Intact observation of secondary xylem and parenchyma cells in woody plants using a combination of scanning electron microscopy and broad argon ion beam milling

Abstract

The secondary xylem of woody plants consists of fragile living cells and rigid cell walls. However, xylem structures are easily damaged during mechanical cross-sectioning for electron microscopy analysis. A broad argon ion beam (BIB) milling technique is commonly employed for analysis using scanning electron microscopy (SEM) in material science to generate a large and distortion-free cross-section. However, BIB milling is rarely used in plant science. In this study, SEM combined with the BIB milling technique was validated as an accurate tool for structural observation of xylem tissues of two woody plants, Pinus densiflora (Japanese red pine) and Quercus phillyraeoides (Ubame oak), in comparison to cutting with the classical microtome. 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 observation using SEM with BIB milling was found to be useful for imaging hard and soft plant tissues, particularly the ultrastructure of fragile secondary xylem cells. Thus, we propose a new imaging method for detailed observation of plant tissue structures.