The preparation and X‐ray microanalysis of bulk frozen hydrated vacuolate plant tissue

Abstract

SUMMARYA series of modifications have been devised which allow the peak to background ratio X‐ray analytical method to be used more effectively to measure elemental concentrations in large vacuolate plant cells. Planar, frozen‐hydrated fracture faces of bulk plant tissue are coated with a thin film of evaporated chromium, which prevents surface charging. Provided the film is sufficiently thin, c. 5–10 nm, there is no attenuation of the electron beam and only a small absorption of soft X‐rays. The chromium makes a small but measurable contribution to the spectral background and suitable corrections may be made to the quantitative results. An improved back‐scattered imaging system is described, which helps to overcome the problem of spurious X‐ray signals from rough surfaces. The microscope column has been modified to permit a continuous readout of beam current, sensu stricta, during X‐ray microanalysis and to allow rapid exchange of the electron gun assembly during low temperature operation. Calculations are given relating the size of the X‐ray interactive volume to electron penetration and X‐ray emission in both frozen hydrated and frozen dried cells. The problems of X‐ray microanalysis are discussed in relation to the highly vacuolate cells found in most mature plant tissues and an example given of the distribution of four major cations in tobacco leaves.