V shaped dnp silicon nitride cantilevers

For Scanning Electron Microscopy (SEM) characterization a JEOL JSM 6490LA microscope was employed, using a 15 kV accelerating voltage. Prior to SEM evaluation, samples were coated with gold. Then, the specimens obtained were mounted on aluminium stubs, with a double-stick carbon tape.
Single mycelium fibers were characterized in a liquid environment of phosphate buffered saline (PBS) by atomic force microscopy (AFM). A Nanowizard III AFM head (JPK Instruments, Germany) mounted on an Axio Observer D1 (Carl Zeiss, Germany) inverted optical microscope was employed. V shaped DNP silicon nitride cantilevers (Bruker, Massachussets, US) with a nominal spring constant 0.24 N/m, resonance frequency in air ranging from 40 kHz to 75 kHz and tip typical curvature radius of >20 nm were used. The spring constant of each cantilever was determined in situ using the thermal noise method. Images were acquired in quantitative imaging (QI) mode. A maximum force of 7 nN was applied on the sample and 256 × 256 force-distance (FD) curves were acquired per each image. Then, FD curves were converted into force-indentation (FI) and the stiffness was extracted from the curves slope through Hertzian fitting.

AFM images were acquired by using a Nanowizard III (JPK Instruments, Germany) mounted on an Axio Observer D1 (Carl Zeiss, Germany) inverted optical microscope. V-shaped DNP silicon nitride cantilevers (Bruker, MA, USA), with a nominal spring constant 0.24 N/m, resonance frequency in air ranging from 40 kHz to 75 kHz and tip typical curvature radius of 20-60 nm were used. Intermittent contact mode AFM images were acquired in the constant-amplitude mode, working in water with an oscillating frequency of 10-20 kHz. The amplitude setpoint was kept above 70% of free oscillation amplitude in all cases. OA and OB were administered to the sample under the AFM head at a final concentration of 12 μM and left standing for 30 min. The difference in thickness between ordered (L_β) and disordered (Lalpha) lipid domains (ΔZ) was determined by considering image height distributions: the values of height per each one of the 512×512 image data points were calculated by using the JPK Data Processing software (JPK Instruments, Germany). Raw data were plotted by using Origin (OriginLab, MA, USA) and the distributions were fitted to the sum of two Gaussian functions. The ΔZ value associated with each image was the separation between the peaks of the two Gaussian curves. This procedure was repeated for at least 10 different images for each experiment.