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The Pennsylvania State University the Graduate School The The Pennsylvania State University The Graduate School The Department of Chemistry NEW ANALYTICAL APPROACHES TO UNDERSTAND BIOLOGICAL SYSTEMS WITH SECONDARY ION MASS SPECTROMETRY A Dissertation in Chemistry by Lauren Marie Jackson 2014 Lauren Marie Jackson Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2014 The dissertation of Lauren Marie Jackson was reviewed and approved* by the following: Nicholas Winograd Evan Pugh Professor of Chemistry Dissertation Advisor Chair of Committee Wayne Curtis Professor of Chemical Engineering Thomas Mallouk Evan Pugh Professor of Materials Chemistry and Physics Associate Head of the Chemistry Department Barbara Garrison Shapiro Professor of Chemistry Head of the Chemistry Department *Signatures are on file in the Graduate School iii ABSTRACT The work of this thesis has focused on investigating biological systems with the most current secondary ion mass spectrometry instrumentation and novel sample preparation techniques. The microalga biofuel candidate, Botryococcus braunii, has been thoroughly investigated by three-dimensional chemical imaging to answer several questions regarding its metabolism and chemical composition. These findings provide a breadth of information beneficial and crucial to the successful advancement of B. braunii as a biofuel. The enhancement effects and cellular vitrification capabilities of trehalose have been explored and confirmed trehalose entry into cells has been found. This work is evidence of the great contributions to the biological realm to which SIMS can contribute. iv TABLE OF CONTENTS List of Figures ........................................................................................................... vii List of Tables ............................................................................................................... xi Acknowledgements ...................................................................................................... xii Chapter 1 Introduction ................................................................................................. 1 1.1. Background of Secondary Ion Mass Spectrometry Imaging and Instrumentation .............................................................................................. 1 1.2. Research Overview ........................................................................................ 4 1.3. References ...................................................................................................... 7 Chapter 2 The Use of Botryococcus braunii as a Simple Biological Model Sample for Diverse Secondary Ion Mass Spectrometry Analysis ..................................... 20 2.1. Introduction .................................................................................................... 20 2.2. Experimental .................................................................................................. 21 2.2.1. Algal Culture Conditions ..................................................................... 21 2.2.2. Algal Sample Preparation for SIMS Analyses .................................... 21 + 2.2.3. SIMS and SEM Algal Analyses with C60 Primary Ion Beam ............ 22 2.2.4. SIMS and SEM Algal Analyses with Argon Cluster Primary Ion Beam ....................................................................................................... 22 2.3. Results and Discussion .................................................................................. 23 2.3.1. The Tolerance of B. braunii Cells to the Vacuum Environment ......... 23 2.3.2. Three-Dimensional Analysis without the Effects of Charging ........... 26 2.3.3. The Variety of Chemical and Morphological Features in the B. braunii Colony ....................................................................................... 28 3.3.4. The Chemical Diversity of B. braunii observable by SIMS ............... 30 2.4. Conclusions.................................................................................................... 35 2.5. References ...................................................................................................... 37 Chapter 3 Understanding the Behavior of the Triterpene Hydrocarbons of Botryococcus braunii during SIMS Depth Profiling Experiments ....................... 46 3.1. Introduction .................................................................................................... 46 3.2. Experimental .................................................................................................. 48 3.1.1. Algal Culture Conditions ..................................................................... 48 3.1.2. Algal Sample Preparation for TOF-SIMS Analyses ........................... 49 3.1.3. Squalene standard sample preparation for depth profiling by SIMS analysis ................................................................................................... 49 v 3.1.3. SIMS depth profiling conditions for squalene standard and B. braunii analysis ...................................................................................... 50 3.3. Results and Discussion .................................................................................. 50 3.4. Conclusions.................................................................................................... 56 3.5. References ...................................................................................................... 56 Chapter 4 Elucidation of the Metabolic Processes of Three Dimensional Chemical Imaging of the Algal Biofuel Candidate, Botryococcus braunii .......................... 60 4.1. Introduction .................................................................................................... 60 4.2. Experimental .................................................................................................. 62 4.2.1. Algal Culture Conditions ..................................................................... 62 4.2.2. Algal Sample Preparation for TOF-SIMS Analyses ........................... 63 4.3.3. Instrument Parameters for SEM and TOF-SIMS Algal Analysis ....... 63 4.3. Results and Discussion .................................................................................. 64 4.4. Conclusion ..................................................................................................... 77 4.5. References ...................................................................................................... 78 Chapter 5 Trehalose as a biologically-relevant matrix for secondary ion enhancement and quantitative detection of purines for SIMS analysis ................ 82 5.1 Introduction ..................................................................................................... 82 5.1.1. The de novo Purine Biosynthetic Pathway and Associated Metabolites .... 84 5.2. Semi-Quantitative Detection of Purines ........................................................ 86 5.2.1. Experimental ........................................................................................ 86 5.2.2. Results and Discussion ........................................................................ 89 5.2.3. Conclusions ......................................................................................... 94 5.4. Determining the Enhancement Effect of Trehalose for Purine Detection ..... 96 5.4.1. Experimental ........................................................................................ 96 5.4.2. Results and Discussion ........................................................................ 98 6.4.3. Conclusions ......................................................................................... 104 5.5. New Trehalose Vitrification Technique for Three-Dimensional Single Cell Studies .................................................................................................... 105 5.5.1. Experimental ........................................................................................ 105 5.5.2. Results and Discussion ........................................................................ 107 5.5.3. Conclusions ......................................................................................... 116 5.6. References ...................................................................................................... 116 Chapter 6 Conclusions and Future Directions ............................................................. 121 6.1. Ongoing and Future Research ....................................................................... 121 6.1.1. The Potential Symbiotic Role of the Flavonoids of B. braunii ........... 121 6.1.2. Wax Monoester Expression and the Maintenance of an Axenic Line of B. braunii Race B ...................................................................... 122 6.1.3. Physiological Engineering of the Oil Bodies of B. braunii ................. 122 vi 6.1.4. Time-Course Chemical Imaging of Mammalian Cells ....................... 123 6.1.5. Guiding Principles for the Development of Biological Analyses with SIMS .............................................................................................. 124 6.2. Conclusory Remarks ...................................................................................... 125 6.3. References ...................................................................................................... 125 vii LIST OF FIGURES Figure 2-1: A comparison of Race A UTEX #2441 and #572 out- and in-vacuum environment of the SIMS instrument. Optical microscopy images
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