Samples were fixed immediately upon recovery in 2.5% glutaraldehyde in PBS at 4°C for 48 h, then immersed in a 3% hydrogen peroxide solution for 48 h at room temperature (for bone marrow removal), and then rinsed with distilled water. Samples were then sonicated in a sonic device [13 ] in distilled water at room temperature, rinsed with distilled water, and dehydrated in acetone series. Samples were finally dried using a critical point dryer (Emitech K850, Emitech, Corato, Italy), mounted on aluminum stubs, platinum coated using an Emitech K 550 sputter coater (Emitech, Corato, Italy), and observed by a Hitachi FE SEM S 4000 operating at 7 kV. SEM micrographs were acquired with a DISS5 Digital Image Scanning System (point electronic, Germany).
Emitech k 550 sputter coater
Manufactured by Quorum Technologies
Sourced in United Kingdom
The Emitech K 550 is a sputter coater, a device used to deposit thin metallic films on samples. It is a versatile piece of lab equipment suitable for a range of applications that require a conductive coating on samples, such as scanning electron microscopy and material science research.
Automatically generated - may contain errors
Lab products found in correlation
Fei xl 30 feg high resolution sem, by Thermo Fisher Scientific (2 mentions)
Emitech k850, by Quorum Technologies (1 mentions)
S 4000 fe sem, by Hitachi (1 mentions)
S 3400n scanning electron microscope, by Hitachi (1 mentions)
Nano z, by Malvern Panalytical (1 mentions)
S 4800 microscope, by Hitachi (1 mentions)
Esem quanta 200 scanning electron microscope, by Thermo Fisher Scientific (1 mentions)
S 2700, by Hitachi (1 mentions)
Prism 6, by GraphPad (1 mentions)
7 protocols using emitech k 550 sputter coater
1
Ultrastructural Bone Marrow Analysis
2
Electrospun Composite Nanofibers Characterization
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The Hitachi S-3400N Scanning Electron Microscope (Hitachi, Tokyo, Japan) was used to examine the surface morphology of the electrospun composite nanofibers fabricated from gel-based blends of HW-DESs_PVA. For SEM observation, the electrospun nanofibers were previously cut into small sections, affixed onto aluminum stubs using Araldite glue, and then received a thin gold layer through an Emitech K550 sputter coater (Emitech Ltd., Ashford, UK) to heighten image contrast. To maintain consistency, all the samples were simultaneously sputtering coating for the same period of time, aiming to minimize variations introduced during this process. The SEM images were captured at a magnification of ×5000 and an accelerating voltage of 20 kV.
3
Cryogenic Specimen Preparation for SEM
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Fresh specimens were frozen in solidifying propane at approximately −188°C and fractured under liquid nitrogen at −196°C in order to obtain clean, untouched surfaces. The specimens were then thawed in Karnowski solution and dehydrated in graded ethanol and hexamethyldisilazane.
Other fragments were briefly washed in 0.1 M phosphate buffer, thermally treated at 400°C for 24 h to remove the cells and the soft matrix and allowed to slowly return to room temperature.
All specimens were mounted on appropriate stubs with a colloidal silver glue, coated with 10 nm gold–palladium in an Emitech K550 sputter‐coater (Emitech) and observed with a FEI XL‐30 FEG high‐resolution SEM (now Thermo Fisher). Images were directly obtained as 8bpp, 1424 × 968 TIFF files.
Other fragments were briefly washed in 0.1 M phosphate buffer, thermally treated at 400°C for 24 h to remove the cells and the soft matrix and allowed to slowly return to room temperature.
All specimens were mounted on appropriate stubs with a colloidal silver glue, coated with 10 nm gold–palladium in an Emitech K550 sputter‐coater (Emitech) and observed with a FEI XL‐30 FEG high‐resolution SEM (now Thermo Fisher). Images were directly obtained as 8bpp, 1424 × 968 TIFF files.
