Scanasyst fluid cantilever

AFM experimentation was conducted as previously(Eskandarian et al., 2017 (link)). In short, polydimethylsiloxane (PDMS) – coated coverslips were prepared by spin-coating a mixture of PDMS at a ratio of 15:1 (elastomer:curing agent) with hexane (Sigma 296090) at a ratio of 1:10 (PDMS:hexane) (Koschwanez et al., 2009 (link); Thangawng et al., 2007 (link)). A 50 µl filtered (0.5 µm pore size PVDF filter – Millipore) aliquot of bacteria grown to mid-exponential phase and concentrated from 2 to 5 ml of culture was deposited onto the hydrophobic surface of a PDMS-coated coverslip and incubated for ~20 min to increase surface interactions between bacteria and the coverslip. 7H9 medium (~3 ml) was supplied to the sample so as to immerse the bacterial sample and the AFM cantilever in fluid. The AFM imaging mode, Peak Force QNM, was used to image bacteria with a Nanoscope five controller (Veeco Metrology) at a scan rate of 0.5 Hz and a maximum Z-range of 12 µm. A ScanAsyst fluid cantilever (Bruker) was used. Height, peak force error, DMT modulus, and log DMT modulus were recorded for all scanned images in the trace and retrace directions. Images were processed using Gwyddion (Department of Nanometrology, Czech Metrology Institute). ImageJ was used for extracting bacterial cell profiles in a tabular form.

The antheridia were treated with 30% sucrose for 10 min to remove turgor pressure and then attached to a glass slide using transparent nail polish. The antheridium was scanned using a BioScope Resolve atomic force microscope (Bruker, Billerica, MA, USA). The probe was a standard pyramidal silicon nitride ScanAsyst‐Fluid cantilever (Bruker) with 0.7 N m⁻¹ spring constant and 20 nm tip radius. The “PeakForce Quantitative Nanoscale Mechanical (QNM) in fluid” operation mode was used to record peak force error and the DMT modulus. Peak force frequency was set at 1 kHz and peak force set‐point at 3 nN. The images were acquired at 15 by 15 µm. The scanning rate was 0.5 Hz. The cell wall Young's modulus was measured using an indentation depth of 150 nm. Data were analyzed with NanoScope Analysis version 1.8. For statistical analysis of AFM, the position with the highest modulus in the outer walls was initially selected, and subsequently an 8‐µm × 8‐µm area surrounding this highest modulus position was selected. The average elastic modulus within this selected 8‐µm × 8‐µm area was then measured.

The fibre surface was investigated by digital microscopy with a digital microscope VHX-100 (Keyence, Osaka, Japan) and by scanning electron microscopy (SEM) with the scanning electron microscope FE-SEM Ultra 55 (Carl Zeiss SMT AG, Oberkochen, Germany) with an Everhart–Thornley detector. AFM measurements were carried out by a Dimension nanoscope (Bruker-Nano, USA) in the tapping mode under ambient conditions (ScanAsyst fluid + cantilevers, Bruker-Nano, Billerica, MA, USA).

Surface roughness of the goethite surface immobilized on Tempfix was measured using AFM in the ScanAsyst mode with ScanAsyst-Fluid cantilevers with 0.4 N m⁻¹ nominal spring constant (Bruker) in deionized water. The goethite surface was imaged at five randomly chosen positions and surface plots were made to provide a three-dimensional perspective of the surface, from which the average roughness (R_a) and root-mean-square (RMS) roughness (R_q) were calculated. The R_a is the average deviation of the height values from the mean line/plane, and similarly the R_q is the root-mean-square deviation from the mean/plane, i.e. the standard deviation from the mean.