The morphology of the nanoporous alumina membranes, including top, bottom and cross-sections views, was studied by scanning electron microscopy (SEM) in a JEOL-5600 Scanning Microscope (Akishima, Tokyo, Japan). Prior to SEM observation, the membranes were coated with a thin gold layer deposited by sputtering (Polaron SC7620, Quorum Technologies, Laughton, UK), in order to improve their electrical conductivity. The geometrical parameters (nanopore size and interpore distance) were measured by computer assisted image analysis using ImageJ (v 1.51j8) [30 (link)].
Polaron sc7620
Manufactured by Quorum Technologies
Sourced in United Kingdom
The Polaron SC7620 is a sputter coater designed for the deposition of conductive and non-conductive thin films onto samples for scanning electron microscopy (SEM) and other applications. It features a rotary vacuum pump and digital thickness control.
Automatically generated - may contain errors
Lab products found in correlation
Jsm 6510 scanning electron microscope, by JEOL (1 mentions)
Synapt hdms mass spectrometer, by Waters Corporation (1 mentions)
Merlin, by Zeiss (1 mentions)
Freezone, by Labconco (1 mentions)
Synapt g1 hdms, by Waters Corporation (1 mentions)
Masslynx v4, by Waters Corporation (1 mentions)
7k mwco zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Spin column, by Bio-Rad (1 mentions)
Synapt g2 s mass spectrometer, by Waters Corporation (1 mentions)
Jsm 5500 sem, by JEOL (1 mentions)
11 protocols using polaron sc7620
1
Nanoporous Alumina Membrane Characterization
2
Native Mass Spectrometry of Protein Oligomers
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Spectra were acquired using a Synapt HDMS orthogonal acceleration quadrupole-time-of-flight mass spectrometer (Micromass UK Ltd, Waters Corp., Wilmslow, UK). Oligomer samples isolated from size-exclusion chromatography were button-dialyzed against 100 mm ammonium acetate, pH 6.8, whereas the fused Cp samples were analyzed in the same solution at pH 9.5. For the disassembly reaction analyzed without prior size-exclusion separation, fused Cp capsids were dialyzed into 4 m urea, 50 mm ammonium acetate, pH 9.5, 5 mm DTT, and incubated at 4 °C for ∼16 h. The sample was then dialyzed into 50 mm ammonium acetate, pH 9.5, 5 mm DTT. The samples were electrosprayed from gold/platinum-plated borosilicate capillaries fabricated in-house using a P-97 micropipette puller (Sutter Instrument Company, Novato, CA) and a sputter coater (Polaron SC7620; Quorum Technologies Ltd, Kent, UK). The electrospray capillary voltage was set at 1.7 kV, and the sample cone voltage at 40–60 V. To improve the resolution of the oligomer analyte signals, the voltage of the Transfer region of the mass spectrometer was increased to 50 V. The instrument had a source pressure of 3 mbar and a Trap gas (argon) flow rate of 2 ml min−1. Data were processed using the MassLynx (v 4.1) suite of software programs supplied with the instrument.
3
SEM Sample Preparation Protocol
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For SEM, SSC were dried by a graded ethanol series (40%, 50%, 60%, 70%, 80%, 90%, 100%, 15 min each) and by increasing ethanol–hexamethyldisilazane (HMDS) series up to 100% (33%, 66%, 100%—1 h each). Subsequently, the specimens were sputter coated with Pd–Au using a Polaron SC7620 sputter coater (Quorum Technologies Ltd., East Grinstead, UK), and examined using a JEOL JSM-6510 scanning electron microscope (Jeol GmbH, Eching/Munich, Germany).
4
Exosome Structural Analysis by SEM
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Pellets containing exosomes were fixed in a 2% EM-grade paraformaldehyde aqueous solution and then dehydrated in ethanol and acetone. Samples were mounted on a scanning electron microscopy (SEM) stage with carbon paste. To make the surface conductive, a coating of 2–5 nm gold–palladium alloy was applied by sputtering (Polaron SC7620; Quorum Technologies, Laughton, UK) using argon as the gas for plasma before imaging by SEM using a ZEISS EVO40 microscope (Carl Zeiss AG, Jena, Germany). Microscopy was performed under low beam energies (10.0 kV), mag = 50,000 KX.
