Sigma sem

X-ray diffraction (XRD) were tested on Bruker SmartLab SE X-ray powder diffractometer. UV–vis spectra were tested on Shimadzu Cary 5000 UV–vis spectrophotometer. Transmission electron microscopy (TEM) photographs were acquired on JEM-1400, while elemental data obtained using Tecnai F30. Scanning electron microscopy (SEM) photographs were characterized using ZEISS Sigma SEM at 15 kV. Hydrodynamic diameter and ζ potential were determined via DLS on Malvern ZEN 3600. Reactive oxygen species was measured with ESR (EMX Bruker-10/12). Fluorescence imaging was recorded using a Leica DM 6000 B, and bacterial counts and area calculations were performed using NIH ImageJ software.

In order to determine the composition of the materials used in the M. bucklandii dentary, samples of plaster from across the specimen were taken and subjected to EDX analysis. A total of seven samples of plaster material from across the specimen based upon their known locations from the CT data were taken, five from one of the suspected plaster materials (M1) and the two from the other (M2). A smaller number of samples were taken from this second material on the advice of the museum conservator, who did not wish to carry out any further destructive sampling on this very visible part of the specimen. These were then affixed to an SEM stub via carbon tape and gold-coated to a thickness of 5 nm. The samples were placed in a Zeiss Sigma SEM at AAMC (WMG-University of Warwick) and their composition analysed using EDX mapping.
A further, larger sample was taken from the plaster of the ventro-posterior portion of the dentary (M1) to explore the bulk composition of the plaster material. This sample was cut and mounted on a glass microscope slide in order for its composition to be examined via light microscopy and X-ray fluorescence spectroscopy (XRF). XRF imaging was done using a Bruker M4 Tornado at AAMC and the slide was examined under plane-polarized and cross-polarized light using a standard petrological microscope to confirm the results of the analysis.

HeLa or A549 cells were seeded in 24 well plates containing 5 × 7 mm silicon chip specimen supports (Ted Pella, Inc., Redding, CA, USA) at 5 × 10⁴ cells per well. Cells were then incubated with 10 µg/mL MSN for 24 h, and then processed for SEM imaging as previously described [13 (link)]. Secondary electron images were acquired under high vacuum, at 20 kV with a spot size of 5, using an FEI Quanta 3D FEG, (FEI, Hillsboro, OR, USA). Alternatively, images were acquired using a Sigma SEM (Carl Zeiss Microscopy, Göttingen, Germany) at 1 kV. Prior to imaging, dehydrated samples were sputter-coated with approximately 5 nm gold-palladium using a ACE600 coater (Leica Microsystems, Wetzlar, Germany). Select images have been pseudo-colored using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA, USA) and gamma levels adjusted to enhance image contrast and brightness.

Immediately after filtration, filters dehydrated in an ethanol series of 30, 50, 70, 80, 90, and 100% (vol/vol) ethanol (dilutions were in deionized water) for 10 min each. Samples were then dried, mounted on stubs with carbon tape, and coated with 5-nm gold. Cells were obtained on a Zeiss Sigma SEM by using a SE2 detector (2 to 2.5 kV, WD = 8 mm).

For electron microscopy, tissue was fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer. For transmission EM (TEM) the tissue was post-fixed in osmium tetroxide, dehydrated in acetone and embedded in epoxy resin. Ultrathin sections (70 nm) were stained with uranyl acetate and lead citrate, and viewed on a CM100 TEM. For serial block face scanning EM (SBFSEM), tissue was incubated in a series of heavy metal solutions before being dehydrated and embedded in hard resin. The resin blocks were glued onto an aluminium pin and placed into a Zeiss Sigma SEM incorporating the Gatan 3view system, which allows sectioning of the block in situ and the collection of a series of images in the z direction. A region containing cone cells was imaged at ×1058 magnification (1750×3500 pixel scan), which gave a pixel resolution of ∼20 nm. Section thickness was 70 nm in the z direction. In the resulting z stack, the cone cells were segmented semi-manually using the watershed brush tool in Microscopy Image Browser (MIB, University of Helsinki, Finland). The segmentations were imported into Amira (FEI) for construction of the 3D model.

Forty biochar particles were extracted from each treatment and examined using a Zeiss Sigma SEM. Detailed analysis of five particles was carried out using a Bruker X-ray Dispersive analyzer (EDS). A Cs-corrected FEI Titan 80/300 STEM, working at 80 keV and equipped with a Gatan imaging filter Tridiem and an EDS analyzer, was used to determine the structure and composition of the organo-mineral clusters that had accumulated on the surface of the aged biochar. Twenty biochar particles were sonicated in ethanol and a sample placed on a lacey carbon grind^{35 (link)}. Detailed examination of two clusters was carried out using EELS and EDS^{35 (link)}.