Multimode 8 afm

Conidial surfaces were analyzed by AFM, using a Multimode VIII AFM (Bruker, Santa Barbara, CA). A. fumigatus conidia were immobilized by mechanically trapping them into porous polycarbonate membranes with a pore size similar to the conidium diameter (it4ip SA, Belgium). After filtering a concentrated suspension of conidia, the filter was rinsed with deionized water, carefully cut and attached to a metallic puck using double-sided sticky tape. The mounted sample was then transferred to the AFM liquid cell. Images were performed in contact mode under minimal applied force, using oxide-sharpened microfabricated Si₃N₄ tips (MSCT, Bruker) with a nominal spring constant of 0.01 N/m. The surface coverage of the amorphous layer and the rodlets were determined visually using ImageJ software.

Nanomechanical mapping of non-porous and porous PCL scaffolds was performed in PeakForce QNM (PF-QNM) mode^{50 ,51 (link)} on a Bruker MultiMode VIII AFM with controller Nanoscope V at ambient conditions in tapping mode. The samples were investigated using a rectangular silicon cantilever with nominal spring constant of 3 N/m. The cantilever was calibrated on the calibration samples (Bruker, USA) - typically low-density polyethylene and polystyrene with Young’s moduli ranging from 100 MPa to 2 GPa (polyethylene) and from 1 to 20 GPa (polystyrene). The spring constant was measured by a thermal tuning method. The oscillation frequency of the Z-piezo was 1 kHz. The tip radius was obtained by tapping mode imaging of a Bruker reference tip-check sample and then analysed by commercial Nanoscope software. The Young’s modulus, E, is obtained by fitting the unloading curve using the Derjaguin, Muller, Toropov (DMT) model^{52 (link)}, which takes into account the adhesive force between the tip and the surface. The analysis of the Derjaguin-Mueller-Toporov (DMT) modulus was performed by the software Nanoscope Analysis.

The cell morphology of Xoo was observed using atomic force microscopy with a previously described method with some modification (Tang et al., 2020 (link)). Cell preparations with 1 × 10⁹ CFU/mL were obtained as described in the method of TEM observation. After treated with 54 μg/mL TKA at 28°C for 24 h, 2.5 μL cell preparation was deposited onto freshly cleaved mica and was dried in a chamber at room temperature. Atomic force microscopy was performed using a Multimode VIII AFM with Nanoscope V controller (Bruker AXS, Germany), and images were pictured in the scanasyst mode under air condition. Methanol (0.3%, v/v) treatment was used as the negative control. At least 50 Xoo cells were detected in each treatment. Three replicates were performed in each treatment, and the experiment was repeated three times.

A drop (1.5 μl) of sacculi suspension was spread onto freshly cleaved mica and air-dried. AFM imaging was carried out using a Multimode VIII AFM with a Nanoscope V controller (Bruker, USA). Silicon cantilevers (XSC11/AI BS, MikroMasch) were used for imaging under ambient conditions. AFM imaging was carried out in ScanAsyst mode in air condition.

For AFM imaging, 30 µL NiCl₂ (10 mm) was deposited on freshly cleaved mica and left to adsorb for 10 min. The mica surface was washed with water and dried with nitrogen. Next, 3 µL of sample solution was deposited onto the mica and left for 3 min. Excess salt was removed using a drop of doubly distilled H₂O, and the sample was dried with nitrogen. AFM imaging was performed under air tapping mode using a Multimode VIII AFM (Bruker, Inc.). A “SCANASYST‐AIR” model cantilever (Bruker) with a spring constant of 0.4 N m⁻¹ and a tip radius of 2 nm was chosen for the measurements. The AFM images were analyzed using NanoScope analysis software.

Scanning Electron Microscopy. SEM images were collected on uncoated samples using a Phenom G2 and a Zeiss (LEO) 1530VP. The roots were fixed with glutaraldehyde 2.5% in PBS and then air dried. To obtain sections, roots were embedded into polydimethylsiloxane (PDMS) and cut. The root was sliced at the level of the mid-elongation zone, in between the apex and the stem.
Atomic Force Microscopy. Substrate surfaces used for AFM imaging were native Silicon Oxide (SiOx) functionalized with 3-Aminopropyltriethoxysilane (APTES). Briefly, the SiOx pieces were first cleaned by the sequential sonication in Acetone, Isopropyl alcohol, and milliQ water; then they were exposed to an oxygen plasma for 5 min. The fragments were then closed in a desiccator containing 10 ul APTES and 10 ul triethanolamine under a mild vacuum for 30 min; 5 ul of CNT solution was spotted onto the APTES-SiOx and dried. Images were performed using a Multimode VIII AFM (Bruker) equipped with a Nanoscope V controller and operated in Peakforce tapping mode; NSG01 probes (NT-MDT) were used. Images were processed by Gwyddion.