Imaging the Separation Distance between the Attached Bacterial Cells and the Surface with a Total Internal Reflection Dark-Field Microscope.

The attachment of bacterial cells to a surface is implicated in the formation of biofilms. Although the surface-related behaviors in this process, such as single cell motility and surface sensing, have been investigated intensively, the precise information of separation distance between the attached cells and the surface has remained unclear. Here, we set a prism-based total internal reflection dark-field microscope (p-TIRDFM) combined with the microfluidic method to image the separation distance of single attached cells. We directly observed that bacterial cells attached to the surface with one nearest touchpoint, and it gradually changed to two touchpoints, respectively, for the two offspring with the cell division. We first monitored the fluctuation of the relative distance on nanometer scale when cells twitch on a surface and further established the relationship between the twitching velocity and the separation distance. The results indicated that the moving cells are a considerable distance apart from the surface and the separation distance fluctuated more widely than immobile cells.