Animals were perfused transcardially with 2.5% glutaraldehyde and 2% PFA in 0.1 M PBS. After post fixation for 24 h, nerves were isolated and stained with 1% osmium tetroxide reduced in 1.5% potassium ferrocyanide. Tissues were washed in four changes of 0.1 M sodium cacodylate buffer, dehydrated in an ascending ethanol series and two changes of 100% propylene oxide, infiltrated with Araldite 502/EMbed812 resin (Electron Microscopy Sciences), and finally polymerized at 60 °C for 48 h. Tissue blocks were oriented to cut either cross or lateral ultrathin sections (~60 nm). Sections were collected on pioloform-coated slot grids and stained with 2% uranyl acetate and Reynold’s lead citrate. Electron micrographs were obtained with a JEM-1400 transmission electron microscope (JEOL) operating at 120 kV. Micrographs were collected with an Ultrascan 1000 camera (Gatan) at a resolution of 1.6 nm per pixel.
Ultrascan 1000 camera
Manufactured by Ametek
Sourced in United States
The UltraScan 1000 is a high-performance camera designed for laboratory applications. It features a sensitive image sensor capable of capturing detailed images. The camera is optimized for use in controlled environments and provides reliable performance for various scientific and research tasks.
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Lab products found in correlation
Tecnai g2 20 twin 200 kv lab6 tem, by Thermo Fisher Scientific (4 mentions)
Jem 1400 transmission electron microscope, by JEOL (2 mentions)
Ultracut 7 ultramicrotome, by Leica (2 mentions)
Carbon support film, by Quantifoil (2 mentions)
Jem 1400 tem, by JEOL (2 mentions)
Formvar coated copper slot grids, by SPI Supplies (2 mentions)
Em utc ultramicrotome, by Leica (2 mentions)
Formvar coated slot grids, by SPI Supplies (2 mentions)
Lead citrate, by Leica (2 mentions)
Tecnai 20, by Thermo Fisher Scientific (1 mentions)
Fei tecnai g2 20 twin 200 kv lab6 tem, by Thermo Fisher Scientific (1 mentions)
Em pact2, by Leica (1 mentions)
2100 tem instrument, by JEOL (1 mentions)
Em uc7 ultramicrotome, by Leica (1 mentions)
Xeuss 3.0 instrument, by Xenocs (1 mentions)
Empyrean x ray diffractometer, by Malvern Panalytical (1 mentions)
V 770 spectrophotometer, by Jasco (1 mentions)
Fluorolog 3, by Horiba (1 mentions)
Tecnai t12 g2 tem, by Thermo Fisher Scientific (1 mentions)
Lab6 electron gun, by Ametek (1 mentions)
16 protocols using ultrascan 1000 camera
1
Ultrastructural Analysis of Peripheral Nerves
2
Quantitative Cristae Ultrastructure Analysis
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Cells were washed in PBS, fixed with 2.5% glutardialdehyde and 2% formaldehyde in a buffered solution and postfixed in 1% osmium tetraoxide that had been reduced with 1% potassiumhexacyanoferrate34 (link). The cells were dehydrated in an ascending ethanol series, embedded in TAAB embedding resin, and sectioned on a Leica Ultracut 7 ultramicrotome using a Diatome diamond knife. The sections were counter stained using platinum blue (IBIlabs) and lead citrate (Leica) and visualized in an FEI Tecnai 20 transmission electron microscope. They were photographed at ×27,000 magnification with a Gatan ultrascan 1000 camera. To quantitatively analyze the electron microscopically images a line was drawn in imageJ manually into the cristae starting with the cristae junction proceeding into the cristae volume as far as the image quality was sufficient for image quantification, the curvature of the cristae was not crossing the line or a maximum of 130-nm length was reached. Along the drawn line every 2 nm orthogonally line plots with a width of 10 nm were measured with an ImageJ macro starting from the CJ. Each line plot was halved and the position of the minimal intensity was determined for both sides. The distance of both minimal intensities was set as the distance between opposing sides of the cristae.
