Bridge C will carry out basic research with the objective to develop mass spectrometric techniques that can be used to generate images of the specific location and relative abundance of lipids in tissues and cells. In part, this research program builds on the success during the first award period of using matrix assisted laser desorption ionization (MALDI) coupled to a quadrupole tandem time-of-flight mass spectrometer and application of matrix by sublimation onto thin tissues slices (10 um) to enhance ion yields following irradiation of small areas of tissue (approximately 100 um2) with a focused laser beam. The resultant secondary ions emitted are then mass analyzed and stored as the mass and intensity values at that pixel. The adjacent areas are irradiated sequentially to build a large database that can be used to generate images specific to the lipid ion (m/z) as a function of position and ion abundance. Sublimation of matrix was found to minimize lipid lateral diffusion in tissues and allow generation of detailed images of tissues based upon mass spectrometric pixels. Secondary ion mass spectrometry (SIMS) using Buckminster fullerene (C60 +) ion beams will be systematically studied to bring lateral resolution of lipid imaging below 1 um so that lipids in single cells can be detected and analyzed. Specific goals include development of derivatization chemistry to enhance lipid secondary ion yield in MALDI and SIMS imaging experiments to allow discovery of novel lipids. New tissue embedding materials will be developed and explored that will be specifically suited for mass spectrometric imaging. The reproducibility of lipid anatomical images will be assessed by analysis of sequential tissue slices as well as of identical tissue regions from different animals. Threedimensional rendering of lipid images from sequential slices will be pursued using software developed for MALDI and SIMs data sets. A major focus will be imaging of aortas and atherosclerotic plaques as part of the LIPID MAPS coordinated studies to reveal anatomical locations of specific lipids including but not limited to phospholipids, sphingolipids, and oxidized neutral lipids. In addition, this research will develop novel and powerful research methods that can be used to examine tissues (biopsies) taken from human subjects for molecules that mark unique disease processes such as atherosclerosis and diabetes.
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