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

Johns Hopkins University, Baltimore MD

Investigators

Abstract

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.