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Respired particles accumulated in the epithelial regions of trachea and bronchi were identified and characterised using micro-PIXE elemental mapping of thin frozen sections carried out at the Oxford Nuclear Microprobe facility. Isolated particles with diameters of 2-10 mu m could be detected, mainly atthe trachea epithelial surface. In bronchi respiratory mucosa, granular regions can also be observed that may correspond to particle agglomerations (2-4 mu m diameters) and/or inclusions inmacrophages. Particles, observed in the upper regions of the respiratory tract consist mainly of earth crust elements such as Al, Si, Ca and Fe. Occasionally, Ti and Zn are also present. Particles observedin the bronchi have a more varied chemical composition. Elements such as V, Cr, Mn, Fe, Cu, Zn were detected, mainly in association with S, K, Ca or Si.

Immobilized cells of a Citrobacter sp. can remove heavy metals from wastewaters by deposition ofmetals with enzymatically liberated phosphate. Nickel is not removed effectively by this technique, but Ni2+ can be intercalated into cell-bound, crystalline HUO2PO4 previously deposited enzymatically. This technique for efficient removal of Ni from solution has been generically termed microbially enhanced chemisorption of heavy metals(MECHM). The nickel uranyl phosphate deposits bound to Citrobactersp. cells immobilized in polyacrylamide gel (FAG) were analyzed using scanning transmission electronmicroscopy with electron probe X-ray microanalysis (EPXMA) and proton-induced X-ray emissionanalysis(PIXE). Both methods gave the molar ratios of nickel, uranium, and phosphorus in the depositsas close to 1:2:2 in all analyzed parts of the sample. EPXMA proved that the deposits were localized onthe surface of cells inside FAG particles as well as those immobilized on the edge. Small deposits ofnickel uranyl phosphate were also found in FAG between the cells, indicating the possible involvementof extracellular polymeric substances (EPS) in the creation of intercellular deposits. These findings confirm the mechanism of MECHM and show that this mechanism operates throughout the immobilized cell matrix. The use of two independent methods of solid-state analysis in a common sample provides validation of both techniques for the spatial and quantitative analysis of biomass-bound elements.

To respond to varying environmental demands the bone tissue in the body is under continual reconstruction throughout life. It is known that metallic elements are important for maintaining normal bone structure, bur their roles are not well understood. More information about the effects of metal excess or deficiency is needed to help in the development of metallic bone implants and to improve the treatment of bone fractures and defects. The Oxford Scanning Proton Microprobe (SPM) is being applied in two studies involving metal ions in bone: (1) bone regrowth and bonding to titanium bone implants may be influenced by diffusion of Ti ions into the bone. We are using microPIXE to determine the metal ion content of bone developing in contact with implants of pure Nb, Ti and Ti alloys. (2) Bone lengthening as a surgical procedure is induced by fracturing the bone and allowing it to heal with a small gap between the fractured ends created by the use of external fixators. The gap can be slowly increased during the healing process to stimulate the production of new bone. The enzymes and other constituents of the developing bone need certain metals for their function. Using experimental animals we have studied the concentrations of the metals and whether a deficiency of trace metals limits the optimum rate of bone lengthening.

The nuclear microprobe was used to study elemental distributions in cultured insulin-producing cellsand in pancreatic tissue. To see whether insulin producing cells are polarized, the cells were stimulatedin the presence of Sr as a Ca analogue. The cells were stimulated to secrete insulin and Sr was detected in cells after momentary stimulation. We did not detect a polarized distribution of Sr in the RINm5F-cells. The distributions of Ca and Zn were affected when beta-cells (ob/ob) from a primary culture flattened out onto a substrate. In uprounded beta-cells (ob/ob) the distribution of Ca and Zn was polarized. Pancreatic tissue (ob/ob) was prepared for elemental analysis using freeze-substitution intetrahydrofurane (THF) followed by embedding in Araldite. The resulting samples can be sectioned at room temperature, easing physiological studies.

A new freeze-substitution method using tetrahydrofuran (THF) as organic solvent was tested in the preparation of pancreas for determination of elemental content and distribution. Particle induced X-rayemission (PIXE) allowed analysis at the cellular level and X-ray microanalysis (EPMA) at the subcellular level. Using PIXE allowed detection of Zn, Ca and Fe, not detectable in the EPMA spectra. The ratios of Cl and K to P and to S obtained from analysis using both PIXE and EPMA agreed well. A good preservation of the in vivo ionic content was manifested in high K to Na and K to Cl ratios.

The most mobile and biological and chemically active fractions of trace metals in sediments are the dissolved components present in pore waters. Measuring metals in pore waters is complicated by the requirement for anoxic handling procedures. Due to the dynamic nature of sediment, steep concentration gradients extending over as little as 1 mm may develop at the sediment-water interface. New procedures for measuring metals in pore waters using polyacrylamide gels as in-situ probes are described. The gel can be used to establish a diffusive equilibration in a thin-film (DET). Because the film is typically less than 1 mm thick, equilibration is achieved within five minutes and insertion of the gel assembly causes minimal disturbance of sediment. An alternative procedure is to use a diffusive gradient in a thin-film (DGT), whereby a monolayer of chelating resin is incorporated at one side of the gel. Such a technique provides a kinetic measurement of labile species in solution. If the supply of metal from solid phase sediment to pore waters is fast enough, DGT provides a quantitative estimate of labile metal concentration. Alternatively, it measures directly the rate of supply of metal from solid phase to pore waters. As both DET and DGT are simple procedures capable of submillimetre spatial resolution, they provide previously unobtainable information on trace metal concentrations and fluxes. Furthermore, DGT has the potential to be used as a long-term monitor, providing mean concentrations of metals in sediment pore waters over periods of days, weeks, or even months.

Elemental redistribution induced by insulin secretion, was investigated by electron and proton probe X-ray microanalysis. In particular, ion fluxes following immediately upon stimulation were studied. As the sensitivity of the electron probe was insufficient, the proton microprobe was employed. In order to see whether the cell is asymmetric with respect to Ca2+ influx, the cells were stimulated in the presence of Sr2+ (as a Ca2+ analog). Insulin-secreting cells (RINm5F cells and isolated mouse beta-cells) were cultured on grids and shock-frozen at 2-30 seconds after stimulation. In a large number of cells, the major elements and and large fluxes were analyzed by the electron microprobe. In the proton microprobe, selected cells were analyzed and elemental maps were compared with electron micrographs of the same cells. The proton microprobe, but not the electron microprobe, could detect an influx of Sr in response to K+-stimulation for 2 seconds, in RINm5F cells. No polarization of Sr2+ uptake in RINm5F-cells could be detected, and the beta-cells did not respond to high K+ by uptake of Sr. Momentary stimulation of beta-cells also resulted in a significant increase in Na, detected by the electron probe. Spreading of the beta-cells on the substrate appears to influence the subcellular elemental distribution. Thus, the proton probe has potential to detect small changes in elements such as those occurring after short-time stimulation.