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Title:Mass spectrometry imaging for probing molecular distributions in the nervous system
Author(s):Tucker, Kevin R.
Director of Research:Sweedler, Jonathan V.
Doctoral Committee Chair(s):Sweedler, Jonathan V.
Doctoral Committee Member(s):Bailey, Ryan C.; Scheeline, Alexander; Lisy, James M.
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Mass Spectrometry Imaging
Abstract:Biological systems include a highly intertwined and complex set of interactions in which molecules vary spatially and temporally in addition to being present in concentrations that extend over many orders of magnitude. These chemical entities belong to numerous classes including inorganic salts, metabolites, lipids, peptides and proteins, each presenting its own set of analytical challenges. Here, these challenges were addressed via mass spectrometry. Different mass spectrometric technologies have been applied to probe the identity and spatial distribution of analytes within spinal cord from Rattus norvegicus and neurons from Aplysia californica. Mass spectrometry imaging (MSI) is a technique that allows for mass measurement of analytes, while also providing their spatial location and does not require a priori knowledge of the analytes to be imaged. MSI is capable of generating ion distribution images of many analytes within a single experiment as a result of the multiplexed detection capabilities that record a full mass spectrum at each position across the sample. MSI has been performed using secondary ion mass spectrometry (SIMS) for single cell imaging and matrix-assisted laser desorption/ionization mass spectrometry for the imaging of tissue sections. The combined use of these two complementary techniques allows for probing molecular distributions over a wide range of mass-to-charge ratios at spatial resolutions, ranging from submicron to hundreds of microns. Several method development projects were undertaken to improve the spatial resolution obtainable and to increase the information content that is collected during MSI experiments. MSI of rat spinal cord determined the localization of lipids, peptides and proteins within a single experiment using enzyme-modified beads mounted to a stretchable membrane. These beads were used to perform in situ digestion of spatially isolated tissue fragments generating digest peptide used to identify the proteins present. In a similar application, tissue was mounted directly on the stretchable membrane, which was then stretched, fragmenting the tissue prior to analysis using MSI. This stretched tissue mounting resulted in a 25-fold increase in pixel density, allowing for the visualization of smaller tissue features. Additionally, protocols were developed for the treatment of cultured neurons in order to preserve cellular morphology and improve signal from different analyte classes during SIMS imaging. A SIMS instrument using a primary cluster ion source, which produces more secondary molecular ions than primary atomic ion sources do, was used to perform analyses of single cells. The secondary molecular ions from the single cells were then fragmented using tandem mass spectrometry, a new feature for a SIMS instrument, in order to provide confident analyte identification. In summary, these new methods improved accurate characterization of the spatial distributions of a wide range of analytes within the neuronal systems of Rattus and Aplysia. Additionally, the developed methods will aid researchers in addressing increasingly complex questions about neuroscience.
Issue Date:2012-02-01
URI:http://hdl.handle.net/2142/29419
Rights Information:Copyright 2011 Kevin R. Tucker
Date Available in IDEALS:2014-02-01
Date Deposited:2011-12


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