Jpk nanowizard 1 afm

A JPK Nanowizard 1 AFM (JPK Instruments Ltd., Germany) was used to obtain 10 × 10 µm high‐resolution topography images (1024 × 1024 pixels) from each segment (I–II–III, Figure 1a) of the Lower and Higher gradient gels and the Control gel. Gels were kept for 24 h in phosphate buffered saline (PBS) and rinsed using distilled water. Imaging was conducted using MSNL‐10‐A tip cantilevers (Bruker Ltd., France) with the following characteristics: resonant frequency of ≈22 kHz and spring constant ≈0.07 N m⁻¹. JPK/SPM data processing software was used to extract data.

Adherent cells were cultured on 70% ethanol-cleaned glass slides in 6-wells culture plates, rinsed to remove unbound cells and fragments and mounted in a temperature-controlled chamber (Biocell, JPK Instruments, Berlin, Germany) set to 37 °C. Cells were indented with a JPK Nanowizard 1 AFM (JPK Instruments), using the force mode with a closed loop 15 μm range piezo. The AFM sits on an Axiovert 200 microscope equipped with a Colibri 2 diode illumination system (Zeiss, Oberkochen, Germany) and a CoolSnap HQ2 camera (Photometrics, Tucson, AZ, USA). A glass sphere of diameter 10 μm was glued by micromanipulation (using a homemade micropipette/biomembrane force probe setup) to a gold-coated triangle-shaped MLCT cantilever (Bruker Instruments, Billerica, MA, USA), using UV polymerizable glue (Dymax OP-29) in order to measure cell mechanics on similar scales as in the microindentation experiments. The decorated AFM cantilever was calibrated in situ prior to the experiments using the thermal noise method implemented in the JPK SPM control software and found to be 11.5 nN/μm, compatible with the nominal data provided by the manufacturer (10 nN/μm). The approach and retract speeds of the indenter were 1 μm/s over a distance of 5 μm and the maximal applied force was set between 3 and 6 nN. The acquisition frequency was set at 1024 Hz.

A JPK Nanowizard 1 AFM (JPK Instruments Ltd, Germany) was used to obtain example 50 × 50 µm, 10 × 10 µm and 3 × 3 µm AFM images from each biofilm covered HAP disc under PBS conditions. These were conducted using MSNL-10 cantilevers (Bruker Ltd, France) after 1 hour in PBS.