(Left) The procedure of an optical tweezers force measurement. Mounting and measuring procedures. (1) First, we mount a bacterium to a 10-m poly-L-lysine coated bead that is immobilized on the coverslide. The coated bead serves as a mount for a bacterium. A bacterium is thereafter trapped in the optical tweezers and attached to the coated bead. (2) Second, a 2-m probe bead is trapped and the trap stiffness is calibrated using the power spectrum method.25After calibration, the probe bead is brought close (~1–2 m) to the mounted bacterium using a piezo-stage. When a pilus attaches, the motion of the probe bead changes from the equilibrium position, which is seen in the live camera image and the real-time force response. (3) Finally, with a pilus attached, we move the piezo-stage at 0.1 m/s while keeping the trap stationary. This applies tensile force to a pilus as the bacterium and probe bead are separated. A force response of pili is shown in (A). First, two pili are bound, so the force is double. After one pilus detached (~3.5 m, the unwinding force, and filament extension, are for one pilus.
