Omcl tr4

Gold-coated glass coverslips and cantilevers (OMCL-TR4, Olympus Ltd., Tokyo, Japan; nominal spring constant, ∼0.02 N m⁻¹) were immersed overnight in an ethanol solution containing 1 mM 10% 16-mercaptododecahexanoic acid–90% 1-mercapto-1-undecanol (Sigma), rinsed with ethanol, and dried with N₂. Substrates and cantilevers were then immersed for 30 min into a solution containing 10 mg ml⁻¹N-hydroxysuccinimide (NHS) and 25 mg ml⁻¹ 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC [Sigma]), rinsed with Ultrapure water (ELGA LabWater), incubated with 0.1 mg ml⁻¹ vWF (Merck) for 1 h, rinsed further with PBS buffer, and then immediately used without dewetting.

Gold cantilevers (OMCL-TR4, Olympus Ltd., Tokyo, Japan) with a nominal spring constant of ~0.02 N×m^-1 were functionalized with fibrinogen using thiols chemistry as previously described for substrates. The spring constants of the cantilevers were measured using the thermal noise method. Bacteria from exponential phase culture were immobilized by mechanical trapping into porous polycarbonate membranes (Millipore, Billerica, USA) with a pore diameter of 0.8 μm. After filtering a cell suspension, the membrane was rinsed with PBS, cut into piece (1 x 1 cm²) and attached to a steel sample puck using a small piece of double-face adhesive tape. The mounted sample was transferred into the AFM liquid cell while avoiding de-wetting. Bare tips were first used to localize and image individual cells and then replaced by functionalized tips. Adhesion maps were obtained by recording 32-by-32 force-distance curves on areas of 500 x 500 nm², using an applied force of 250 pN, a constant approach-retraction speed of 1 μm×s^-1 and a contact time of 100 or 500 ms. Data were analyzed using the Nanoscope software from Bruker (Santa Barbara, USA). Adhesion forces were calculated considering the last peak for each curve and adhesive events are displayed as light pixels. For each condition, experiments were repeated for at least 3 times with independent cultures.

Gold-coated glass coverslips and cantilevers (OMCL-TR4; Olympus; nominal spring constant, ~0.02N m^-1) were immersed overnight in an ethanol solution containing 1 mM 10% 16-mercaptododecahexanoic acid—90% 1-mercapto-1-undecanol (Sigma), rinsed with ethanol, and dried with N₂. Substrates and cantilevers were then immersed for 30 min in a solution containing 10 mg ml^-1 NHS and 25 mg ml^-1 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Sigma), rinsed with ultrapure water (ELGA LabWater), incubated with 0.1 mg ml^-1 of fibrinogen for 1 h, rinsed further with PBS buffer, and immediately used without de-wetting.