Single Cell Analysis and Tissue Imaging by Laser Ablation and Mass Spectrometry

B. Shrestha

Columbian College of Arts and Sciences| The George Washington University
Dissertation for Doctor of Philosophy, January 31, 2010



The direct analysis of biochemicals in tissues and single cells is critical for understanding living organisms. Due to excellent selectivity and sensitivity, mass spectrometry (MS) has proven to be an invaluable tool for the analysis of the biomolecules. Recent developments in atmospheric pressure direct ionization sources have enabled the in situ analysis of biological samples without external influences (e.g., purification, extraction, matrix addition etc.) that might alter their biochemical makeup. The work presented in this dissertation shows my efforts to utilize two novel atmospheric pressure (AP) direct ionization methodologies, AP infrared (IR) matrixassisted laser desorption ionization (MALDI) and laser ablation electrospray ionization (LAESI) MS, for metabolomics, tissue imaging and single cell analysis.

Chapter 1 introduces analytical techniques used for the analysis of tissues and single cells. The fundamental aspects of IR laser ablation and its utilization in two direct ionization techniques, AP IR-MALDI and LAESI, are reviewed.

Chapter 2 introduces AP IR-MALDI for MS. It presents proof-of-principle molecular imaging of mock peptide samples at atmospheric pressure. The utility of AP IR-MALDI for plant tissue imaging and metabolomics are also discussed.

Chapter 3 describes the AP IR-MALDI analysis of various pharmaceuticals directly in their commercial formulations, as well as endogenous metabolites, exogenous drug metabolites and synthetic polymers in urine.

Chapter 4 presents the application of mid-infrared laser ablation for molecular imaging. The dynamics of the ablation plume and ion production in AP IR-MALDI and LAESI are compared.

In Chapter 5 metabolites and lipids are identified in mouse brain sections using MS with AP IR-MALDI and LAESI ion production. Reactive LAESI relies on interactions between the laser ablated species and reactants, e.g., Li+, introduced through the electrospray. This new modality of LAESI enables the analysis of samples with otherwise low ion yields.

Chapter 6 discusses the metabolic analysis of single cells by MS at atmospheric pressure. This breakthrough is made possible by the tight focusing of mid-IR laser light through an etched optical fiber tip and accurate aiming of cells for ablation through visualization and micromanipulation. Similar to conventional LAESI, the ablated plume is postionized by an electrospray.

Chapter 7 surveys the major challenges in the field of atmospheric pressure ion production based on mid-IR laser ablation. The need for the analysis of smaller cells, reactive LAESI-MS, ultrahigh resolution LAESI-MS, and the potential application of LAESI-MS in laser surgery are discussed.