Freeze stabilization and cryopreparation technique for visualizing the water distribution in woody tissues by X-ray imaging and cryo-scanning electron microscopy

The protocol of freeze stabilization and cryopreparation techniques to examine the distribution of water in living tree stems by X-ray imaging and cryo-scanning electron microscopy have been developed and described. The brief procedures are as follows. Firstly, a portion of transpiring stem is frozen in the standing state with liquid nitrogen to stabilize the water that is present in the conducting tissue. After filling with liquid nitrogen, discs are then collected from the frozen portion of the stem and stored in liquid nitrogen. In a low-temperature room, the samples for X-ray imaging are sectioned with a fine handsaw, and the trimmed sample blokes for cryo-scanning electron microscopy are cleanly planed using a sliding microtome. Finally, the frozen sections are irradiated in a soft X-ray apparatus, and the small blocks are examined in cryo-scanning electron microscope after freeze-etching and metal coating.