Computational Separation of Conformational Heterogeneity using Cryo-Electron Tomography and 3D Sub-Volume Averaging

1793-Pos Board B685 Detecting Microparticles in Human Intestine with Synchrotron Based X-Ray Beamline Ronghua Zhao1, Emil Hallin2, Lachlan MacLean2, Renfei Feng2, Jennifer Jones1. College of Medicine, Saskatoon, SK, Canada, Canadian Light Source, Saskatoon, SK, Canada. Purpose: Microparticles (MP) are non-biological inorganic bacterial-sized particles (0.1-0.7mm) from endogenous formation (calcium phosphate) or dietary intake (AlSi and Ti02), which may contribute to the pathogenesis of inflammatory bowel disease (IBD). This study was to establish the method for MP detection in human intestine. Methods: Biopsy tissue samples were collected from 5 IBD and 5 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. They were mapped with micro-X-ray fluorescence spectroscopy to determine the spatial location of the MPs at an X-ray excitation energy of 13 keV. The beam had a spot size of 4 mm with an 8 mm step-size and 15 second dwell time. Tested sample conditions include formalin fixation, section thickness, mounting substrate (regular glass slide and Lexan film), and embedding medium (OCT). Results: Randomly selected areas in the mucosa or submucosa were observed. There was significant contamination in formalin-fixed tissue samples causing non-specific background. VESPERS exhibited greatest sensitivity with tissue sections of 20 um in thickness and demonstrated the best signal versus noise ratio. Specific signals of MPs from samples on Lexan film were much better with relatively lower background than those on 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.