Stomatal Characterization of Grass Leaves by Four Preparation Techniques

Four leaf preparation techniques (air drying, tetramethylsilane air drying, critical point drying, and freeze substitution) used in scanning electron microscopy (SEM) were evaluated with respect to the degree of cellular distortion they produce in stomatal guard cells of leaves of Dactylis glomerata and Elymus canadensis. Surface morphological distortion and cuticle disruption in the air-dried and tetramethylsilane air-dried leaves, and cuticle disruption within the critical point-dried tissue made it difficult to obtain measurements.The freeze-substituted tissue experienced little cuticle disturbance, and the cellular morphology appeared normal. The length of the guard cells did not significantly differ between the air-dried, tetramethylsilane air-dried, critical point-dried, or freeze-substituted samples. Widths did significantly vary, with the freeze-substituted tissue having lower values than tissues treated with the other treatments. Freeze substitution methodology produced SEM images that appear to be less distorted and allow easy and precise measurement.