Single molecule super-resolution imaging of bacterial cell pole proteins with high-throughput quantitative analysis pipeline.

Bacteria show sophisticated control of their cellular organization, and many bacteria deploy different polar landmark proteins to organize the cell pole. Super-resolution microscopy, such as Photo-Activated Localization Microscopy (PALM), provides the nanoscale localization of molecules and is crucial for better understanding of organization and dynamics in single-molecule. However, analytical tools are not fully available yet, in particular for bacterial cell biology. For example, quantitative and statistical analyses of subcellular localization with multiple cells from multiple fields of view are lacking. Furthermore, brightfield images are not sufficient to get accurate contours of small and low contrast bacterial cells, compared to subpixel presentation of target molecules. Here we describe a novel analytic tool for PALM which integrates precisely drawn cell outlines, of either inner membrane or periplasm, labelled by PALM-compatible fluorescent protein fusions, with molecule data for >10,000 molecules from >100 cells by fitting each cell into an oval arc. In the vibrioid bacterium Vibrio cholerae, the polar anchor HubP constitutes a big polar complex which includes multiple proteins involved in chemotaxis and the flagellum. With this pipeline, HubP is shown to be slightly skewed towards the inner curvature side of the cell, while its interaction partners showed rather loose polar localization.