A combination of scanning electron microscopy and broad argon ion beam milling provides intact structure of secondary tissues in woody plants

“The secondary tissues of woody plants consist of fragile cells and rigid cell walls. However, the structures are easily damaged during mechanical cross-sectioning for electron microscopy analysis. Broad argon ion beam (BIB) milling is commonly employed for scanning electron microscopy (SEM) of hard materials to generate a large and distortion-free cross-section. However, BIB milling has rarely been used in plant science. In the present study, SEM combined with BIB milling was validated as an accurate tool for structural observation of secondary woody tissues of two samples, living pine (Pinus densiflora) and high-density oak wood (Quercus phillyraeoides), and compared with classical microtome cross-sectioning. The BIB milling method does not require epoxy resin embedding because of prior chemical fixation and critical point drying of the sample, thus producing a three-dimensional image. The results showed that xylem structures were well-preserved in their natural state in the BIB-milled cross-section compared with the microtome cross-section. The observations using SEM combined with BIB milling were useful for wide-area imaging of both hard and soft plant tissues, which are difficult to observe with transmitted electron microscopy because it is difficult to obtain sections of such tissues, particularly those of fragile reaction woods.”

Xylem to phloem radial cross-section prepared by BIB milling

Figure 1a shows a BIB-milled radial cross-section of P. densiflora. Before BIB milling, we performed chemical fixation and critical point drying of the samples. Figure 1b is a schematic depiction of the BIB milling process. The BIB milling process created a distortion-free cross-section from the xylem to the bark. Because pine resin, rich in unsaturated fatty acids, reacts with osmium tetroxide33, the resin canals showed high contrast in the back-scattered electron image. Mechanical cross-sectioning frequently separates the cambium; however, this separation did not occur in the BIB-milled cross-section (Fig. 1a).

Figure 1
figure 1

Scanning electron microscopy results of the broad argon ion beam (BIB)-milled radial sections of Pinus densiflora (a) and illustration of the BIB milling process (b). Using the BIB milling process, a broad cross-section from phloem to xylem was obtained without cutting defects and distortions. The resin canal can be easily distinguished from the neighboring tracheids as the resin shows high contrast in the back-scattered electron image. A red-boxed area corresponds to Fig. 2a. Ca, cambium; Ph, phloem; R, resin canal; T, tracheids; Xy, xylem. Scale bar = 100 µm.

Structure of the radial parenchyma secondary phloem cells prepared by BIB milling and microtome methods

Figure 2 shows the radial cross-sections of the parenchyma cells in the secondary phloem prepared by the BIB milling and microtome methods. The BIB milling method resulted in fine structural preservation of cell organelles and intracellular storage materials (Fig. 2a). The nuclei, starch granules, and oil bodies were observed in the BIB-milled cross-section without epoxy resin embedding. Additionally, because no epoxy resin was embedded, the oil bodies showed a natural spherical shape. The protoplasm was observed as 3D network structure that fixed intracellular storage material. The vacuoles appeared as voids owing to the leakage of the cell sap containing inorganic salts and water34,35 during the critical point drying process. In the microtome cross-section, starch granules were detached from the epoxy resin by mechanical stress during cutting (Fig. 2b). With epoxy resin embedding, the oil bodies were deformed to an amorphous sphere, and protoplasmic 3D networks and vacuoles were observed as planar images (Fig. 2b). In contrast, by not requiring epoxy resin embedding, the BIB milling method yielded 3D information of xylem tissues.

Figure 2
figure 2

Radial sections of parenchyma cells in Pinus densiflora secondary phloem. (a) Radial section of broad argon ion beam milling (red-boxed area in Fig. 1a). Nuclei, protoplasmic 3D networks, and intracellular storage materials such as starch granules and oil bodies show good structural preservation without milling damage. The oil bodies exhibit a natural spherical shape, and vacuoles are void because epoxy resin embedding was not performed. (b) Radial microtome section. Starch granules were separated from the protoplasm by mechanical stress during the microtome cutting. The oil bodies were deformed by epoxy resin embedding, therefore presenting irregular shape, and the SEM image of the cell structure lost its depth information. N, nucleus; O, oil bodies; P, protoplasm; S, starch granules; V, vacuole. Scale bars = 20 µm.

Transverse sections of resin canals

Figure 3 shows transverse cross-sections of resin canals and parenchyma cells (epithelial, ray, and axial cells) prepared using the BIB milling and microtome methods. In the BIB-milled cross-section prepared without mechanical stress, we observed a broad area encompassing the phloem, cambium, and xylem, including resin canals (Fig. 3a). The cellular contents were readily identifiable because no epoxy resin embedding was performed (Fig. 3a). Further magnification of the resin duct revealed that the thin walls of parenchyma cells were not squashed and the inside of cells was filled with many spherical oil bodies and starch granules (Fig. 3b). The resin canal and void spaces enclosed within the thin cell walls retained their structure (Fig. 3b). On the contrary, in the microtome cross-section, the resin canal and void spaces were crushed by mechanical stress, and oil bodies appeared as amorphous spheres due to epoxy resin embedding (Fig. 3d). Moreover, in the microtome cross-section, the epoxy resin had high contrast due to pine resin infiltration, and SEM images of intracellular storage materials were obscured (Fig. 3d). In addition, the epoxy resin deteriorated by pine resin bleeding was detached from the tracheids (Fig. 3c,d).

Figure 3
figure 3

Transverse sections of Pinus densiflora resin canals and xylem parenchyma cells. (a,b) Broad argon ion beam (BIB)-milled cross-section. BIB milling created a broad and smooth cross-section from the phloem, cambium, and xylem, including the resin canal. (b) Parenchyma cells (epithelial, ray, and axial cells) contain many oil bodies (O) and have thinner cell walls than neighboring tracheids. No crushing of the thin cell walls or detachment of starch granules is observed in the BIB-milled cross-section. (c,d) Microtome cross-sections. The oil bodies are deformed into blurred outlines due to epoxy resin embedding. The epoxy resin is detached from the tracheids due to pine resin bleeding. Scanning electron microscopy images of intracellular storage materials are obscured by resin infiltration into epoxy resin. The resin canal and void spaces enclosed by thin cell walls are crushed by mechanical stress. Ca, cambium; N, nucleus; O, oil bodies; R, resin canal; S, starch granules; V, void space. Scale bars = 100 µm (a and c) and 20 µm (b and d).

Cross-sectional observation of the reaction wood

Figures 4 and 5 show the transverse cross-sections of tracheids in compression wood and opposite wood (non-reaction wood) of P. densiflora. Opposite wood is defined as the xylem located opposite to the reaction wood formed at the leaning trunk36.

Figure 4
figure 4

Transverse sections of tracheids in the compression wood and opposite wood of Pinus densiflora. (a,b) Broad argon ion beam (BIB)-milled cross-section of compression wood. The tracheids have a round shape with a thick cell wall. Many intercellular spaces can be observed among tracheids. No cutting damage, such as lightning bolt cracks or detachment between the S1 and S2 layers of the secondary wall, as observed in microtome cross-sections, are present in the BIB-milled section. (c,d) BIB-milled cross-section of opposite wood (non-reaction wood). Tracheids have rectangular or hexagonal shapes with relatively thin cell walls. Arrows indicate bordered pits. (e,f) Microtome cross-section of compression wood. The structures of cell walls and helical cavities are extended in the cutting direction. (g,h) Razor blade-cut cross-section of compression wood. Several cracks are generated in the S2 layer of the secondary wall (e,h). The S2 layers are delaminated from the S1 layers due to cutting stress. Scale bars = 50 µm (aceg) or 10 µm (bdfh).

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