Mice were perfused with 4% PFA in 1x PBS, the inner ears were isolated, and the stapes footplate was removed. The ear was flushed with, then fixed overnight in 4% paraformaldehyde (PFA), and 2.5% glutaraldehyde in 1x PBS. After washing in ddH2O for 3X in 1-hour, the samples were post-fixed with 1% osmium tetroxide for approximately 1 hour. The sample was washed in PBS before decalcifying for 3–4 days in 0.25 M EDTA at 4°C with daily solution changes. The cochleae were microdissected, the tectorial membranes removed and gradually dehydrated using 30%, 50%, 70%, 80%, 90%, 100% ethanol, 2:1 ethanol/ hexamethyldisilazane (HMDS) (Thermo Scientific #A15139 AE), 1:2 ethanol/HMDS, and finally 100% HMDS. Samples were transferred to an open well plate in HMDS and allowed to air dry overnight in a fume hood. Samples were then mounted on aluminum stubs (Ted Pella #16111) using double-sided carbon tape (EMS #77817–12) and stored in a specimen mount holder (EMS #76510) and sealed in a desiccator until sputter-coated with Au/Pd (Emitech Sputter Coater K550). Samples were viewed and images captured under SEM (Hitachi S-4800- University of Iowa Central Microscopy Research Facilities). An accelerating voltage of 5-kV and 11-kV were used (Supplement Fig. 10 (S10) ). Images were compiled using CorelDRAW X7 graphic software.
K550 sputter coater
Manufactured by Emitech
Sourced in United Kingdom, Italy
The K550 sputter coater is a laboratory equipment used for the deposition of thin films onto various substrates. It utilizes a vacuum chamber and a high-voltage electrical field to sputter material from a target and deposit it onto the substrate. The core function of the K550 is to provide a controlled and consistent method for creating thin film coatings for various applications.
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Lab products found in correlation
S 4800, by Hitachi (2 mentions)
A15139 ae, by Thermo Fisher Scientific (2 mentions)
Smz1500, by Nikon (2 mentions)
Hp deepsee multi photon laser, by Spectra-Physics (2 mentions)
Spectrum one spectrometer, by PerkinElmer (2 mentions)
Auriga fib sem, by Zeiss (2 mentions)
Evo sem, by Zeiss (2 mentions)
S 4000 fe sem, by Hitachi (2 mentions)
S 2700 scanning electron microscope, by Hitachi (2 mentions)
Fei xl 30 feg high resolution sem, by Thermo Fisher Scientific (2 mentions)
Sylgard 184, by Dow (1 mentions)
Evo ma10, by Zeiss (1 mentions)
Nuclepore polycarbonate membrane, by Cytiva (1 mentions)
Whatman, by Cytiva (1 mentions)
2700 scanning electron microscope, by Hitachi (1 mentions)
Inca edx system, by Oxford Instruments (1 mentions)
S 4800 scanning electron microscope, by Hitachi (1 mentions)
S 4800 sem, by Hitachi (1 mentions)
Quanta 400, by Thermo Fisher Scientific (1 mentions)
S 2700, by Hitachi (1 mentions)
48 protocols using k550 sputter coater
1
Ultrastructural Analysis of Mouse Inner Ear
2
Engineered Extracellular Matrix Surfaces
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PDMS (Sylgard-184, Dow Corning) was mixed at a curing agent to elastomer ratio of 1:10, de-gassed, cast in a 10 cm2 Petri dish (no patterning, for adhesion assays) or on a nickel shim with nano-fabricated obstacles of 1.05 or 1.5 μm (Kelvin Nanotechnology, Glasgow, UK) and cured for 2 h at 60°C. Chemical modification of PDMS surfaces: UVO3 treatment for 30 min (Jelight) or gold sputtering at 25 mA for 1 min with a Sputter Coater K550 (EMITECH) with overnight coating with Collagen I (0.05 mg ml−1 in phosphate-buffered saline (PBS) pH 7.0), Collagen IV (0.05 mg ml−1 in PBS pH 7.0), poly-L-lysine (0.01% in ddH2O) or fibronectin (0.05 mg ml−1 in ddH2O) as indicated. Substrates were dried for 24 h at RT. Surface modification was validated by quantifying changes in contact angle using the sessile drop method on a goniometer (First Ten Angstroms FTA1000, V2.1), and by X-ray photoelectron spectroscopy of key samples (see Supplementary Methods). Glass substrates for asymmetry experiments were silanized by acid washing in 1.0 M HCl and treating with 5% dimethylsilane in xylene for 5 min before drying, further washing and sterilizing at 200°C. The mean contact angles [± standard deviation (SD)] of untreated or silanized glass were 68 ± 6.4° and 91 ± 4.5° respectively.
