High-resolution measurement of near-surface bacterial swimming reveals bimodal cell-surface separations

Bacterial interactions with solid boundaries initiate surface sensing, adhesion, and biofilm formation, yet the dynamics of near-wall swimming remain difficult to quantify because the cell–surface gap is challenging to measure with high precision in real time. Here, we introduce a calibrated, exclusion-based fluorescence microscopy method that converts fluorescence intensity into absolute height using in situ microsphere calibration, enabling reconstruction of three-dimensional trajectories of swimming Escherichia coli with tens-of-nanometer axial precision over a ∼400-nm range. We observe that steady-state gap heights are bimodal: cells occupy two preferred separation regimes, a short-range state at ∼67 nm and a long-range hovering state at ∼240 nm. Increasing ionic strength enriches the short-range population and shifts it toward the surface, consistent with electrostatic screening predicted by DLVO theory, whereas the hovering state remains essentially unchanged, indicating a predominantly hydrodynamic origin. We further quantify how gap height covaries with swimming speed and trajectory curvature. This approach reconciles disparate estimates of cell–surface distances reported previously and offers a robust, non-invasive tool for investigating the mechanisms of bacteria–surface interactions.