Analyzing Calcification in Atherosclerotic Plaques

One cm in diameter and 1 cm in length of 5 plaques with macroscopic evidence of an eruptive calcification and 5 fibrocalcific plaques were fixed in in 4% (v/v) paraformaldehyde for 24h. The samples were treated as previously described [27 (link)]. Critical point drying (Agar Scientific, Essex, UK, Elektron Technology UK Ltd., Cambridge, UK) with supercritical CO₂ was then performed to prevent cell deformation. EDX microanalysis was performed by using a liquid N₂-cooled Si detector with a super-ultrathin Be window on unpost-fixed samples placed on specific copper stubs. Spectra were collected by a SEM LEO 1450VP (Carl Zeiss Meditec, Oberkochen, Germany) scanning electron microscope at acceleration voltage of 5 KeV employing an area scan mode (640 × 640 μm sampling area), 300 s acquisition time, and 32-37% detector dead time. Analysis of acquired spectra was performed under a nonstandard mode using atomic number-absorption-florescence correction (ZAF) methods using Inca Energy software (Oxford Instruments Ltd., High Wycombe, UK; Si(Li) detector, ATW - atmospheric thin window, resolution 133 eV for MnKα at 10 000 counts). For each specimen, we acquire 5 spectra on 8 mm² of calcification surface in total.