Jem 2000ex

Characterization was performed using the supernatants after the centrifugation of the LCNFs before and after enzymatic hydrolysis. Polarizing optical microscopy (POM, Nikon Optiphot-Pol, Nikon Corp., Tokyo, Japan) and TEM (JEM-2000EX, JEOL, Japan) were used to observe the morphology of the hydrolyzed products. To observe TEM images, a drop of a diluted bamboo LCNF suspension was deposited on a carbon-coated grid and allowed to dry at room temperature. The length and diameter of individual LCNFs were measured at least 50 times from POM images and TEM images, respectively.
The crystalline characteristics of LCNFs were determined using XRD (RINT 2000, Rigaku Corporation, Tokyo, Japan). For X-ray analyses, disks that were 1 cm in diameter, 0.8 mm in thickness, and 0.1 g in weight were prepared from the freeze-dried samples. Ni-filtered Cu Kα radiation (λ = 0.1542 nm) was employed at an accelerating voltage of 200 kV and a current of 40 mA. The diffracted intensity (I) was determined in the 2θ range of 5°–40° at a rate of 2°/min. The crystallinity index (CrI) was calculated using Segal’s method (formula (1)) [29 (link)].
$\mathrm{Crystallinity}\mathrm{index}(\%)=\frac{\left({\mathrm{I}}_{200}-{\mathrm{I}}_{\mathrm{amor}}\right)}{{\mathrm{I}}_{200}}\times 100$
where I₂₀₀ is the crystalline intensity and I_amor is the amorphous intensity.

After treatment with 5 mM Met or NH₄Cl for 36 h, PRECs were fixed successively with 1% osmic acid fixation solution and 2.5% glutaraldehyde for more than 2 h. Then, the cells were washed with phosphate buffer (0.1 mM) and dehydrated with a gradually increasing concentrations of acetone. After this, they were coated with epoxy resin, the cell samples were sliced. A transmission electron microscope (JEM-2000EX, JEOL Co, Tokyo, Japan) was applied to collect the electron images [17 (link)].

The HeLa and SiHa cells (2 × 10⁵ cells/mL) were plated in 6-well plates, treated with dioscin, harvested, and fixed overnight at 4 °C in 2% glutaraldehyde. The samples were implemented as previously described [27 (link)]. The obtained sections were then stained and observed using a transmission electron microscope (JEM-2000EX, JEOL, Tokyo, Japan).

The morphology and structure were characterized by field emission scanning electron microscope (SEM, Hitachi S4800, Tokyo, Japan). The graphitic structure was analyzed by high-resolution transmission electron microscopy (HRTEM, JEM 2000EX, JEOL, Tokyo, Japan). The surface chemical composition was determined by X-ray photoelectron spectroscopy (XPS, Thermo ESCALAB 250XI, Waltham, MA, USA). Raman spectrometer was performed on a Raman Microscope (LabRAM HR800, Paris, France) with YAG solid-state laser under wavelength of 532 nm. An X-Ray Diffractometer (XRD, D8 Advance Bruker, Germany) was used to study the crystallinity. The magnetic properties of samples were examined using a vibratory probe sample magnetometer (VSM-7300, Quantum design Lakeshore, San Diego, CA, USA). The specific surface areas were determined using the Brunauer–Emmett–Teller (BET, MicrotracBEL Corp, Shanghai, China).

EV Extraction Kit (4484450, Invitrogen Life Technologies, CA) was used to extract plasma-EVs. The plasma samples stored at − 80 °C were taken out, and placed in a 37 °C water bath until completely melted. The samples were put on ice, and centrifuged at 2000×g for 20 min to remove cells and debris. The supernatant was harvested and transferred to a new centrifuge tube using a pipette, followed by centrifugation at 10,000×g for 20 min at ambient temperature to remove debris. The supernatant was removed into a new centrifuge tube, which was placed on ice for separation. The obtained precipitate was EVs and stored at − 80 °C.
The morphology of EVs was then observed under a transmission electron microscope (TEM; JEM-2000EX; JEOL, Tokyo, Japan). Nanoparticle tracking analyzer (NS300, Malvern Instruments, Malvern, UK) was used to measure the size distribution of EVs. The expression of EV markers (CD63, CD81, Alix and Calnexin) was detected with the help of western blot analysis, with the used antibodies including anti-CD63 (1:1000, 25682-1-AP, Proteintech ProteinTech Group, Chicago, IL), anti-CD81 (1:1000, 66866-1-Ig, Proteintech), Alix (1:1000, 12422-1-AP, Proteintech) and Calnexin (1:1000, ab133615, Abcam).

The size and shape of nanoparticles were inspected using transmission electron microscopy (TEM: JEM-2000EX; JEOL, Tokyo, Japan) at 80 keV. Images were captured with a Morada 11 megapixel camera (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Droplets of nanoparticles solutions were placed onto formvar-coated copper grids (Agar Scientific, Stansted, UK) and immediately after air-drying the grids were inspected by TEM (Fig 1).