Detection of embolization of vessels by a double staining technique

Air seeding and water vapor embolization of vessels were induced in branches of Acer rubrum by freezing-thawing experiments. A technique has been developed for detecting embolized vessels at the microscopical level. In this technique, one dye is used to label functioning vessels prior to freezing while the second dye is offered after a freezing-thawing cycle at vacuum pressure or at atmospheric pressure in order to reveal water vapor embolization or recovering at 1 bar. Double staining is achieved by differential staining of vessel walls and of their particular contact pits facing paratracheal contact cells. The water potential of the leaves and the rate of uptake of the dye solutions was recorded during the experiments using a Scholander pressure bomb and a potetometer, respectively. While almost 100% of the vessels proved to be functioning in the outer 5 to 7 annual rings, one freezing-thawing cycle inhibited water conduction in up to 82 to 98% of the vessels provided vacuum (0.13 bar) was applied. Rapid recovering however is observed, if the second dye is introduced at atmospheric pressure.