Nonlinear Imaging of Bacterial Membranes during Adhesion

Electrostatic interactions between bacteria and abiotic surfaces induce changes that propagate from the cell exterior to perturb the inner membrane and associated cellular processes. Direct optical readouts that disentangle the potentially coupled effects from these interactions remain limited. Here we use simultaneous second-harmonic generation (SHG) and two-photon fluorescence (TPF) microscopy of the styryl dye FM 1-43 to probe how adhesion to substrates of different surface charge alters the inner-membrane environment of Escherichia coli. From negatively charged glass to positively functionalized surfaces, the initial SHG and TPF signals exhibit opposing substrate-dependent trends. The SHG readout is consistent with previously proposed alterations in inner membrane potential due to charge regulation but also reveals a substrate-dependent spatially heterogeneous response. In contrast, the magnitude and time dependence of the TPF signals cannot be explained by membrane potential alterations but instead implicate a probe population associated with mechanosensitive channel activation and subsequent adaptation. Together, these results establish concurrent SHG/TPF imaging as an integrated tool for directly capturing how surface electrostatics bridge bacterial adhesion with inner membrane physiology.