Single-Cell Probing of Nanoscale Bacterial Adhesion in Real-Time Using Optical Tweezers

cells, a dominant early colonizer in environmental biofouling, focusing on its holdfast, a strong nanoscale adhesive organelle that mediates irreversible attachment within seconds of contact. To characterize the time-dependent mechanical properties of holdfast, we developed the Trapezoid, a custom optical tweezers platform that combines nanometer spatial precision with millisecond temporal control of cell position, contact timing, and applied force on defined surfaces. We implemented a trapezoidal temporal profile of these programmed contact cycles, in which a single cell is brought into contact with the surface, maintained for a defined duration, retracted, and subjected to controlled pulling forces. This approach enables real-time quantification of nanoscale adhesion onset, holdfast deployment kinetics, and influence of surface chemistry at the level of individual live adhesion events. Our results dissect the physical and biochemical determinants of bacterial adhesion, providing a quantitative framework for the rational design of antiadhesive coatings, nanoscale biofouling control strategies, and bioinspired adhesives functional in wet environments.