3
Transmission Electron Microscopy of Peptide Fibrils
4
Lignin Staining of Resin-Embedded Sections
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Thin (60 nm) resin-embedded sections were positioned on 0.5% Formvar-coated copper slot grids (SPI Supplies, West Chester, PA, USA). Grids were post-stained for 6 minutes with 2% aqueous uranyl acetate and for 6 minutes with 1% aqueous KMnO4 to selectively stain for lignins. Images were taken with a 4 mega-pixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA, USA) on a FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI, Hilsboro, OR, USA).
5
Cryo-Preservation and Embedding of Arabidopsis Stems for TEM
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Samples of Arabidopsis stem were prepared for transmission electron microscopy (TEM) by high-pressure freezing and freeze substitution according to the literature (Donohoe et al., 2006 (link)). Briefly, samples were cryo-preserved using high-pressure freezing in a Leica EM-Pact2 and freeze substituted in 1% OsO4 over several days in a Leica AFS2 (Leica, Wetzlar, Germany). Samples were infiltrated with Eponate 812 (EMS, Hatfield, PA) by incubating at room temperature for several h to overnight in increasing concentrations of resin (15, 30, 60, 90%, 3 × 100% resin, diluted in acetone). The samples were transferred to capsules and the resin polymerized in an oven at 60°C overnight. Resin embedded samples were sectioned to approximately 50 nm with a Diatome diamond knife on a Leica EM UTC ultramicrotome (Leica, Wetzlar, Germany). Sections were collected on 0.5% Formvar coated slot grids (SPI Supplies, West Chester, PA). Grids were post-stained for 4 min with 2% aqueous uranyl acetate and for 2 min with Reynold's lead citrate. Images were taken with a 4 mega-pixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA) on a FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI, Hillsboro, OR).
6
Negative Staining and Transmission Electron Microscopy
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A small portion
(ca. 1 mg) of the lyophilized solid was suspended in a 50% (v/v) ethanol/water
mixture. Negative staining was done by adapting standard procedures36 as follows. A 5 μL drop of the sample
suspension was placed on a 300-mesh nickel-covered carbon film TEM
grid (FCF300-Ni Formvar Carbon Film) from Electron Microscopy Sciences
(Hatfield, PA), allowed to evaporate for 30 s, and then blotted with
filter paper. A 5 μL drop of 1% uranyl acetate dye was then
placed on the grid and blotted immediately. A JEOL 2100 TEM instrument
(JEOL Ltd., Tokyo, Japan) was used with a LaB6 beam source,
a beam strength of 200 kV, and a current density of 30–60 pA/cm3. Images were captured with an Ultrascan 1000 camera (Gatan
Inc., Pleasanton, CA) and processed to derive lamellar spacings with
Digital Micrograph software (ver. 2.11.14.04.0, Gatan Inc.). At least
eight spacing measurements were made at different locations for each
of the self-assemblies and plant phellem tissues, allowing us to determine
mean values and standard errors for each value.
(ca. 1 mg) of the lyophilized solid was suspended in a 50% (v/v) ethanol/water
mixture. Negative staining was done by adapting standard procedures36 as follows. A 5 μL drop of the sample
suspension was placed on a 300-mesh nickel-covered carbon film TEM
grid (FCF300-Ni Formvar Carbon Film) from Electron Microscopy Sciences
(Hatfield, PA), allowed to evaporate for 30 s, and then blotted with
filter paper. A 5 μL drop of 1% uranyl acetate dye was then
placed on the grid and blotted immediately. A JEOL 2100 TEM instrument
(JEOL Ltd., Tokyo, Japan) was used with a LaB6 beam source,
a beam strength of 200 kV, and a current density of 30–60 pA/cm3. Images were captured with an Ultrascan 1000 camera (Gatan
Inc., Pleasanton, CA) and processed to derive lamellar spacings with
Digital Micrograph software (ver. 2.11.14.04.0, Gatan Inc.). At least
eight spacing measurements were made at different locations for each
of the self-assemblies and plant phellem tissues, allowing us to determine
mean values and standard errors for each value.
