Cryoplaning technique for visualizing the distribution of water in woody tissues by cryoscanning electron microscopy

The protocol of cryoplaning techniques that to examine the distribution of water in living tree stems by cryoscanning electron microscopy have been developed and described. In brief, the procedures are as follows: First, 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. The surface of disc is cleanly cut using a sliding microtome in a low temperature room at -20 degrees C. Finally, the frozen sample is examined in a cryoscanning electron microscope after freeze-etching and metal coating.