E 1010 ion sputter

Seeds after mucilage extraction were air dried, and dry seeds were mounted on aluminum stubs (Hitachi Ion Sputter E-1010, HITACHI), sputter-coated with 40 nm of gold-palladium and viewed using a Hitachi S-4800 FESEM.

The leaf tips, leaf middle, leaf base of the young leaves (third leaf counting from the tip), and the mature leaves (the 9th leaf) were taken from 4-month-old wild-type and 35S::PtoCYCD3;3 plants. The samples (0.2–0.3 cm) were fixed in FAA solution (70% ethanol: glacial acetic acid: formaldehyde = 18:1:1) for 24 h at 4 °C. Subsequently, the samples were dehydrated in graded ethanol series (70%, 80%, 90%, 95%, and 100% ethanol) for 15 min, and then incubated at 4 °C overnight. The 100% ethanol was replaced sequentially and separately through an isoamyl acetate series (ethanol: isoamyl acetate (3:1), ethanol: isoamyl acetate (1:1), ethanol: isoamyl acetate (1:3), and 100% isoamyl acetate) for 15 min. The samples were then dried using a Critical Point Dryer (HCP-2; Hitachi, Tokyo, Japan) with liquid CO₂ and coated with platinum using an Ion Sputter (E-1010; Hitachi, Tokyo, Japan). The coated samples were observed and photographed with a scanning electron microscope (S-4800 FESEM; Hitachi, Tokyo, Japan) at 10 keV. All statistical analyses were performed using SPSS v16.0. Independent-Samples T-test was used.

Scanning electron microscopy (SEM) was conducted to observe the morphology of the gelatin-BVSM explants. A Hitachi E-1010 ion sputter (Tokyo, Japan) was used to coat the specimens with gold and a Hitachi S3000N SEM was used to obtain micrographs at a 5-kV accelerating voltage.

The biofilms of each bacterial strain were developed on 1 × 1 cm size of cover slips with all treatments as described above and all the slides were visualized under SEM. The developed biofilms were fixed on glass cover slips using 2.5% of glutaraldehyde at 37 °C for 30 min. Afterwards, cover slips were rinsed and washed with PBS solution 3 times and dehydrated from a graded series of 30%, 50%, 70%, 90%, and 100% of ethanol for 15 min in each. At the end, ethanol was replaced with isoamyl acetate and the samples were freeze-dried. Coverslips were kept on the aluminum holder, covered with gold via E-1010 ion sputter (Hitachi^®, Tokyo, Japan), and observed under SEM (S-34002N SEM, Hitachi^®, Tokyo, Japan).

The membranes were snap-frozen in liquid nitrogen and then dried in a vacuum freeze dryer (RLE-103, Kyowa Vacuum Engineering. Co., Ltd., Tokyo, Japan) at 298 K for 24 h. Then, the dried membranes were sputter-coated with a thin Pt membrane, using a sputter-coater (E-1010 Ion Sputter, Hitachi, Ltd., Tokyo, Japan). Finally, cross-sectional images of the membranes were obtained using a scanning electron microscope (SEM) (Miniscope TM-1000, Hitachi, Ltd., Tokyo, Japan).

Mitochondria morphology was visualized with transmission electron microscopy. Cells were fixed in 2.5% glutaraldehyde, post-fixed in 1% OsO₄, dehydrated in a graded series of ethanol solution, washed in acetone, and embedded in resin mixture. Sections were obtained by EM UC7 ultratome (Leica, Germany), and then stained by uranyl acetate and lead citrate. Images were taken by H-7650 transmission electron microscope (Hitachi, Japan). The ultrastructure of cilium in cells was observed using scanning electron microscopy. After dehydrated through an ethanol series and washed with pure ethanol, cell samples were inserted into HCP-2 critical point dryer (Hitachi, Japan) until dry, coated with gold-palladium in E−1010 ion sputter (Hitachi, Japan) for 4–5 min and observed in the SU-8010 scanning electron microscope (Hitachi, Japan).

Samples were placed into a piece of aluminum foil and dried in an oven at 120 ± 2 °C until a constant weight was achieved. The sample fracture surface was sputter-coated with gold by using an ion sputter (Hitachi E-1010 Ion Sputter, Japan), and then a Hitachi S-3400N (Hitachi Science System, Ibaraki, Tokyo, Japan) scanning electron microscope was used to observe the fracture surface of the resultant adhesives.