Variation in stomatal aperture in leaves of Avena fatua L. observed by low-temperature scanning electron microscopy

Abstract. A novel technique to record the variability of stomatal aperture over the leaf surface is described. This combines observations of leaf surfaces using low-temperature scanning electron microscopy (LTSEM), with digital image analysis to produce the most accurate aperture measurements obtained to date. Leaf samples are rapidly immobilized by cryo-fixation in liquid nitrogen and stored in a purpose-built cryo-storage system. Specimens can be collected in the field, remote from the cryopreparation system, and stored for up to several weeks before being examined on the LTSEM. The advantages of this method are that the time frame of the measurements is accurately known, and is identical for all stomatal apertures in a sample, and the precision of the measurements is not limited by the resolving power of the microscope. Measurements of stomatal aperture were obtained from leaves of field grown Avena fatua using the above procedure. Leaf surface conductance (g_sur) was determined by porometry immediately before cryo-fixation of the same region of the leaf. Measurements of aperture size showed a high degree of variability within each specimen, with coefficients of variation similar to those found in previous studies. Stomatal conductance (g_s) was calculated from stomatal dimensions using formulae derived elsewhere. A linear regression between the computed values of g_s and porometric estimates of g_sur showed good agreement with the regression line passing through the origin with a slope of 1.0 (R²=0.96). Applications of the experimental system are discussed.