X-ray fluorescence microscopy (XFM) facilitates high-sensitivity quantitative imaging of trace metals at high spatial resolution over large sample areas and can be applied to a diverse range of biological samples. Accurate determination of elemental content from recorded spectra requires proper calibration of the XFM instrument under the relevant operating conditions. Here, we describe the manufacture, characterization, and utilization of multi-element thin-film reference foils for use in calibration of XFM measurements of biological and other specimens. We have used these internal standards to assess the two-dimensional distribution of trace metals in a thin tissue section of a rat hippocampus. The data used in this study was acquired at the XFM beamline of the Australian Synchrotron using a new 384-element array detector (Maia) and at beamline 2-ID-E at the Advanced Photon Source. Post-processing of samples by different fixation techniques was investigated, with the conclusion that differences in solvent type and sample handling can significantly alter elemental content. The present study highlights the quantitative capability, high statistical power, and versatility of the XFM technique for mapping trace metals in biological samples, e.g., brain tissue samples in order to help understand neurological processes, especially when implemented in conjunction with a high-performance detector such as Maia.

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The moisture uptake and molecular mobility of freeze-dried powders containing whey protein isolate-carbohydrate matrices (1WPI:2maltodextrin; 1WPI:1maltodextrin:1d-glucose; and 1WPI:1maltodextrin:1l-glucose) and encapsulated Lactobacillus rhamnosus GG (LGG) in these matrices were investigated at 25 °C and 33% and 70% relative humidity (RH). The inactivation rate constant for probiotics in freeze-dried matrices were positively correlated (R(2) = 0.98) to moisture uptake and molecular mobility measured by NMR relaxometry. The stability of probiotics in glassy protein-carbohydrate matrices was dependent on the composition of the matrix. The partial substitution of maltodextrin with glucose (d- or l-) which improved microbial survival at 33% RH was related to the reduced molecular mobility and lower water uptake of the matrix. This study suggests that moisture uptake properties and molecular mobility of the matrix composition, as opposed to the relative humidity of the environment, are better determinants of probiotic viability during storage. Dynamic vapour sorption and NMR relaxometry are promising tools to assist in the selection of protein-carbohydrate matrices for enhancing probiotic viability during storage.