamics of bacterial cytoskeletons in single living E. coli cells. Based on the newly developed photoactivated localization microscopy (PALM), this research pushes PALM imaging to its limit by developing novel strategies to accommodate high-speed, high density multicolor imaging in single living E. coli cells. If successfully developed, this technique will transform current studies of bacterial cytoskeletons and enable researchers to uncover structural and dynamic details of bacterial cytoskeletons at a spatial-temporal resolution level that is not available with current approaches. The direct outcome from this work will be: (1) optimized imaging strategies for single-, two- and three-color superresolution imaging in living E. coli cells; (2) a complete package of PALM imaging protocol and reconstruction algorithm that allow high-speed, high-density mapping of molecule positions; and (3) elucidation of time-dependent structural evolution of bacterial cytoskeletons at the nanometers resolution level during the cell cycle in single living E. coli cells. Broader Impacts: The development of a live-cell, multicolor superresolution imaging technique will be far-reaching in many ways. First, the new imaging technique will be particularly powerful for bacterial cell biologists. In the past scientists have to rely on diffraction-limited immunofluorescence or conventional FP fluorescence imaging methods to visualize cellular structures in small bacterial cells because EM are its related high resolution imaging techniques are not fruitful in identifying bacterial cellular structures. Second, although specifically tailored to E. coli cells, the basic imaging concepts and principles of the new technique can be generalized to allow its application to higher eukaryotic cells. Third, once developed, the imaging protocols and post-imaging analysis algorithms will be disseminated and distributed to a wide range of research communities. Finally, successful completion of this work requires an interdisciplinary team that has expertise in biology, chemistry, physics and engineering, providing excellent training opportunities for undergraduate, graduate and postdoctoral students.This project is co funded by the Genes and Genome Systems and the Cellular Systems Clusters within the Division of Molecular and Cellular Biosciences.