Chapter 9 Laser Ablation Electrospray Ionization for Atmospheric Pressure Molecular Imaging Mass Spectrometry Peter Nemes and Akos Vertes Abstract Laser ablation electrospray ionization (LAESI) is a novel method for the direct imaging of biological tis- sues by mass spectrometry. By performing ionization in the ambient environment, this technique enables in vivo studies with potential for single-cell analysis. A unique aspect of LAESI mass spectrometric imag- ing (MSI) is depth profiling that, in combination with lateral imaging, permits 3D molecular imaging for the first time under native conditions. With current lateral and depth resolutions of ∼100 and ∼40 μm, respectively, LAESI MSI helps to explore the molecular architecture of live tissues. Key words: Mass spectrometry, imaging, ambient, direct analysis, depth profiling, three- dimensional, in vivo, tissue imaging. 1. Introduction Traditional mass spectrometric imaging (MSI) methods, such as matrix-assisted laser desorption ionization (MALDI) and sec- ondary ion mass spectrometry (SIMS), have become important tools for the investigation of molecular distributions in tissues due to their high ionization efficiencies and excellent lateral and depth resolutions. Invasive sample preparation and the need for vacuum conditions, however, are incompatible with the analysis of live samples. Novel ionization methods in ambient mass spectrometry (1) overcome these limitations by performing imaging under native conditions. Desorption electrospray ionization (2), atmospheric pressure (AP) mid-infrared (mid-IR) MALDI (3), laser ablation S.S. Rubakhin, J.V. Sweedler (eds.), Mass Spectrometry Imaging, Methods in Molecular Biology 656, DOI 10.1007/978-1-60761-746-4_9, © Springer Science+Business Media, LLC 2010 159