4
SEM Specimen Preparation Protocol
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Specimens for SEM were fixed in Karnowski’solution, dehydrated in graded ethanol and hexamethyldisilazane, mounted on appropriate stubs with a colloidal silver glue, gold coated with an Emitech K550 sputter-coater (Emitech, Montigny-le-Bretonneux, France) and observed with a FEI XL-30 FEG high resolution SEM (now Thermo Fisher, Waltham, MA, USA) operated in secondary electron imaging at an acceleration voltage of 7 kV. At least three fragments were observed for each sample. Images were directly obtained as 8bpp, 1424x968 TIFF files.
5
Microsphere Characterization by DLS and SEM
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Microsphere size distribution was determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern, Westborough, MA, USA). Chitosan-NTX microspheres were dispersed in absolute ethanol to prevent swelling and aggregation, while parameters of viscosity (1.061 cp), dielectric constant (24.3), and refractive index (1.361) for absolute ethanol [24 ,25 ,26 (link)] were adjusted on the instrument before analysis. Zeta potential was determined at 25 °C by laser doppler micro-electrophoresis technique.
Scanning electron microscopy (SEM) was performed to evaluate the microsphere shape and surface morphology using a Hitachi-S4800 microscope (Hitachi, Tokyo, Japan) at 5-kV acceleration voltage. Samples were prepared using double-coated carbon conductive tapes on stubs and were sputter-coated under vacuum before analysis with gold/platinum using an Emitech K550 sputter coater (Quorum Technologies Ltd, Laughton, UK). Fiji (ImageJ) software [27 ] was used to determine microsphere diameters of 100 randomly selected microspheres from the photomicrographs acquired with the SEM.
Scanning electron microscopy (SEM) was performed to evaluate the microsphere shape and surface morphology using a Hitachi-S4800 microscope (Hitachi, Tokyo, Japan) at 5-kV acceleration voltage. Samples were prepared using double-coated carbon conductive tapes on stubs and were sputter-coated under vacuum before analysis with gold/platinum using an Emitech K550 sputter coater (Quorum Technologies Ltd, Laughton, UK). Fiji (ImageJ) software [27 ] was used to determine microsphere diameters of 100 randomly selected microspheres from the photomicrographs acquired with the SEM.
6
Scanning Electron Microscopy of Ossicles
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Samples (four ossicles) were observed with the ESEM Quanta-200 Scanning Electron Microscope (Fei Company, Hillsboro, OR, USA). The samples were fixed for 20 min in 4% paraformaldehyde in PBS (pH 7.4) and dehydrated through a graded series of alcohol (50%, 75%, 95%, 100%) for 10 min each. The specimens were finally dried overnight and coated with gold-palladium (10 nm thickness) using an Emitech K550 sputter coater (Quorum Technologies, South Stour Avenue, Kent, UK). SEM images were collected under low and high vacuum conditions using a large field detector (LFD) and an Everhart Thornley detector (ETD), respectively. Finally, two selected samples were also analyzed via X-ray microprobe (X-ray energy dispersion spectroscopy, X-EDS) using the ESEM software INCA Suite (version 4.07, Oxford Instruments Analytical, Tubney Woods, Abingdon, Oxon, UK) acquiring five spectra for each SO.
7
Nanofiber Morphology Characterization
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The surface morphology of the electrospun nanofibers of the top layer (PCL) and bottom layers (PVA_CS-TPP and PVA_CS-TPP_CA) of the developed double-layered nanocomposite membranes was observed using scanning electron microscopy (SEM) (S2700, Hitachi, Tokyo, Japan) at an accelerating voltage of 20 kV. First, the samples were mounted on aluminum stubs and sputter-coated with a thin gold layer in an Emitech K550 sputter coater (Quorum Technologies Ltd., Laughton, East Sussex, UK) for better conductivity during imaging. The fiber diameters were measured from the obtained SEM images using ImageJ software (National Institutes of Health, MD, USA) and the size-frequency distributions constructed with GraphPad Prism 6 software (GraphPad Software, La Jolla, CA, USA).
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