5
Native Mass Spectrometry of Proteins
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All nanoESI-TWIMS-MS protein measurements were carried out using a Synapt HDMS mass spectrometer (Waters Corp., Wilmslow, UK). Samples were introduced to the mass spectrometer using in-house pulled borosilicate capillaries (Sutter Instrument Co., Novato, CA, USA) coated with palladium using a sputter coater (Polaron SC7620; Quorum Technologies Ltd., Kent, UK). All protein samples were analyzed in positive ESI mode. The m/z scale was calibrated using 10 mg/mL aqueous caesium iodide (CsI) clusters across the acquisition range (typically m/z 500–15,000).
Protein samples were dialyzed into 150 mM aqueous ammonium acetate before being infused into the Synapt HDMS instrument. nESI-MS and nESI-TWIMS-MS experiments were conducted under the following settings: capillary voltage, 1.5 kV; sample cone, 30 V; extraction cone, 4 V; source temperature, 60–80 °C; backing pressure, 3.0–5.0 mBar; trap voltage, 10–40 V; trap/transfer gas flow, 1.5 mL/min; IMS nitrogen gas flow, 20 mL/min, IMS wave height (ramped), 5–30 V; and traveling wave speed, 300 ms.
All data were processed and analyzed with the MassLynx v4.1 and Driftscope software, supplied with the mass spectrometer.
Protein samples were dialyzed into 150 mM aqueous ammonium acetate before being infused into the Synapt HDMS instrument. nESI-MS and nESI-TWIMS-MS experiments were conducted under the following settings: capillary voltage, 1.5 kV; sample cone, 30 V; extraction cone, 4 V; source temperature, 60–80 °C; backing pressure, 3.0–5.0 mBar; trap voltage, 10–40 V; trap/transfer gas flow, 1.5 mL/min; IMS nitrogen gas flow, 20 mL/min, IMS wave height (ramped), 5–30 V; and traveling wave speed, 300 ms.
All data were processed and analyzed with the MassLynx v4.1 and Driftscope software, supplied with the mass spectrometer.
6
Arsenic Impact on Tetrahymena Morphology
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The effects of different As species on the morphological characteristics of T. pyriformis cells were identified by performing scanning electron microscopy on cells cultured in 40 μM Na3AsO4 in media containing P concentrations at 3 or 30 mg·L−1. As in the experiment described above, the cells were incubated for 21 h, then arsenate was added and the cells were cultured at 25 °C for 20 h. Each culture was then centrifuged and the supernatant discarded. The cells were fixed with 2.5% glutaric dialdehyde overnight, then washed with 0.1 M phosphate buffer and dehydrated using ethanol. Each cell sample was then dried and coated with metal using a Polaron SC7620 sputter coater (Quorum Technologies, Ashford, UK), then observed using a Hitachi S-2000N scanning electron microscope (SEM) (Hitachi High-Technologies, Tokyo, Japan).
7
Structural Analysis of Hydrogel Pores
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To visualize pores in the obtained gels, cross-sections of samples were analyzed using a scanning electron microscope (SEM). The SEM images were taken with a Zeiss Merlin (Zeiss, Jena, Germany) field-emission instrument. Before taking the micrographs, the hydrogel samples were dried using the lyophilization method. The samples were first frozen in liquid nitrogen to maintain their porous structure and then freeze-dried at −82 °C under 0.05 mbar pressure in a Lyophilizer Labconco FreeZone apparatus (Labconco, Kansas City, MO, USA). Before the imaging, the samples were coated with a 3 nm layer of sputtered Au–Pd alloy using a Polaron SC7620 (Quorum, Hertfordshire, UK) mini sputter coater.
8
Native Mass Spectrometry of β2-Microglobulin
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Samples prepared
for native MS were
buffer exchanged twice into 150 mM ammonium acetate (pH 7.4) using
7k MWCO Zeba spin desalting columns (Thermo Scientific, Hemel Hempstead,
UK). The final sample was diluted to a concentration of 10 μM,
calculated based on the absorbance at 280 nm and the Beer–Lambert
law, using an extinction coefficient of 20065 M–1 cm–1 for both wild-type and D76N β2m. Samples were then loaded into borosilicate glass capillaries pulled
in-house (Sutter Instrument Company, Novato, CA) and coated with palladium
using a sputter coater (Polaron SC7620, Quorum Technologies Ltd.,
Kent, UK).
