Nanoscope 5 controller and e scanner

About 120–150 µm thick sections of live rat tibia bones were obtained by vibratome (Thermo Fisher Scientific) and mounted onto glass discs using silicone grease from the Bruker fluid cell accessory kit (Bruker). The force spectroscopy measurements were performed using a MultiMode eight atomic force microscope with a Nanoscope V controller and E scanner (Bruker). The regions of cells of interest for the acquisition of force-distance curves were selected under the optical microscope in combination with the AFM instrument.
The force-distance curves were acquired employing CP-PNP-BSG colloidal probes (NanoandMore GmbH, Germany) with a 5 µm borosilicate glass microsphere attached to the 200 µm cantilever. The spring constants of the cantilevers (measured by the thermal tune procedure) were 0.06–0.09 N/m.
All measurements were conducted at 25°C and all tissue was handled in DMEM/F12 HEPES-containing medium on ice. At least 70 individual force-distance curves were acquired for each type of cell by ramping over the surface and a total of 30 cells were measured from three different animals. These force-distance curves were processed with the NanoScope Analysis v.1.10 software (Bruker). Utilizing retract curves, the elastic modulus E was extracted from these force-distance curves by fitting in accordance with the Hertzian model of contact mechanics.

Were acquired on air in the PeakForce Tapping® Mode, using a MultiMode 8 atomic force microscope with a Nanoscope V controller and E scanner (Bruker). AFM imaging was conducted with RTESPA-300 probes (Bruker) with a nominal spring constant of 40 N/m, nominal frequency of 300 kHz and a nominal tip radius of 8 nm. 10 × 10 µm images were obtained at a scan rate of 1 Hz and a 512 × 512 pixels resolution. The raw AFM images were processed using the NanoScope Analysis v.1.10 software (Bruker).

AFM images were acquired from the tissue sections mounted on standard microscope slides, deparaffinized and left unstained and non-coverslipped. The imaging was performed on air in the PeakForce Tapping® mode, using a MultiMode 8 atomic force microscope with a Nanoscope V controller and E scanner (Bruker, USA). The regions of interest for AFM scanning were selected following the preliminary histological assignment and coordinated to the sample’s view in the optical microscope combined with the AFM instrument. At least 5 images from different regions of each section were acquired. AFM imaging was conducted with RTESPA-150 and -300 probes (Bruker) with a nominal spring constant of 5 and 40 N/m, nominal frequency of 150 and 300 kHz, respectively, and a nominal tip radius of 8 nm. Detailed 5 × 5 μm images were obtained at a scan rate of 1 Hz and a 512 × 512 pixels resolution. The raw AFM images were processed using the NanoScope Analysis v.1.8 software (Bruker, USA).