Three‐Dimensional Super‐Resolution Imaging of the Midplane Protein FtsZ in Live Caulobacter crescentus Cells Using Astigmatism

Abstract

Single‐molecule super‐resolution imaging provides a non‐invasive method for nanometer‐scale imaging and is ideally suited to investigations of quasi‐static structures within live cells. Here, we extend the ability to image subcellular features within bacteria cells to three dimensions based on the introduction of a cylindrical lens in the imaging pathway. We investigate the midplane protein FtsZ in Caulobacter crescentus with super‐resolution imaging based on fluorescent‐protein photoswitching and the natural polymerization/depolymerization dynamics of FtsZ associated with the Z‐ring. We quantify these dynamics and determine the FtsZ depolymerization time to be <100 ms. We image the Z‐ring in live and fixed C. crescentus cells at different stages of the cell cycle and find that the FtsZ superstructure is dynamic with the cell cycle, forming an open shape during the stalked stage and a dense focus during the pre‐divisional stage. 3D is here to stay: Three‐dimensional super‐resolution astigmatic optical imaging provides images of the FtsZ Z‐ring in bacterial cells in various stages of the cell cycle. The picture shows, left to right: live stalked cell, live pre‐divisional cell, fixed stalked cell, fixed pre‐divisional cell. Scale bar: 200 nm.