Distribution of selected elements in calcific human aortic valves studied by microscopy combined with SR-µXRF: influence of lipids on progression of calcification.

Calcified heart valves display a significant imbalance in tissue content of trace and essential elements. The valvular calcification is an age-related process and there are data suggesting involvement of lipids. We studied elemental composition and lipid distribution in three distinct regions of calcified human aortic valves, representing successive stages of the calcific degeneration: normal, thickened (early lesion) and calcified (late lesion), using SR-µXRF (Synchrotron Radiation Micro X-Ray Fluorescence) for elemental composition and Oil Red O (ORO) staining for demonstration of lipids. Two-dimensional SR-µXRF maps and precise point spectra were compared with histological stainings on consecutive valve sections to prove topographical localization and colocalization of the examined elements and lipids. In calcified valve areas, accumulation of calcium and phosphorus was accompanied by enhanced concentrations of strontium and zinc. Calcifications preferentially developed in lipid-rich areas of the valves. Calcium concentration ratio between lipid-rich and lipid-free areas was not age-dependent in early lesions, but showed a significant increase with age in late lesions, indicating age-dependent intensification of lipid involvement in calcification process. The results suggest that mechanisms of calcification change with progression of valve degeneration and with age.

Enhancement in statistical and image analysis for in situ µSXRF studies of elemental distribution and co-localization, using Dioscorea balcanica.

Synchrotron-based X-ray microfluorescence (µSXRF) is an analytical method suitable for in situ investigation of the distribution of micronutrient and macronutrient elements in several-micrometres-thick unstained biological samples, e.g. single cells and tissues. Elements are mapped and quantified at sub-p.p.m. concentrations. In this study the quantity, distribution and grouping/co-localization of various elements have been identified in straight and twisted internodes of the stems of the monocotyledonous climber D. balcanica Kosanin. Three different statistical methods were employed to analyse the macronutrient and micronutrient distributions and co-localization. Macronutrient elements (K, P, Ca, Cl) are distributed homogeneously in both straight and twisted internodes. Micronutrient elements are mostly grouped in the vasculature and in the sclerenchyma cell layer. In addition, co-localization of micronutrient elements is much more prominent in twisted than in straight internodes. These image analyses and statistical methods provided very similar outcomes and could be applied to various types of biological samples imaged by µSXRF.

A confocal set-up for micro-XRF and XAFS experiments using diamond-anvil cells.

A confocal set-up is presented that improves micro-XRF and XAFS experiments with high-pressure diamond-anvil cells (DACs). In this experiment a probing volume is defined by the focus of the incoming synchrotron radiation beam and that of a polycapillary X-ray half-lens with a very long working distance, which is placed in front of the fluorescence detector. This set-up enhances the quality of the fluorescence and XAFS spectra, and thus the sensitivity for detecting elements at low concentrations. It efficiently suppresses signal from outside the sample chamber, which stems from elastic and inelastic scattering of the incoming beam by the diamond anvils as well as from excitation of fluorescence from the body of the DAC.