Detection of Metals in Multiple Sclerosis Brain Tissue using Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray (EDX) Analyses Rebecca Stearns 1 , Charles R.G. Guttmann 2 , Rohit Bakshi 3 , Umberto DeGirolami 4 , Brice Due 4 and John J. Godleski 1,4 1 Harvard School of Public Health, Dept. of Environmental Health, Boston MA 02115 2 Brigham and Woman’s Hospital, Harvard Medical School, Dept. of Radiology, Boston, MA 02115 3 Brigham and Woman’s Hospital, Harvard Medical School, Dept. of Neurology, Boston, MA 02115 4 Brigham and Woman’s Hospital, Harvard Medical School, Dept. of Pathology, Boston, MA 02115 Iron deposition in the brain of patients with multiple sclerosis (MS) has been suggested by previous magnetic resonance imaging (MRI) [1] and histologic studies [1, 2]. Previous studies have relied on light microscopy (LM) techniques and have focused on white matter deposition, while MRI studies suggest that high levels of iron are present in the gray matter, as well [1]. These findings are of interest for further study due to the potentially neurotoxic role of iron in neurologic diseases [3]. The primary goal of this work was to assess the feasibility of EDX for the localization and elemental confirmation of iron suggested by histochemical staining and MRI as well as to assess the presence of other elements in various locations in brain tissue. Since access to autopsy material from MS patients is limited and difficult to control, the feasibility of retrospective EDX characterization of paraffin embedded tissue is of significant interest. By working in the backscatter (BS) mode in SEM, foci of material of higher atomic number may be imaged in relationship to tissue comprised of lower atomic number elements such as C, N, and O [4]. Foci containing significant concentrations of elements with high atomic number such as Fe, our primary target, should be readily detectable, if present. Sections of paraffin-embedded brain tissue (7 µ thick) from autopsies of a MS patient and one age- matched control were mounted on to 2.5 cm carbon planchettes (EMS Fort Washington, Pa). Uncoated samples were analyzed in BS-mode with a LEO 1450VP Scanning Electron Microscope (LEO Electron Microscopy, Inc., Thornwood, New York) and Oxford INCA 300 microanalysis system (Oxford Instruments America, Inc., Concord, MA) with a silicon detector and by using Oxford’s Inca software. The tissue sections were systematically searched for foci of material highlighted in BS, and then identified foci were analyzed by EDX. Each section had at least 4 fields analyzed. The parameters for x-ray analysis were kept constant: magnification = 750x, accelerating voltage = 15 kV, and live time = 120 secs. Figure 1a is a hemotoxylin and eosin stain (H&E) of control with no evidence of inorganic material. In the control with SEM-EDX, four foci were found, containing calcium and sulfur (Fig.1c, e). The foci in the MS patient (Fig.1b, d, f) contained iron along with barium in 3 foci in association with small vessels as well as in the neuropil. Correlation of histochemical iron stain of MS (Fig. 1b) correlated well with the SEM-EDX findings (Fig.1d and f). This study shows the feasibility of using SEM-EDX to detect and characterize abnormal iron deposition in the brain in MS. The SEM-EDX approach will also be applied to random fields to assess the potential of detecting low concentrations Microsc Microanal 10(Suppl 2), 2004 Copyright 2004 Microscopy Society of America DOI: 10.1017/S1431927604883685 1342