Use of electron microprobe x-ray analysis for determination of low calcium concentrations across leaves deficient in calcium.

An electron microprobe with wavelength-dispersive x-ray spectrometry (WDS) was found to be useful for the determination of Ca concentrations in leaf tissue deficient in Ca. WDS effectively detected Ca concentrations as low as 0.2 mg/g dry wt in the presence of high levels of K and Mg (120 and 50 mg/g dry wt, respectively). Leaf specimens were prepared for analysis by quick-freezing in liquid nitrogen and freeze-drying at -20 degrees C to maintain elemental integrity within the tissue. Because dry material was analyzed, sample preparation was simple and samples could be stored for long periods before analysis. A large beam diameter of 50 gm was used to minimize tissue damage under the beam and analyze mineral concentrations within several cells at one time. Beam penetration was between 50 and 55 microns, approximately one-third of the thickness of the leaf. For analysis of concentrations in interveinal areas, analyses directed into the abaxial epidermis were found most useful. However, because of limited beam penetration, analyses of veinal areas would require use of cross sections [correction of crosssections]. Solid mineral standards were used for instrument standardization. To prevent measurement errors resulting from differences between the matrix of the mineral standards and the analyzed tissue, concentrations in leaves were corrected using gelatin standards prepared and analyzed under the same conditions. WDS was found to be useful for documenting that very low Ca levels occur in specific areas of lettuce leaves exhibiting the Ca deficiency injury termed tipburn.