1 μm Thick Frozen Hydrated/Dried Sections for Analysing Pericellular Environment in Transport Epithelia; New Results from Old Data

Abstract

During the period 1973–84, we carried out electron probe X-ray microanalysis (EPXMA) of 1/um thick frozen-hydrated sections on a variety of ion and water transporting epithelia. The instrumentation and methodology have been described in a number of previous publications (Gupta et al., 1977; Gupta and Hall, 1979, 1981b, 1982; Hall and Gupta, 1983). Hall (1986) has discussed the advantages of using ∼1/um thick rather than ultrathin (∼200 nm) cryosections for EPXMA, in spite of the limited structural image resolution afforded, especially in a scanning microscope operating at 45–50 keV (JEOL JXA-50A). However, most of our biological results published up to 1983 were obtained without the facility of a fully comprehensive X-ray data reduction and conversion into mM kg⁻¹ wet or dry mass or mM 1⁻² H₂O, required by Hall’s continuum normalization procedure, especially with respect to actual Z²/A correction for every microvolume analyzed. Corrections for all the X-rays from extraneous sources may not have been as accurate because of limitations of computation facilities. The completion of the Link System QUANTEM/FLS soft-ware together with its dedicated computer and fast on-line print-out facilities (Gupta and Hall, 1982) allowed us to obtain more refined data on many tissues analyzed previously. The availability of a comprehensive standards file both for the chemical elements and the mass thickness (= section thickness under the beam) in the soft ware allowed us not to use bath Ringer + Dextran as peripheral standard in our in vitro preparations, as done previously; although we always established that 10–20 % Dextran (Mr 250,000) added to bath Ringers surrounding the epithelia in vitro did not measurably effect the transport function (Barnard et al., 1984). However, a Ringer’s solution with 20 % Dextran frozen and cut with every tissue block was still required as a ccperipheral standard’ in every section in order to establish the exact level of hydration of the sections. In the studies reported here, this ‘peripheral standard’ was added on the Ringer-surrounded piece of tissue (on the serosal side in ileum, Table 1) immediately before rapid freezing a 1a Münich group (Rick et al., 1982). Local dry mass and hence water fraction for every analyzed field were obtained by using formulations involving mas fractions of all the nuclides analyzed in every microvolume (Hall and Gupta, 1982; Table 2). Great care was taken that local continuum counts from the sections after drying were collected from an area of the subcompartment not previously analyzed in a frozen-hydrated state and hence had not suffered the latent mass loss due to beam damage. Here I report results concerning the ion and water composition of various pericellular compartments which in the normal living tissues always contain a ‘matrix’ of mucoid substances constituting a local dry mass of some 5% to 30%, depending on the site and the physiological state of the transport function, even though such pericellular compartments usually appear ‘empty’ in conventionally prepared material for light and electron microscopic histology without special precautions to fix mucoids. A more comprehensive discussion of the subject has recently been published (Gupta, 1989).