No abstract

K E Y W O R D S . Scanning electron microscopy (SEM), resin histology, transmission electron microscopy (TEM), small intestine, frozen hydrated, mouse, cryomicroscopy, ice crystal damage. S U M M A R YEtched frozen hydrated specimens of mouse small intestine have been examined with low temperature scanning electron microscopy as a preliminary to X-ray microanalysis. Recognizable images have been obtained of most of the known histological features of the gut. Nuclear and cytoplasmic details were often seen. Ice crystal damage was evident, although the degree of artefact depended on the cell type being examined and also varied from cell to cell or within cells.The same specimens were later examined with resin light microscopy and transmission electron microscopy. These two techniques confirmed that preservation was adequate for identification of cells and tissues, although cavities were seen, representing ice crystal damage.These preliminary results indicate that SEM of etched, frozen, hydrated specimens provides adequate identification of cellular detail to allow further work using X-ray microanalysis to be carried out.

Although sections of frozen salt solutions have been used as standards for x-ray microanalysis, such solutions are less useful when analysed in the bulk form. They are poor thermal and electrical conductors and severe phase separation occurs during the cooling process. Following a suggestion by Whitecross et al we have made up a series of salt solutions containing a small amount of graphite to improve the sample conductivity. In addition, we have incorporated a polymer to ensure the formation of microcrystalline ice and a consequent homogenity of salt dispersion within the frozen matrix. The mixtures have been used to standardize the analytical procedures applied to frozen hydrated bulk specimens based on the peak/background analytical method and to measure the absolute concentration of elements in developing roots.

Low temperature x-ray microanalysis (LTXM) of solid biological materials has been documented for Lemna minor L. root tips. This discussion will be limited to a demonstration of LTXM for measuring relative elemental distributions of P,S,Cl and K species within whole cells of tobacco leaves.Mature Wisconsin-38 tobacco was grown in the greenhouse at the University of California, Berkeley and picked daily from the mid-stalk position (leaf #9). The tissue was excised from the right of the mid rib and rapidly frozen in liquid nitrogen slush. It was then placed into an Amray biochamber and maintained at 103K. Fracture faces of the tissue were prepared and carbon-coated in the biochamber. The prepared sample was transferred from the biochamber to the Amray 1000A SEM equipped with a cold stage to maintain low temperatures at 103K. Analyses were performed using a tungsten source with accelerating voltages of 17.5 to 20 KV and beam currents from 1-2nA.