Tm3000 microscope

To observe the mechanical bending and twisting of PMA-III, scanning electron microscopy was performed by using TM3000 microscope (Hitachi) with an accelerating voltage of 15 kV. The crystals were attached on a double-sided carbon tape.

The shape and surface aspect of particles were accessed using the scanning electron microscopy (SEM) images taken in TM 3000 Microscope Hitachi (Tokyo, Japan). The particles were dried and mounted on metal stubs using double-sided adhesive carbon tape and analyzed at the voltage of 20.0 kV. The mean diameter and the size distribution of the microparticles was determined using dynamic light scattering (DLS) in a Nanotrac NPA252 (Montgomeryville, PA, USA) with Flex software 10.4.3. The amount of 4.0 mg of powder was dispersed in 15.0 mL of aqueous solution of polysorbate 80 at 0.5% w/v. The cumulative diameter of 10, 50 and 90% in the particle size distribution were determined in triplicate. The index span was calculated by the equation: SPAN = D90 − D10/D50 [28 (link)].
The surface area, pore volume and pore size of the chitosan microparticles were determined following the method of Brunauer-Emmett-Teller (BET), using the liquid N2 adsorption and desorption isotherms, measured at 77 K temperature with an ASAP 2420 surface area analyzer (Micromeritics Instrument, Norcross, USA). All samples were degassed and stored at vacuum at room temperature overnight prior to measurements. The experiments were repeated at least three times using fresh powder.

On day 6, the culture medium was removed from the wells, and cells were fixed with 2.5% glutaraldehyde (Panreac, Barcelona, Spain) and with 0.1% OsO₄ (Sigma-Aldrich, St. Louis, MO, USA). After washing, cell samples were passed through a battery of alcohols of increasing concentration. Samples were sprayed with gold in magnetron EmitechK575XD (Quorum Technologies, Lewis, UK), and analyzed using a TM 3000 microscope (HITACHI, Tokyo, Japan).

For drought treatment, the transgenic tomato plants ectopically expressing MdPIP1;3 and the wild type plants weren’t watered in greenhouse. After 20 days without watering, the soil volumetric moisture content (ϴ_V) dropped from 43 to 45% (well-watered condition) to 0.5-1% (water deficit stress), the plants were re-watered every two days until the ϴ_V increased to 43-44% to detect their recovery capability. The ϴ_V was detected by a Soil Moisture Sentor (NC®, Beijing; Type: SU-LB). The phenotype was observed and recorded using photography. The experiment was repeated three times.
40-mm (in length) leaves of transgenic lines ectopically expressing MdPIP1;3 and wild type plants were detached, placed on the filter paper for 15 h at room temperature (20 ± 2 °C) for an in vitro dehydration rate experiment. The weight of leaves was measured. The mass ratio, as an indicator of dehydration rate, was calculated by the weight after drought treatment divided by the original weight before treatment. The experiment was repeated three times.
Five leaves were collected from transgenic and wild type plants under normal condition and drought treatment, respectively. Leaf stomata observation was performed using a TM3000 microscope (HITACHI, Tokyo, Japan). Stomata were observed randomly in 50 visual sections of the same area [37 (link)]. The experiment was repeated in triplicate.

The surfaces and cross-sections of the shot composite samples were subjected to macro and microscopic examination. Microstructure analysis after firing was performed using light microscopy with a Nikon Eclipse MA200 microscope and SEM on a Hitachi TM-3000 microscope equipped with an EDS system. The observations were made on surfaces of the hole created after the projectile’s passage, on the surfaces of radial and internal cracks. Moreover, analysis of microstructural changes in the areas directly affected by the projectile’s impact occurring inside the material directly below the penetration surface was performed.