3
Scanning Electron Microscopy of Inner Ear
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Mice were perfused with 4% PFA in 1x PBS, the inner ears were isolated, and the stapes footplate was removed. The ear was flushed with, then fixed overnight in 4% paraformaldehyde (PFA) and 2.5% glutaraldehyde in 1x PBS. After washing in ddH2O for 3X in 1 h, the samples were post-fixed with 1% osmium tetroxide for approximately 1 h. The sample was washed in PBS before decalcifying for 3–4 days in 0.25 M EDTA at 4 °C with daily solution changes. The cochleae were microdissected, the tectorial membranes removed, and gradually dehydrated using 30%, 50%, 70%, 80%, 90%, 100% ethanol, 2:1 ethanol/ hexamethyldisilazane (HMDS) (Thermo Scientific #A15139 AE), 1:2 ethanol/HMDS, and finally 100% HMDS. Samples were transferred to an open well plate in HMDS and allowed to air dry overnight in a fume hood. Samples were then mounted on aluminum stubs (Ted Pella #16111) using double-sided carbon tape (EMS #77817-12) and stored in a specimen mount holder (EMS #76510) and sealed in a desiccator until sputter-coated with Au/Pd (Emitech Sputter Coater K550). Samples were viewed, and images were captured under SEM (Hitachi S-4800- University of Iowa Central Microscopy Research Facilities). An accelerating voltage of 5-kV and 11-kV was used (Supplement Fig. 10 (S10) ). Images were compiled using CorelDRAW X7 graphic software.
4
Morphological Analysis of Prepared Samples
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Morphology study of the prepared samples was carried out by SEM (ZEISS EVO MA 15, EVO-ZEISS, Cambridge, UK). The samples were gold-sputtered up to a thickness of 20 nm by means of an Emitech K-550 sputter coater (Ashford, Kent, UK). In order to perform the analysis, an accelerating voltage of 20.00 KV and a working distance of 15 mm was used. Scans were carried out at different magnifications ranging from 500× to 25,000×.
5
Adhesive Failure Analysis in Dental Restorations
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In addition, to observe the type of failure at the adhesive interfaces and post-curing adaptation of adhesive resin or luting cement in the different groups, the specimens were firstly visually examined under a stereomicroscope (Nikon H550L, Nikon, Tokyo, Japan) and then subjected to scanning electron microscopy (SEM) observation. Prior to SEM observation, all the specimens were gold-coated using a sputter coater in a vacuum evaporator (Emitech K550 Sputter Coater, New York, NY, USA). For the SEM evaluation, a low vacuum scanning electron microscope (Joel, Joel Co., Tokyo, Japan) with a voltage of 15 kV, using secondary electrons (SE), was used. Failures were classified as follows: A: Adhesive failure at the dentin–cement interface; C: Cohesive failure within the restorative material; M: Mixed failure at the dentin–cement interface and cement–restorative material interface.
6
Microscopic Specimen Preparation Techniques
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Some specimens were cleared in Nesbitt’s fluid, and after washing for one hour in 70% Ethyl alcohol, they were mounted in Hoyer’s medium for the compound microscope observation in a phase contrast and DIC. Some specimens were decapitated, immersed in a saturated solution of KOH and emptied before being mounted on the slides.
Some specimens were examined using a Scanning Electron Microscope (SEM). Specimens from 70% ethyl alcohol were slowly rehydrated in a decreasing series of concentrations (60%, 50%, 40%, 30%, 20%, 10%, distilled water) over 24 hours. Once in distilled water, they were fixed in 4% glutaraldehyde in a cacodylate buffer for 24 h and then transferred to sucrose 0.25 M for 24 h. The specimens were then dehydrated up to 100% ethyl alcohol. Complete desiccation was achieved using the CO2 critical point technique. Samples were then covered with a 16-nm thin layer of molecular gold using an Emitech K550 sputter coater. Observations were performed using a Zeiss Digital Scanning Microscope 940 A.
