Xe 100 atomic force microscope

An XE-100 atomic force microscope (Park Systems, Suwon, Korea) was used for the imaging of fibrils. Surface imaging was obtained in non-contact mode using silicon/aluminum coated cantilevers (SSS-NCHR 10M; Park Systems) 125 μm long with a resonance frequency of 204 to 397 kHz nominal force constant of 42 N/m and a typical tip radius 2 nm (<5 nm max). Here we used a low tip radius probe to improve measurements of fibril widths. The scan frequency was typically 0.5 Hz per line. When necessary, the AFM images were processed by flattening, in order to remove the background slope, and the contrast and brightness were adjusted. As done in [38 (link)], for sample preparation, muscovite mica with a surface area of ~1 cm² was used as the substratum. The mica was freshly cleaved using adhesive tape prior to each deposition in order to ensure its cleanliness. The dried samples were dissolved in 10 mM phosphate buffer, pH7. 2 μL aliquots of protein were deposited on the substrates and the samples were dried by evaporation at room temperature under a ventilated fume hood. For washed samples, two min after deposition, the surfaces were gently washed with deionized water. Finally, the samples were dried as described above.

Crystal shape and size were also investigated by atomic force microscopy (AFM). Briefly, 50 μl of CaOx crystals, obtained as mentioned above, were layered on glass surfaces and dried by nitrogen flow. The XE-100 atomic force microscope (AFM; PARK Systems Inc.) was used in this study; the instrument was equipped with a 50μm scanner controlled by XEP 1.8.1 software. The AFM was set in non-contact mode, with the X–Y stage in closed loop and high voltage modes. The Z scanner was set in closed loop and high voltage modes and resolution set to 1.8Å. The speed scan was 0.25Hz for large images and 1Hz for the 1x1μm acquisition. The tips used in the present study were the NCHR type with a nominal spring constant of 42 N/m and a typical resonant frequency between 250 and 300kHz. The data acquisition was performed in air at controlled temperature. In addition to topography, amplitude and phase signals were acquired. The amplitude signal was used as a unique drive for the Z feedback circuit, thus rendering the phase signal to exclusively depend on chemical and nano-mechanical proprieties of the surfaces. The phase signal evaluation (Phase Detection Microscopy PDM) was carried out randomly on CaOx particles by using a 1x1μm scanning area, with around 25 images for each condition analysed. AFM images were analysed using XEI software (PARK Systems Inc.).

AFM imaging was carried out by using an XE-100 atomic force microscope (Park systems Corporation, Suwon, Korea). The analyses were carried out in a true noncontact mode under ambient conditions using silicon cantilevers with a spring constant of 42 N m⁻¹ (PPP-NCHR, Park Systems Corp.) and images were recorded in topography mode. Images were collected from 15 to 20 arbitrary areas on the film and were recorded in 256 × 256 pixel resolution. Particles were then grouped at 5 nm intervals based on measured heights.