- Chemical fixation method

Leaves and other organs were fixed with 2% (v/v) glutaraldehyde/1% (w/v) paraformaldehyde in 50 mM sodium phosphate, pH 7.2, at 4°C overnight. The fixed samples were washed with 50 mM sodium phosphate, pH 7.2, fixed with 1% (w/v) osmium tetroxide in 50 mM sodium phosphate, pH 7.2 for 3 h at 4°C, dehydrated in acetone and embedded in Spurr’s resin (Polysciences, Inc.). Thin sections (70 m) were obtained using a diamond knife mounted on an ultramicrotome (ULTRACUT E, Leica), then stained with 4% (w/v) aqueous uranyl acetate for 18 min and with lead citrate solution for 7 min.
Plant Cell Physiol (2014) 55: 358-369.

- High pressure freezing / frozen substitution method

For morphological observation of Arabidopsis thaliana epidermal cells, seeds were planted in 0.8% (w/v) agar plates for 2 weeks and then moved to 2% sucrose on papers for 24 h to inhibit the formation of ice-crystal formation during HPF/FS. The roots and cotyledons were cut into 1-mm rectangles and frozen in a high-pressure freezer. Root tips and leaves were placed on a flat specimen carrier and frozen in a high-pressure freezer (EM-PACT; Leica). For morphological observations with tannic acid staining, the frozen samples were fixed with anhydrous acetone containing 2% osmic acid (OsO4) for 3 to 4 d at −80°C. The tubes containing the frozen samples were warmed at 3°C/h to a temperature of −20°C, at 1°C/h to a temperature of 4°C and kept for 2 h at 4°C using an automatic freeze-substitution system (EM-AFS; Leica). The samples were washed with 100% acetone and then embedded in epoxy resin. The blocks were trimmed using a trimming diamond knife (Ultratrim; Diatome) and then cut by a 35° 2 mm diamond knife (Ultra; Diatome). The serial ultrathin sections were placed on formvar-coated 1-slot copper grids. The grids were stained by a 4% uranyl acetate solution for 10 min and a lead citrate solution for 1 min.
Plant Cell (2009) 21: 1212-1229
Plant Cell Physiol (2014) 55: 1544-1555

- Construction of high-resolution gigapixel TEM pictures

Digital TEM images were captured by a custom auto-acquisition system and merged by an auto-image tiling program using the ImageJ engine on a PC (Precision T7500; Dell). Briefly, the TEM (JEM-1400: JEOL) and the attached CCD camera were controlled by JEM ToolBox (System In Frontier Inc.) using MATLAB (release 2011a; MathWorks). The TEM pictures (3,000–15,000 digital pictures of 1,360×1,024 pixels; overlap ratio, 30%; magnification, ×15,000) were merged with the originally developed ImageJ engine programmed by using the particle swarm optimization algorithm and Photoshop CS5 Extended (Adobe).
Plant Cell Physiol (2014) 55: 1544-1555