Some specimens were examined using a Scanning Electron Microscope (SEM). Specimens from 70% ethyl alcohol were slowly rehydrated in a decreasing series of concentrations (60%, 50%, 40%, 30%, 20%, 10%, distilled water) over 24 hours. Once in distilled water, they were fixed in 4% glutaraldehyde in a cacodylate buffer for 24 h and then transferred to sucrose 0.25 M for 24 h. The specimens were then dehydrated up to 100% ethyl alcohol. Complete desiccation was achieved using the CO2 critical point technique. Samples were then covered with a 16-nm thin layer of molecular gold using an Emitech K550 sputter coater. Observations were performed using a Zeiss Digital Scanning Microscope 940 A.
7
Scaffold Morphology Analysis by FEG-SEM
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Scaffold morphology was analyzed by FEG-SEM (LEO Supra 1535). Samples were mounted on aluminum stubs, coated with a conductive layer of sputtered gold (Emitech K550 sputter coater) and observed at 5 kV accelerating voltage. Average filament diameter and spacing were calculated from SEM images (IMAGE J; National Institutes of Health, Bethesda, MD, USA) and expressed as a mean value ± standard deviation (n>20).
8
Scanning Electron Microscopy of Plant Tissues
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Samples of flower pedicels, bracts, and receptacles (each object n = 6 from different flowers and trees) were fixed in 4% (v/v) glutaraldehyde in 0.1 M phosphate buffer with pH 7.0. Next, the samples were dehydrated in an ethanol series, critical point dried in liquid CO2 (Bal-Tec CPD 030 critical point dryer), and coated with gold–palladium with an EMITECH K 550 × sputter coater. Finally, the samples were viewed under a TESCAN/VEGA LMU scanning electron microscope at an accelerating voltage of 30 kV.
9
Chromium Toxicity in Phototrophic Microbes
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Phototrophic microorganisms cultures were contaminated at different Cr(NO3)3 concentrations, 1, 5, 10, 25, 50, 100, and 200 μM Cr(III), and incubated under the same conditions as mentioned above for a period of 9 days.
For SEM analysis, cultures were filtrated in Nuclepore polycarbonate membranes (Whatman, Ltd.) and then were fixed in 2.5% glutaraldehyde diluted in Millonig phosphate buffer (0.1 M pH 4) at 4°C for 2 hours and washed four times in the same buffer, dehydrated in increasing concentrations of ethanol (30%, 50%, 70%, 90%, and 100%), and dried by critical-point (CPD 030 Critical Point Drier, BAL-TEC GmbH, 58579 Schalksmühle). Finally, samples were mounted on aluminium metal stubs and coated with a 5 μm gold layer (K550 Sputter Coater, Emitech, Ashford, UK) for better image contrast. A Zeiss EVOMA 10 scanning electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany) was used to view the images.
For EDX microanalysis, cells were homogenously distributed and filtered on polycarbonate membrane filters. These filters were fixed, dehydrated, and dried by critical-point drying and then coated with gold. An EDX spectrophotometer Link Isis-200 (Oxford Instruments, Bucks, England) coupled to the microscope operating at 20 kV was used. Finally, EDX-SEM spectra from individual cells were obtained.
For SEM analysis, cultures were filtrated in Nuclepore polycarbonate membranes (Whatman, Ltd.) and then were fixed in 2.5% glutaraldehyde diluted in Millonig phosphate buffer (0.1 M pH 4) at 4°C for 2 hours and washed four times in the same buffer, dehydrated in increasing concentrations of ethanol (30%, 50%, 70%, 90%, and 100%), and dried by critical-point (CPD 030 Critical Point Drier, BAL-TEC GmbH, 58579 Schalksmühle). Finally, samples were mounted on aluminium metal stubs and coated with a 5 μm gold layer (K550 Sputter Coater, Emitech, Ashford, UK) for better image contrast. A Zeiss EVOMA 10 scanning electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany) was used to view the images.
For EDX microanalysis, cells were homogenously distributed and filtered on polycarbonate membrane filters. These filters were fixed, dehydrated, and dried by critical-point drying and then coated with gold. An EDX spectrophotometer Link Isis-200 (Oxford Instruments, Bucks, England) coupled to the microscope operating at 20 kV was used. Finally, EDX-SEM spectra from individual cells were obtained.
10
Scanning Electron Microscopy of C. albicans
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