Spectra were acquired using a Synapt G1 HDMS (Waters
Corp., Wilmslow, UK) in positive ESI mode. MS and IMS settings were
as follows: cone voltage: 70 V, backing pressure: 2.1 mbar, T-wave
velocity: 300 ms–1, IMS T-wave height: 4–10
V ramp (100% cycle). Data were analyzed using MassLynx v4.1 and Driftscope
v3.0 software (Waters Corp., Wilmslow, UK).
for native MS were
buffer exchanged twice into 150 mM ammonium acetate (pH 7.4) using
7k MWCO Zeba spin desalting columns (Thermo Scientific, Hemel Hempstead,
UK). The final sample was diluted to a concentration of 10 μM,
calculated based on the absorbance at 280 nm and the Beer–Lambert
law, using an extinction coefficient of 20065 M–1 cm–1 for both wild-type and D76N β2m. Samples were then loaded into borosilicate glass capillaries pulled
in-house (Sutter Instrument Company, Novato, CA) and coated with palladium
using a sputter coater (Polaron SC7620, Quorum Technologies Ltd.,
Kent, UK).
Spectra were acquired using a Synapt G1 HDMS (Waters
Corp., Wilmslow, UK) in positive ESI mode. MS and IMS settings were
as follows: cone voltage: 70 V, backing pressure: 2.1 mbar, T-wave
velocity: 300 ms–1, IMS T-wave height: 4–10
V ramp (100% cycle). Data were analyzed using MassLynx v4.1 and Driftscope
v3.0 software (Waters Corp., Wilmslow, UK).
9
Structural RNA Analysis by nESI-TWIMS-MS
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All nanoESI-TWIMS-MS RNA measurements were carried out using a Synapt G2-S mass spectrometer (Waters Corp., Wilmslow, UK). Samples were introduced to the mass spectrometer using in-house pulled borosilicate capillaries (Sutter Instrument Company, Novato, CA, USA) coated with palladium using a sputter coater (Polaron SC7620; Quorum Technologies Ltd., Kent, UK). All RNA samples were analyzed in negative ESI mode. The m/z scale was calibrated using 10 mg/mL aqueous caesium iodide (CsI) clusters across the acquisition range (typically m/z 500–15,000).
On receipt, the two 35-nucleotide RNAs (PDB structures 2PCV and 2DRB) were diluted to 200 μM in RNAse-free MilliQ water (Millipore UK, Watford, UK), separated into 100 μL aliquots and stored at –80 °C. When ready for analysis, these samples were diluted with 50 mM ammonium acetate to a concentration of 40 μM, desalted using spin columns (BioRad, Hemel Hempstead, UK) and then diluted to 8 μM with 50 mM ammonium acetate. nESI-TWIMS-MS experiments were conducted under the following settings: capillary voltage, –0.8 kV; sample cone, 30 V; source temperature, 60–80 °C; IMS sheath gas, He 2.42e-2 bar; IMS buffer gas, Ar 3.01 bar; IMS wave height 40 V; IMS wave velocity 400–750 m/s per scan.
On receipt, the two 35-nucleotide RNAs (PDB structures 2PCV and 2DRB) were diluted to 200 μM in RNAse-free MilliQ water (Millipore UK, Watford, UK), separated into 100 μL aliquots and stored at –80 °C. When ready for analysis, these samples were diluted with 50 mM ammonium acetate to a concentration of 40 μM, desalted using spin columns (BioRad, Hemel Hempstead, UK) and then diluted to 8 μM with 50 mM ammonium acetate. nESI-TWIMS-MS experiments were conducted under the following settings: capillary voltage, –0.8 kV; sample cone, 30 V; source temperature, 60–80 °C; IMS sheath gas, He 2.42e-2 bar; IMS buffer gas, Ar 3.01 bar; IMS wave height 40 V; IMS wave velocity 400–750 m/s per scan.
10
SEM Analysis of Phytolith Diversity
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SEM was performed on extracted phytoliths from Sample E to demonstrate the variety of silica structures obtained in the isolation protocol. A sample was mounted on carbon tape on an aluminum stub and sputter coated (Polaron SC7620, Quorum Technologies, Newhaven, East Sussex, England) with gold/palladium to a thickness of ~3 nm. Microscopy was performed on a Phillips XL30 Field Emission Scanning Electron Microscope (FEI Company, Hillsboro, OR, USA) at an accelerating voltage of 2 kV. Planter E was chosen to demonstrate the phytolith morphology because it gave the highest yield of sample material and also exhibited a large variety of silica forms.
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