The resin embedded bone-implant blocks were wet polished with 400–4000 grit SiC grinding paper. The samples were air-dried overnight prior to low-vacuum backscattered electron scanning electron microscopy (BSE-SEM) imaging in a Quanta 200 environmental SEM (FEI Company, The Netherlands) operated at 20 kV and 0.5 Torr water vapour pressure. Five test and control pairs (from the same animal) were used. For each sample, the first thread located below the level of the original cortical bone (first endosteal thread) was imaged in order to ensure that only de-novo-formed bone was being analysed.
Quanta 200 environmental sem
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands
The Quanta 200 environmental scanning electron microscope (ESEM) is a versatile imaging tool designed for high-resolution analysis of a wide range of materials and samples. It utilizes an environmental chamber that allows for the examination of specimens in their natural state, without the need for extensive sample preparation. The Quanta 200 ESEM provides high-quality, high-magnification images by using an electron beam to scan the surface of the sample.
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Lab products found in correlation
Phosphotungstic acid, by Merck Group (1 mentions)
Aqueous osmium tetroxide, by Agar Scientific (1 mentions)
Inca edx system, by Oxford Instruments (1 mentions)
Em cpd300 critical point dryer, by Leica (1 mentions)
E 1010 ion sputter, by Hitachi (1 mentions)
Vertex ft ir spectrometer, by Bruker (1 mentions)
Raman spectrophotometer, by Horiba (1 mentions)
7 protocols using quanta 200 environmental sem
1
Bone-Implant Interface Characterization
2
Collagen Fiber Diameter Quantification
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Gels were transferred to glass containers in 2X Karnovsky's fixative overnight with an equal volume of culture medium. After washing in phosphate buffered saline (PBS), samples were post-fixed in 1% aqueous osmium tetroxide (Agar Scientific, UK) for 1 hour. Following further washing in PBS, samples were dehydrated in graded ethanol solutions of 70, 80 and 90% for 1 hour each. For the final dehydration steps, 1% w/v phosphotungstic acid (Sigma-Aldrich, UK) in 100% ethanol was used for 30 mins followed by another 30 mins in 100% ethanol. Gels were then chemically dried using hexamethyldisilazane (HMDS) (Agar Scientific, UK) in a graded ethanol/HMDS series (1∶3, 1∶1 and 3∶1) before air-drying and mounting on a self-adhesive carbon pad. Gold/palladium was splattered onto the samples to give a coating of ∼70 Å thick. Images were acquired with a Quanta 200 environmental SEM (FEI Company, Netherlands) operated in high vacuum mode. Collagen fiber diameter was measured in three different fields of view using the line measure function in Image J at the mid point of the fiber length.
3
Iodine Staining for SEM Imaging
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Selected resin embedded blocks were polished using 800–2000 grit SiC paper. A stock solution of iodine in potassium iodide (Lugol solution, I2/KI, Sigma-Aldrich) was diluted 1:1 in ethanol, and pipetted (~1000 μL) directly onto polished resin embedded blocks for ~15 min. The stain was washed off with deionized water. Backscattered electron scanning electron microscopy (BSE-SEM) was performed in a Quanta 200 environmental SEM (FEI Company, The Netherlands) operated at 20 kV accelerating voltage, 1 Torr water vapour pressure, and 10 mm working distance.
4
Enamel Prism Visualization via SEM Imaging
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Backscattered electron (BSE) imaging and energy dispersive X-ray spectroscopy (EDX) were performed in a Quanta 200 environmental SEM (FEI Company, The Netherlands) equipped with an INCA EDX system (Oxford Instruments GmbH, Wiesbaden, Germany) operated at 1 Torr water vapour pressure, 20 kV accelerating voltage, 0–10 keV spectral energy range, and 10 mm working distance (NaOCl+ ; 1 spot per sample, n = 6). To visualise enamel prisms, deproteinised hemi-mandibles were etched with 85% H3PO4 (90 s at room temperature) and Au sputter coated (~ 15 nm thickness). Secondary electron (SE) imaging was performed in an Ultra 55 FEG SEM (Leo Electron Microscopy Ltd, UK) operated at 5 kV accelerating voltage.
5
Scanning Electron Microscopy of Colonized Rocks
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Small colonized rock fragments were broken off from sandstones and fixed in 3.0% formaldehyde, 1.5% glutaraldehyde, 5 mM Ca2+, 2.5% sucrose, in 0.1M sodium cacodylate, pH 7.4 at room temperature for 30 min. Afterward, samples were washed thrice in 0.1M sodium cacodylate, 10 min each. Samples were stained with 1% OsO4 in 0.1M sodium cacodylate in the dark for 45 min, followed by three quick washes in ddH2O. Stained samples were dehydrated in a cold ethanol gradient: 50, 75, 90, 95%, then three 15 min changes in 100% ethanol at room temperature, followed by a final dehydration in hexamethyldisilazane for 2 min. Finally, samples were coated with a 1.5 nm Pt layer before imaging in a FEI Quanta 200 Environmental SEM. Imaging was done at 15 kV, spot size 1.5, 6 mm working distance for high-magnification imaging and 20 kV, spot size 2.5, 10 mm working distance for low-magnification imaging.
6
SEM Imaging of S. aureus Biofilms
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S. aureus biofilm cell morphology was observed by SEM after octanoic acid or antibiotic treatments. The biofilm to be tested was cultured on a plastic film and fixed with 4% formaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (PBS), pH 7.0, at room temperature for 1 h. Biofilms were rinsed three times for 10 min with 0.1 M PBS and then fixed with 2% osmium tetroxide in PBS at room temperature for 1 h. The samples were then dehydrated through an alcohol concentration gradient (30%–100%) for 10 min at each concentration. Critical point drying was performed with a Leica EM CPD300 critical point dryer. A Hitachi E−1010 ion sputter was used for sample coating. A FEI Quanta 200 environmental SEM at 20 KV was used for observation and imaging.
7
Comprehensive Materials Characterization
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Scanning electron microscope (SEM) micrographs and energy dispersive X-ray (EDX) spectra were recorded using a FEI Quanta 200 Environmental SEM. Raman spectra were obtained at room temperature by a Raman Spectrophotometer Horiba Jobin-Yvon equipped with a liquid nitrogen-cooled CCD detector. FT-IR characterizations were obtained using a Bruker Vertex FT-IR spectrometer (Bruker, Germany) equipped with a Mercury cadmiumtelluride (MCT) detector and an attenuated total reflectance (ATR) germanium crystal.
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