Detecting Microparticles in Human Intestine in Patients with Inflammatory Bowel Disease with Synchrotron Based X-ray Beamline

Abstract

Purpose: Microparticles (MP) are non-biological inorganic bacterial-sized particles (0.1-0.7 μm) resulting from endogenous formation (calcium phosphate) or dietary intake (AlSi and Ti02). MPs may contribute to the pathogenesis of inflammatory bowel disease (IBD). To elucidate the actual role of MPs in IBD, it is important to directly evaluate the quantity and distribution of MPs at the cellular or sub cellular level in diseased and normal intestinal tissues, particularly the M-cell rich lymphoid aggregates (Peyer's patches), the major sites of MP entry into intestinal tissue. The aim of this study was to establish the method for MP detection in human intestinal tissue. Methods: Intestinal biopsy tissue samples were collected from 5 IBD and 5 colorectal cancer screening patients undergoing colonoscopy. Tissue sections were cut at variable thickness (5, 10 and 20 um) prior to analysis on the Very Sensitive Elemental and Structural Probe Employing Radiation from a Synchrotron (VESPERS) microprobe beamline at the Canadian Light Source (CLS). Tissue sections were mapped with micro-X-ray fluorescence (μXRF) spectroscopy in order to determine the spatial location of the MPs at an X-ray excitation energy of 13 keV. The X-ray beam had a spot size of 4 μm with an 8 μm step-size and 15 second dwell time. More in-depth chemical and structural analysis were performed on regions of interest in the XRF maps by micro-X-ray Diffraction (μXRD) and micro- X-ray absorption near-edge structure (μXANES) spectroscopy. Sample conditions that may have affected the results include formalin fixation, tissue section thickness, and the contents of the mounting substrate (regular glass slide and Lexan film) and embedding medium (OCT). This study was approved by Biomedical Research Ethics Board of University of Saskatchewan. Results: Randomly selected areas in the mucosa or submucosa were observed. There was significant contamination in formalin-fixed tissue samples resulting in a non specific background. VESPERS exhibited greatest sensitivity with tissue sections of 20 um in thickness and demonstrated the best signal versus noise ratio vs. 5 or 10 um thickness. Specific signals of Ti and Si from samples mounted on Lexan film were much better with relatively lower background than those on regular glass slide. The OCT was free of contamination. Conclusion: Synchrotron-based X-ray microprobe techniques can be used for detecting and mapping MPs in normal and IBD affected human intestine. This will allow us further elucidation of the immunopathological role of MPs in IBD patients. Frozen fresh tissue sections of 20 um thickness mounted on Lexan film is the superior method of sample processing.