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.

Figure 1

Visibility times for FtsZ-Dendra2 in A) live C. crescentus cells and B) fixed C. crescentus cells.

Figure 2

False-color two-dimensional SR reconstruction of FtsZ-Dendra2 (red) in live C. crescentus cells overlaid on a reversed contrast white-light transmission image of the cells. The red box highlights the midplane of a stalked cell; here, the arrow points to the FtsZ ring typically observed in stalked cells. The blue box highlights the midplane of a pre-divisional cell; here, the arrow points to the FtsZ constriction typically observed in pre-divisional cells. Scale bar: 2 μm.

Figure 3

2D Projections of 3D SR images of FtsZ-Dendra2 in a live stalked cell oriented lengthwise along the y axis in A) the xy-plane (i.e. top view; typical of 2D SR images) and B) the xz-plane (i.e. cross-sectional view). Scale bar: 200 nm.

Figure 4

2D Projections of 3D SR images of FtsZ-Dendra2 in a live pre-divisional cell oriented lengthwise along the y-axis in A) the xy-plane i.e. top view; typical of 2D SR images) and B) the xz-plane (i.e. cross-sectional view). Scale bar: 200 nm.

Figure 5

2D Projections of 3D SR images of FtsZ-Dendra2 in a fixed stalked cell oriented lengthwise along the y-axis in A) the xy-plane (i.e. top view; typical of 2D SR images) and B) the xz-plane (i.e. cross-sectional view). Scale bar: 200 nm.

Figure 6

2D Projections of 3D SR images of FtsZ-Dendra2 in a fixed pre-divisional cell oriented lengthwise along the y axis in A) the xy-plane (i.e. top view; typical of 2D SR images) and B) the xz-plane (i.e. cross-sectional view). Scale bar: 200 nm.