Map 3d software

Paraffin sections (20-µm thick) obtained after plunge freezing and freeze substitution with two needles were deparaffinized in xylene, rehydrated, stained with 1.0% uranyl acetate in 70% methanol for 5 min, washed with distilled water, and stained with Reynolds’ lead citrate solution for 3 min. After washing with distilled water, the specimens were dried at room temperature for 2 h. The microscope slides were placed on the wide stage of the specimen holder using adhesive conductive tape and then placed in an LVSEM (TM4000Plus; Hitachi High-Tech, Tokyo, Japan) operating at 15 kV. Topographic images were reconstructed using Hitachi map 3D software (Hitachi High-Tech).

The sections were set onto the wide stage of specimen holder (Fig. 1e) using adhesive conductive tape, to be placed in a low-vacuum scanning electron microscope (LvSEM) (TM4000Plus or TM3030Plus, Hitachi High-Technologies, Tokyo, Japan). After evacuation of the specimen chamber a few minutes, the sections were observed under the electron beam accelerating voltage of 5 kV, 10 kV, or 15 kV. The observation angle was changed using the optional 30° or 45° tilt-holder. The LvSEM images were taken by the four-segmented BSE detector, and those four images were averaged into one image. Topographic images were reconstructed using Hitachi map 3D software (Hitachi High-Technologies), subtracting the difference among four-segmented shadow images in which the three-dimensional undulation had been reflected according to the segmented angle of each detector^{12 (link)}.