Jem 1200 exii

The isolation of cellulose nanofibrils from the treated agave pulp was carried out using a colloid mill (Super Mascolloider Microprocessor, from Masuko Sangyo Co., Ltd., Kawaguchi, Japan). The pulp suspension, with a consistency of 2%, was filtered five times through two discs at 1500 rpm, with a distance between discs of 0.1 mm.
The obtained cellulose nanofibrils were analyzed with a Transmission Electron Microscope (JEOL JEM 1200EX-II from JEOL Ltd. Tokio, Japan) coupled with a Gatan CCD high-resolution camera (Orius SC1000B). The microfibers were previously diluted at 0.002 wt% in distilled water, sonicated for 10 min, and placed on a palladium grid.

A morphological analysis of NPs was performed using a transmission electron microscope (TEM; JEOL JEM1200EXII, Jeol, Tokyo, Japan). NP suspensions were diluted 10-fold in PBS (pH 7.4) and dropped onto carbon-coated copper grids (FC300Cu, EM Resolutions, Saffron Walden, UK) with the liquid being quickly removed by touching the edge of the grid with filter paper. Negative staining was performed by exposing samples to 2% uranyl acetate for 30 s, which was also removed by filter paper absorption. Samples were stored in a desiccator after air drying until being viewed in the TEM. At least three batches of each NP were prepared. Representative TEM photos presented in the manuscript were taken from batches prepared on the same date to maximize comparing/characterize NPs. Average NP size was determined from the measurement of 100 randomly selected NPs from different locations on the samples.

Cells (50,000 cells/well) were seeded in a 24-well plate before treatment with EcTI (100 µM) for 24 h, washed with PBS, and incubated with Versene solution for 5–7 min. Cells were fixed with 2% (v/v) formaldehyde and 2.5% (v/v) glutaraldehyde for 30 min at room temperature and diluted in the buffer cacodylate (0.1 M, pH 7.2). Cells were then embedded in 4% (v/v) agar, and fixed with 2% osmium tetroxide in sodium cacodylate (0.1 M, pH 7.2) for 2 h. Then, the cells were dehydrated in increasing ethanol concentrations (70%, 90% (v/v), and absolute). A mixture of propylene oxide and Epon 612 resin (1:1) was added to the samples overnight. Then, pure Epon was added to the mixture for an additional 2 h under vacuum and polymerized in an oven at 60 °C for 48 h. In the fenestrated copper metallic grid, ultra-thin cuts (70 nm) were placed, and uranyl acetate/plumb citrate was used as a contrast. The images were examined using transmission electron microscopy (TEM, JEOL JEM 1200 EX II, JEOL, Peabody, MA, USA) operated at 80 kV. Micrographs were acquired with a GATAN 791 camera (USA), while brightness, contrast levels, and image resolution were adjusted using ADOBE Photoshop 7.

Microscopic images of the materials were taken using a transmission electron microscope JEOL JEM-1200 EX II (JEOL, Akishima, Tokyo, Japan) operating at 80 kV.

The size and shape of the nanoMIPs for β-blockers (atenolol and labetalol) was determined in water using dynamic light scattering (DLS) analyzer, from Brookhaven Instruments Corporation Ltd. (Holtsville, NY, USA) and based on images of dry nanoMIPs obtained by a transmission electron microscope (TEM), JEOL/JEM 1200 EX II (Tokyo, Japan). The size analyzed by DLS includes data of mean hydrodynamic diameter (D), polydispersity index (PDI), and standard deviation (Std dev) of the nanoMIPs dispersion in milli-Q water. Prior to measurements, nanoMIPs were preconcentrated by evaporation and constant bubbling with N₂ to avoid their drying. DLS was performed in 1 mL of the solution of nanoMIPs in water at 25 °C. The measurements were conducted using 3 cm³ disposable polystyrene cuvettes.
Before TEM analysis, samples were sonicated for 5 min, and then 20 µL of the nanoMIPs dispersion was placed on a carbon-coated copper grid and dried at ambient temperature under a fume hood. After that, thin film of a sample was ready for analysis.

The completely open flowers derived from T54S, T54M and T54C, were dissected and dehydrated through a series of increasing ethanol solutions. Additionally, the materials were then critically point-dried with solvent-substituted liquid carbon dioxide and coated with a thin layer of gold palladium. Photomicrographs were obtained using a JEOL 5800 LV at 20 kV (JEOL USA, Peabody, MA, USA). At maturity, the nectaries of broccoli at bolting stage were cut off and pre-fixed in 1% glutaraldehyde and 4% formaldehyde in phosphate buffer (pH 7.2) for 4 h. The nectaries were then post-fixed in 1.5% O_sO₄ at 4 °C in the same buffer for 4 h, and then, the samples were dehydrated in ascending graded series of acetone and embedded in Spurr’s resin. Ultrathin sections were made using a Reichert ultramicrotome and stained with uranyl acetate and lead citrate [43 (link),44 (link)]. The equipment of JEOL-JEM 1200 Ex II transmission electron microscopy (TEM) was used for identifying all the samples at 85 kV.
In addition, the agronomic traits of the broccoli lines T54S, T54M and 54C were investigated during the harvest, flowering, seed ripening and seed germination stages. All the data were analyzed with SPSS 19.0 software (IBM Co., Ltd., New York, NY, USA), and one-way ANOVA was carried out on the agronomic trait data at p < 0.05.