7
Ultrastructural Sample Preparation for TEM Imaging
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The methodology of sample preparation was referred to previous works [25 (link), 42 (link)]. Samples were fixed in 2.5 % (w/v) glutaraldehyde in 0.2 M pH 7.2 sodium phosphate buffer twice for 6 min (2 min on, 2 min off, 2 min on) at room temperature followed by thoroughly washing with phosphate buffer. Dehydration of samples was achieved by transferring to vials containing a graded water–ethanol series (10 % steps for 30–90 % each of 15 min, 100 % for 30 min). After dehydration, the samples were infiltrated with LR White resin in increasing resin concentrations of 15, 30, 60, and 90 % resin diluted in ethanol and three times in 100 % resin. The resin-infiltrated samples were transferred to gelatin capsules and polymerized at 60 °C overnight. LR White-embedded samples were sectioned to 60 nm with a Diatome diamond knife on a Leica EM UC7 ultramicrotome (Wetzlar, Germany). Sections were collected on formvar-coated grids. Grids were post-stained for 10 min with 1 % aqueous uranyl acetate and 5 min with Reynolds lead citrate. Images were taken with a Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA, USA) on an 80 kV JEM-1400 transmission electron microscope (JEOL, Japan).
8
Selective Lignin Staining for TEM
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Grids were post-stained for three minutes with 2% aqueous uranyl acetate and two minutes with 1% KMnO4 to selectively stain for lignins. Micrographs were captured with a four mega-pixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA) on a FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI, Hilsboro, OR).
9
Characterization of Purified Nanoparticles
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Energy-dispersive X-ray spectroscopy (EDS) and transmission electron microscopy (TEM) images were obtained with a JEOL 2100F equipped with a Gatan ultrascan 1000 camera operating at 200 kV microscope. Samples were prepared by evaporation of diluted solutions of purified nanoparticles on carbon-coated copper grids. Absorbance spectra (ABS) were measured on a JASCO V-770 spectrophotometer. The emission was collected by a spectrofluorimeter FLUOROLOG-3 (by Horiba) using excitation wavelength 400 nm. X-ray diffraction analysis (XRD) was performed with an Empyrean X-ray diffractometer (Malvern Panalytical) using CuKα1,2 radiation in the Bragg–Brentano. Small-angle X-ray scattering (SAXS) measurements were carried out on a XENOCS Xeuss 3.0 instrument. Dynamic light scattering (DLS) measurements were prepared by MALVERN ZETASIZER NANO-ZS device.
10
Transmission Electron Microscopy of Nanoparticles
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TEM images of the HEC coated nanoparticles were obtained with the use of a Philips CM20 Analytical TEM at 80 kV accelerating voltage. Samples were prepared by placing a drop of nanoparticles in aqueous suspension onto carbon coated grids for 1 minute and then exposed to a 1% uranyl acetate solution before being dried and placed in the instrument.
Vitrified specimens were prepared at a controlled temperature and at water saturation in the Vitrobot (FEI, Netherlands), and kept in liquid nitrogen until examination. Cryo-TEM analysis was done with a Tecnai T12 G2 TEM (FEI, Netherlands) operating at 120 kV. Images were recorded digitally on a Gatan UltraScan 1000 camera using the DigitalMicrograph software (Gatan, U.K.). Images are recorded in the low-dose imaging mode to minimize beam exposure and electron-beam radiation damage.20,21 (link)
Vitrified specimens were prepared at a controlled temperature and at water saturation in the Vitrobot (FEI, Netherlands), and kept in liquid nitrogen until examination. Cryo-TEM analysis was done with a Tecnai T12 G2 TEM (FEI, Netherlands) operating at 120 kV. Images were recorded digitally on a Gatan UltraScan 1000 camera using the DigitalMicrograph software (Gatan, U.K.). Images are recorded in the low-dose imaging mode to minimize beam exposure and electron-beam radiation damage.20,21 (link)
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