Tecnai g2 f20

Powder X-ray diffraction patterns were collected on a Rigaku MiniFlex600 X-ray diffractometer with Cu Kα radiation. The X-ray diffraction data was refined by the RIETAN-2,000 Rietveld refinement program42 . SEM images were taken with a JEOL JSM-7500F microscope (operating voltage, 5 kV) equipped with an EDS analyzer. TEM and high-resolution TEM imaging was obtained on Philips Tecnai G2 F20 (acceleration voltage, 200 kV). Chemical composition was determined by EDS and atomic absorption spectrometry (AAS, Hitachi 180-90 spectrophotometer). Surface areas were calculated from the N₂ adsorption/desorption isotherms at 77 K on BELsorp-Mini. Thermogravimetric analysis was carried out on a Netzsch STA 449 F3 Jupiter analyzer. XPS was performed by a Perkin Elmer PHI 1,600 ESCA system. The X-ray absorption near-edge structure spectra were collected on BL14W1 beamline of Shanghai Synchrotron Radiation Facility (SSRF) and analysed with software of Ifeffit Athena.

The ultrastructural features of the spermatozoa from the infertile patient and normal control were analyzed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For the SEM assay, the spermatozoa samples were fixed onto slides of 1 cm diameter using 2.5% glutaraldehyde for 4 h at 4°C. After washing the slides with 1× phosphate buffered saline (PBS) three times and postfixing in 1% osmic acid for 1 h at 4°C, dehydration was performed using 30%, 50%, 75%, 95%, and 100% ethanol sequentially, and the slides were dried using a CO₂ critical‐point dryer (Eiko HCP‐2, Hitachi). Before examination with a field emission SEM Hitachi S3400, the dried specimens were glued to aluminum stubs and Pt sputter‐coated by an ionic sprayer meter (Eiko E‐1020, Hitachi).
For the TEM assay, samples were fixed in 3% glutaraldehyde, phosphate‐buffered to pH 7.4, and postfixed with 1% OsO₄. After dehydration, the samples were incubated in propylene oxide followed by embedding in a mixture of Epon 812 and Araldite. Ultrathin sections obtained by an Em UC6 Ultramicrotome (Leica) were collected on TEM nickel grids and analyzed using TEM (TECNAI G2 F20, Philips) at 120 kV.

The morphologies of the MoS₂ nanosheets modified by ethanethiol and nonanethiol were observed using a Tecnai™ G2 F-20 (Philips, Amsterdam, Netherlands) transmission electron microscope (TEM). Raman spectra and Raman maps were obtained using an NRS5100 (JASCO, Tokyo, Japan) spectrometer. Cross-sectional images were obtained using a JSM-6500F (JEOL, Tokyo, Japan) scanning electron microscope (SEM); and, composite samples were cooled in liquid nitrogen and cut by a scalpel to prepare the samples for backscattered electron (BSE) imaging. Atomic force microscopy (AFM) was performed using a NX10 system (Park, Suwon, Korea). X-ray diffraction (XRD) was performed using a D8 SSS (Bruker, Billerica, MA, USA). UV-Vis spectra were obtained using a V-730 spectrometer (JASCO, Tokyo, Japan). Dynamic mechanical analysis was performed using a Q800 (TA Instruments, New Castle, DE, USA), while stress-strain curves were measured using a TS-2000 with a crosshead speed of 500 mm/min. The oxygen transmission rates were measured according to the ASTM D3985 standard using the OX-TRAN 2/61 (Mocon Inc., Minneapolis, MN, USA) at 23 °C and a relative humidity of 0%; film specimens of 5 cm in diameter and 1 mm in thickness were fixed between two chambers, and oxygen filled the upper chamber while nitrogen filled the lower chamber.

For scanning electron microscopy (SEM), the sperm samples were centrifuged at 400×g for 10 min at room temperature. The supernatants were carefully aspirated, and the pellets were suspended and fixed in 2.5% glutaraldehyde for 30 min at 4°C. Next, the samples were evenly spread onto slides and fixed in 2.5% glutaraldehyde overnight at 4°C. Following primary fixation, the slides were washed three times in 1×PBS and gradient dehydration was performed sequentially with 30%, 50%, 75%, 95%, and 100% ethanol for 10 min. Subsequently, the slides were dried to temperature with a CO2 critical-point dryer (Eiko HCP-2, Hitachi). Finally, all of the dried specimens were mounted on aluminum stubs, sputter-coated by an ionic sprayer meter (Eiko E-1020, Hitachi), and analyzed by SEM (Hitachi S3400).
For transmission electron microscopy (TEM), sperm samples were washed routinely and centrifuged at 400 × g for 15 min. Then, the seminal plasma was removed, and the sperm pellets were fixed in 3% glutaraldehyde. Next, the samples were postfixed in 1% buffered OsO4, dehydrated through gradient acetone solutions, and embedded in Epon 812. Finally, the ultrathin sections (80 nm) were double-stained with lead citrate and uranyl acetate before being observed and photographed via TEM (TECNAI G2 F20, Philips).

For SEM, fresh sperm isolated from cauda epididymis of mice were washed three times using PBS and then fixed on coverslips with 4% Paraformaldehyde for 2 h. Subsequently, the coverslips carrying spermatozoa were washed with PBS and then dehydrated with gradient ethanol of 50, 60, 70, 80, 95, and 100%. Then, the sperm were dried with Quorum K850 Critical Point Dryer (East Sussex, UK) and coated with gold particles using an ion sputter coater (Rotary Pumped Quorum Technologies, Q150RS). The spermatozoa were finally observed under SEM (Hitachi, S-3400N).
For TEM, testis tissues were isolated from mice and then fixed with 2.5% glutaraldehyde for more than 24 h. Subsequently, the samples were further fixed with 1% osmic acid for 120 min. After washing with PBS twice, the samples were dehydrated with gradient acetone of 50, 70, 90, and 100% at 4 °C and then embedded in embedding agents containing 6% butylene phthalate, 1% phenol, 44% dodecyl succinic anhydride, and 56% epoxy resin. Next, ultrathin sections (70–90 nm) were obtained and double-stained with lead citrate and uranyl acetate. Finally, the images were taken under transmission electron microscopy (Philips, TECNAI G2 F20) for further analysis.

Surface morphology and microstructure of the Co@C nanoparticles were characterized using field emission scanning electron microscope (FE-SEM; Hitachi S-4800) and high-resolution transmission electron microscope (HRTEM; Philips Tecnai G2 F20). X-ray diffraction (XRD) patterns of the Co@C nanoparticles were recorded on a powder X-ray diffractometer (Rigaku D/max 2000V/pc) using CuKα radiation within the angle range of 20°–90° (2θ) with a 0.02° step. The interplanar spacing of the graphite layer was calculated using the Digital Micrograph software. Raman spectroscopy was performed using the 532-nm line of Ar⁺ laser as the excitation source on a Thermo Fisher DXR Raman Microscope. Thermal properties of the samples were investigated using thermogravimetric analysis (TGA, TA Instruments SDT Q600 TGA). The samples in TGA were analyzed in platinum pans at a heating rate of 10 °C min⁻¹ from 50 to 700 °C in air with a flow rate of 150 mL min⁻¹. The magnetic properties of the samples were measured using a vibrating sample magnetometer (VSM; Lakeshore 7407) at room temperature.