Bioscope resolve afm

AFM topographic images of LHP ANCs thin film was captured using the Bruker BioScope Resolve AFM using PeakForce Tapping mode in combination with ScanAsyst automatic parameter adjustment functionality. The topographic AFM images were captured using a 1 nm ultra-sharp probe with a peak force amplitude of 50 nm, peak force set-point of 0.1 nN, and a scan rate of 1.4 Hz.

Atomic force microscopy was performed in HEPES‐Ringer buffer at room temperature in PeakForce Tapping mode using a BioScope Resolve AFM (Bruker Nano Surfaces, Santa Barbara, CA, USA). A PeakForce QNM‐Live Cell (PFQNM‐LC) probe (Bruker AFM Probes, Camarillo, CA, USA) (tip length 17 µm, tip radius 65 nm, opening angle 15°) was used to image the cell surface. The spring constant of the cantilever was determined with a vibrometer (OFV‐551, Polytec, Waldbronn, Germany) and found to be 0.0611 N/m. The glass bottom Petri dish in which the MDCK cells were grown was held down through the use of vacuum that has been incorporated into the AFM sample plate while still allowing optical access to the sample from below. The use of vacuum reduces noise and eliminates the “drum” effect that is created when these thin‐bottomed petri dishes are positioned over the optical aperture in the sample stage. Images were taken at 384 × 384 pixels with a PeakForce Tapping frequency of 1 kHz and amplitude of 300 nm. Probe–sample contact time was about 200 μs each cycle. Automatic gain control was used to improve the feedback for surface tracking. Height sensor signal was used to display the cell surface image using Nanoscope Analysis v1.60 (Bruker Nano Surfaces, Santa Barbara, CA, USA).

Before the AFM experiments, the 50,000 cells were seeded per dish and treated for 48 and 72 h with inhibitors. The AFM measurements were performed at 37 °C, using a commercial atomic force microscope Bioscope Resolve AFM (Bruker, Billerica, MA, USA) combined with an inverted optical microscope (Carl Zeiss, Ulm, Germany). The PeakForce QNM-Live Cell cantilevers (PFQNM-LC-A-CAL, Bruker AFM Probes, USA) with a pre-calibrated spring constant (in a range of 0.06–0.08 N/m) and a 70 nm tip radius was used. The deflection sensitivity (nm/V) was calibrated from the thermal using the pre-calibrated value of the spring constant. The nanomechanical maps were acquired in the force volume mode with a typical map size of 80 × 80 microns and 40 × 40 measurement points [73 (link)]. For the force curves, a vertical ramp distance was 3 μm, a vertical piezo speed was 183 μm/s, and the trigger force was 0.5–1 nN. The Young’s modulus (E) was calculated by fitting the force curves with the Hertz model with a bottom-effect correction [73 (link),74 (link)].

Bacterial cells or the nanowire filaments were immobilized on glass slides and loaded on a Bioscope Resolve AFM (Bruker, USA). The AFM was operated in PeakForce QNM mode and using SCANASYST-AIR probes (nominal spring constant 0.4 N/m, nominal tip radius 2 nm) (Bruker AFM Probes, USA). All images were further processed and analyzed by Nanoscope Analysis 1.9 (Bruker, USA). KPFM mode was used to measure the surface potential of GY32 cells and the nanowire appendages with electrically conductive SCM-PIT probes (Bruker AFM Probes, USA). The bacteria samples were loaded on gold-coated mica substrates or highly oriented pyrolytic graphites and then washed with deionized water five times. After being air-dried, the samples were scanned with a lift scan height of 50 nm and a drive amplitude of 2 V. The AFM investigation was performed for three independent cultures of L. varians GY32 all showing similar results.

AFM experiments were performed with a Bioscope Resolve AFM (Bruker) at ∼25–30°C in DMEM. PeakForce QNM Live Cell probes (Bruker) with spring constants of 0.10 ± 0.02 N m^–1 and tip radius of curvature of 65 nm were used. The spring constant of the cantilevers was calibrated with a vibrometer (OFV-551, Polytec, Waldbronn) by the manufacturer. The pre-calibrated spring constant was used to determine the deflection sensitivity (Schillers et al., 2017 ) using the thermal noise method (Hutter and Bechhoefer, 1993 (link)) before each experiment. In fast indentation experiments (Dumitru et al., 2018 (link)), the AFM was operated in PeakForce QNM mode and Force-distance (FD)-based multiparametric maps were acquired using a force setpoint of 300 pN. The AFM cantilever was oscillated vertically at 0.25 kHz with a peak-to-peak oscillation amplitude of 500 nm. Height and Young’s modulus maps were recorded using a scan rate of 0.2 Hz and 256 pixels per line. Slow indentation experiments were performed in Force-Volume (FV) mode. Individual FD curves were recorded in contact mode on the RBC surface with a force setpoint of 300 pN, using ramp speeds of 2 μm s^–1 for a 2 μm ramp size. Measurements were performed in the central region of RBCs to avoid substrate effects. Indentations ranged between 300 nm and 1 μm depending on cell type.