Cypher atomic force microscope

AFM scans were performed under tapping mode in air using a Cypher atomic force microscope (Asylum Research, Goleta, CA). Samples were imaged using a force constant of 0.2 N m^–1, scan rate of 2.44 Hz and scan size of 4 μm × 4 μm. Images were processed and analyzed for Root Mean Square (RMS) surface roughness using Igor Pro software (WaveMetrics, Lake Oswego, OR).

For examination with atomic force microscopy, 25–40 µL of the culture was drop-cast onto a conductive 35 nm platinum-coated silicon wafer as previously described (15 (link)). After 12 min, the excess liquid was wicked off with filter paper, the sample was rinsed with deionized water, and the excess liquid was removed with filter paper. The samples were equilibrated with the chamber humidity (ca. 40%) and visualized with a Cypher atomic force microscope (Asylum Research, Oxford Instruments) as previously described (15 (link)). Initially, the cells and filaments were viewed under tapping mode (AC-air topography) with a Pt/Ir-coated tip (PtSi-FM, NanoWorld) at approximately 2.0 N/m spring force constant and 70 kHz resonance frequency. After filament height determination, contact mode was engaged with the tip gently placed on top of the filament (force 30 ± 1.1 nN) as the translatable top electrode. Quadruplicate amplitude voltage sweeping of ±0.4 V at a frequency of 0.99 Hz was applied and averaged for I–V response curves. The conductance of the filament was calculated using the linear slope between 0.2 V and −0.2 V in the I–V response.

AFM imaging
was done with an Asylum Research Cypher atomic force microscope (Asylum
Research Santa Barbara, CA, USA). Measurements were performed in liquid
using the AC mode. A cantilever “BioLever mini” (Olympus
BL-AC40TS-C2) with a resonance frequency of about 25 kHz in water
and a stiffness of 0.09 N m^–1 has been used. A 10
μL portion of recrystallized S-layer sheets was dropped on freshly
cleaved mica attached to a steel sample puck. They were left undisturbed
for 5 min to promote adhesion. Most of the supernatant was removed
with a pipet and replaced by a droplet of recrystallization buffer
(1.5 mM Tris, 10 mM CaCl₂, pH 8) to keep the sample in
liquid upon transfer to a sample stage on AFM and following the measurement.
The temperature was kept at 25 °C, scan frequency at 0.5 Hz,
and scan angle at 90°.

SEM was used to investigate the morphology of the samples. Bar specimens of PLA-c, PLA-g-TPCS, PLA-GRH, and PLA-g-TPCS-GRH blends were produced by compression molding using pellets from the twin-screw extruder. Bars of PLA-g-TPCS and PLA-g-TPCS-GRH were immersed in liquid nitrogen for ~3 min, then fractured by hand, treated with hydrochloric acid (6 N) for 6 h to remove the TPCS phase, and air dried for 12 h in a fume hood [17 (link)]. Finally, the samples were mounted on aluminum stubs using carbon suspension cement (SPI Supplies, West Chester, PA, USA). Samples of films evaluated before and after tensile testing were also mounted on aluminum stubs with high vacuum carbon tabs (SPI Supplies) and coated with iridium at a thickness of ~2.7 nm. Samples were examined in a JEOL 6610LV (tungsten hairpin emitter) and a JEOL 7500F (field emission emitter) scanning electron microscope (JEOL Ltd., Tokyo, Japan) at various magnifications at 10 and 3.0 kV, respectively.
AFM was conducted using a Cypher™ atomic force microscope (Oxford Instruments Asylum Research, Inc., Santa Barbara, CA, USA) in the contact mode. Roughness parameters, calculated as the root mean square (Rq) and average roughness (Ra), were determined for each type of film and were calculated from the Htr mode image. Images were obtained in the Dfr mode. The film area for the determination of roughness was 900 μm².