Microanatomy of Plant Tissues

Confocal microscopy images of plant antatomical specimens in the Department of Plant Sciences, University of Cambridge.
Images by Jim Haseloff.

A wide variety of staining techniques has been adopted for plant specimens over the past 150 years. Perhaps the most widely used general tissue stains are Safranin O and haematoxylin. These are often accompanied by the use of counterstains such as Fast Green, Orange G, or Alcian Blue. In addition, there is a large variety of more specific staining techniques that have been developed for particular plant materials and organelles. For example, Feulgen staining has been used for the specific labelling of DNA, the periodic acid-Schiff reaction can be used to label carbohydrates, Aniline Blue for callose, Nile Red for oil bodies, and Phloroglucinol for lignin. A multitude of published protocols are available. An excellent published source of procedures can be found in Plant Microtechnique and Microscopy by Steve Ruzin, Oxford University Press, Oxford, UK. 1999.

Interestingly, many of the synthetic dyes used for plant microtechniques are highly fluorescent. This is particularly true for red, orange, and yellow dyes in the azine (e.g., Safranin O), acridine (e.g., Acridine Orange), and xanthene (e.g., Rhodamine) families. Thus, many classical histological techniques unintentionally produce specimens that are intensely fluorescent. In addition, aldehyde fixation, certain mountants, and long-term storage of stained preparations can result in tissue fluorescence, and the high concentration of stains deposited in the sections can lead to metachromasia. In our hands, it is rare to find stained and sectioned botanical material that is not highly fluorescent. The digital controls of a confocal microscope allow for the clean separation of different fluorescent emission signals and the balancing of signal levels in different channels. Thus, fluorescent images of exceptional clarity and vivid color can be easily obtained. In addition, the optical sectioning properties of the confocal microscope can be used to collect clear images from within thick sections and wholemounts.