Phenom prox sem

For SEM imaging, a tiny amount of the white pellet was dispersed in ethanol and 50 µl of the suspension was dropped onto a silicon wafer that was fixed to a standard SEM stub followed by drying in air. The material was then sputter-coated with gold using Turbo sputter coater Emitech K585X Dual (UK) with a thickness about 10 nm. For EDS line scan and elemental mapping, 10 µl of the above suspension was pipetted onto a standard 200 mesh copper TEM grid covered by a 10 nm continuous carbon film. TEM grids were placed in an in-house made sample holder suitable for a SEM stage. SEM–EDS line scan and mapping were performed using SEM Quanta 3D FEG (Thermo Fisher Scientific, USA), at an acceleration voltage of 3 and 10 kV. EDS spot measurements were performed at an accelerating voltage of 10 kV using Phenom proX SEM (Thermo Fisher Scientific, USA). The mean diameters of areolae, and small pores were calculated via in house MATLAB scripts as shown in the Supplementary Figs S1, S2. In addition, length and width of the valves and morphological features such as transapical rib, cross extension, distance between neighboring areolae within an array (Da) and distance between two areolae of two parallel arrays (Dp) were measured using Gatan Digital Micrograph for at least 15 valves pre culture. The area of the valve was determined as an ellipse.

Boron and cerium were determined using inductively coupled plasma optical emission spectroscopy (ICP-OES) with a Varian 710-ES spectrometer (Varian, Mulgrave, Victoria, Australia). The following parameters were used: radio frequency (RF) power 1.0 kW, plasma flow 15 dm³/min, auxiliary flow 1.5 dm³/min, nebuliser pressure 200 kPa, pump rate 15 rpm and emission lines: λ = 208.956, λ = 249.678 and λ = 249.772 nm (for boron) and λ = 407.347, λ = 407.570, λ = 418.659 and λ = 446.021 nm (for cerium). Calibration curves were prepared using standard solutions in the ranges of 0.1–1.5 mg/dm³ and 0.1–5 mg/dm³ for boron and cerium, respectively. The changes in the chitosan structure were determined by a Fourier-transform infrared (FTIR) Spectrum Two spectrometer (Perkin Elmer, Waltham, MA, USA). The scanning electron microscope (SEM) micrographs of the chitosan bio-composite were produced using a Phenom ProX SEM (Phenom-World Bv, Eindhoven, The Netherlands). The phase compositions of the chitosan-based samples were determined using a Seifert 3003TT powder X-ray diffractometer with a Cu X-ray tube: kλ₁ = 1.540598 Å, kλ₂ = 1.544426 Å and kβ = 1.39225 Å (Seifert, Ahrensburg, Germany). Powder samples were analysed three times, and the XRD patterns presented in the manuscript were an average of the scans between 10 and 90° 2Theta.

The phase composition of samples was determined using a powder X-ray diffractometer (Seifert 3003TT) with a Cu X-ray tube (kλ1 = 1.540598 Å, kλ2 = 1.544426 Å, kβ = 139,225 Å). The powder samples were analyzed between 5° and 80° of 2Theta with 0.05° step. In order to validate the crystal structures, the X-ray diffraction patterns that were acquired were compared with the information that was collected from the Joint Committee on Powder Diffraction Standards (JCPDS) [28 (link)]. Morphological features of the sample surfaces were obtained by scanning electron microscope (SEM) images using a Phenom ProX SEM (Phenom-World BV, Eindhoven, The Netherlands). For the SEM imaging, the samples were coated with a thin layer of gold and mounted on a slab using double-sided tape. The elemental analysis of the samples was also carried out by energy dispersive X-ray spectroscopy (EDS) during SEM image acquisition (Phenom-World BV, Eindhoven, The Netherlands). The BET surface of the samples was measured using a Micromeritics ASAP 2020 adsorption analyzer (Micromeritics Inc., Norcross, GA, USA).

A ThermoFisher Scientific (Waltham, MA, USA) Nicolet iS 10 FTIR spectrometer was used for FTIR characterizations. 1D ¹H and ¹³C NMR were performed using a Bruker (Billerica, MA, USA) Avance 300 MHz NMR spectrometer, while 2D ¹H-¹H COSY and ¹H-¹³C HMBC NMR were performed on a Bruker (Billerica, MA, USA) Avance 600 MHz NMR. All NMR spectra were collected in D₂O. Thermogravimetric analysis was performed using a TA Instruments (New Castle, DE, USA) simultaneous thermal analyser (SDT 650) under nitrogen atmosphere. Data were collected from 30 to 800°C at a heating rate of 10°C min⁻¹ after water was removed through equilibration at 110°C for 4 min. Scanning electron microscopy (SEM) images were obtained in backscatter mode using a Phenom ProX SEM (Phenom-World B.V., Eindhoven, Netherlands).