X-ray imaging and cryo-scanning electron microscopy (cryo-SEM) have been used to examine the frozen-hydrated plant tissues. X-ray imaging is useful for surveying a wide range of water distributions at tissue level [1–4]. X-ray imaging visualizes a transmission image of soft X-ray through the sample section (see Fig. 1). The intensity of X-ray on the imaging plane depends on the sample tissue density [5] and the water content [6], because the cell wall and water attenuate X-ray. Some studies represent the highly attenuated region a darker image (see Fig. 1a and also refs. 1, 7, 8) while other reports show it a brighter image [2–4, 9]. The cell wall and water distribution are clearly discriminated when the same sample is observed in both the green state (see Fig. 1b) and the dry state (see Fig. 1c). The resolution of X-ray imaging is sufficient to identify growth-ring boundary in conifers (see Fig. 1d and also refs. 2, 8) and in diffuse-porous hardwood species that have small vessels (see Fig. 1e). In some cases, each wide vessel can be observed (see Fig. 1f and also [7]).

Cryo-SEM is useful for observing the state of cytoplasm at the cellular level without chemical fixation and dehydration [8, 10–14]. The presence or absence of water in the lumina of dead cells is clearly distinguished in each cell (see Fig. 2). To observe the dynamic state of a plant tissue by cryo-SEM, samples are usually prepared by cryo-planning (see Fig. 3a) or freeze-fracturing (see Fig. 3b and also [10, 11, 15, 16]). Freeze-fracturing is simple and permits the cellular ultrastructure of various plant tissues to be revealed [17–22]. However, freeze-fracturing is not adequate for examining the lumina of axial elements such as vessels and tracheids because it is difficult to cleanly expose a cross section of secondary xylem in a tree sample (see Fig. 3b). Although a longitudinal section can be cleanly exposed by freeze-fracture, the frequency of exposure of lumina of axial elements is low because the fracture tends to occur between cell walls of the adjacent cells. In addition, ragged surfaces often cause a substantial margin of error in quantitative morphological analyses. A series of suitable surfaces from the same specimen is usually impossible to obtain [16, 23].

In contrast, the cryoplaning method is useful for examining the state of lumina of axial elements [23]. With this method, 2–3 mm thick sections are cut away from the fractural surface of specimens with a steel blade on a sliding microtome under frozen conditions. It is possible to cleanly expose a wider range of surface of sample blocks (see Fig. 3a) and allows a desired smooth face of hard woody tissue to be cleanly exposed (see Fig. 2 and also [1, 24–27]).

This chapter describes a freeze stabilization and cryo-planning method that we devised. The method enables reproducible observations of the water distribution in the functional xylem of woody plant stems from tissue level to cellar level. In the text that follows, we will explain not only the sample preparation but also the whole process from the sampling procedure in the transpiring woody stems to X-ray imaging and cryo-SEM observation.