Accelerated Articles Ambient Molecular Imaging and Depth Profiling of Live Tissue by Infrared Laser Ablation Electrospray Ionization Mass Spectrometry Peter Nemes, Alexis A. Barton, Yue Li, and Akos Vertes* Department of Chemistry, W. M. Keck Institute for Proteomics Technology and Applications, George Washington University, Washington, D.C. 20052 Mass spectrometry in conjunction with atmospheric pres- sure ionization methods enables the in vivo investigation of biochemical changes with high specificity and sensitiv- ity. Laser ablation electrospray ionization (LAESI) is a recently introduced ambient ionization method suited for the analysis of biological samples with sufficient water content. With LAESI mass spectrometric analysis of chimeric Aphelandra squarrosa leaf tissue, we identify the metabolites characteristic for the green and yellow sectors of variegation. Significant parts of the related biosynthetic pathways (e.g., kaempferol biosynthesis) are ascertained from the detected metabolites and metabolomic data- bases. Scanning electron microscopy of the ablated areas indicates the feasibility of both two-dimensional imaging and depth profiling with a ∼350 μm lateral and ∼50 μm depth resolution. Molecular distributions of some endog- enous metabolites show chemical contrast between the sectors of variegation and quantitative changes as the ablation reaches the epidermal and mesophyll layers. Our results demonstrate that LAESI mass spectrometry opens a new way for ambient molecular imaging and depth profiling of metabolites in biological tissues and live organisms. In the past two decades, mass spectrometry (MS) has provided dramatic new insight into biological processes. The key factor that has enabled these advances is our ability to ionize and identify a wide range of molecular classes with high accuracy, excellent selectivity, and low detection limits. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) have brought peptides, proteins, their noncovalent complexes, and other biomolecular classes within the reach of detailed mass spectro- metric investigations. 1 While ESI calls for solution-phase samples of specific conductivity, MALDI requires the presence of a denaturing matrix and operates in vacuum. These conditions are incompatible with in vivo investigations. Consequently, there is a need for methods that achieve efficient ion generation under nativelike experimental conditions. Technical innovations of recent years have provided an arsenal of ambient ion sources 2 for MS. Many of them apply physical or chemical pretreatments or conditions that are known to disrupt living organisms; thus, the choice of techniques suitable for in vivo MS narrows down to a few. For example, desorption electrospray ionization has been applied successfully for the detection of drugs, metabolites, and explosives on human fingers, 3,4 on untreated Escherichia coli, and on different strains of Salmonella typhimurium cells. 5 Most recently, DESI-mass spectral fingerprints have allowed rapid biodetection for a variety of bacteria. 6 MALDE- SI, 7 a combination of MALDI and DESI, has enabled the characterization of intact polypeptides. 8 Human breath and fruit maturity have been investigated by extractive electrospray ionization. 9,10 Atmospheric pressure infrared MALDI (AP IR- MALDI), 11,12 electrospray-assisted laser desorption/ionization (ELDI), 13 and laser ablation electrospray ionization (LAESI) 14 employ focused laser radiation for ambient sampling. AP IR- MALDI and LAESI have proved particularly useful in targeting primary and secondary plant metabolites with no sample prepara- tion or chemical modification required. Following the introduction * To whom correspondence should be addressed. E-mail: vertes@gwu.edu. Phone: (202) 994-2717. Fax: (202) 994-5873 (1) Siuzdak, G. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 11290–11297. (2) Cooks, R. G.; Ouyang, Z.; Takats, Z.; Wiseman, J. M. Science 2006, 311, 1566–1570. (3) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471–473. (4) Justes, D. R.; Talaty, N.; Cotte-Rodriguez, I.; Cooks, R. G. Chem. Commun. 2007, 2142–2144. (5) Song, Y. S.; Talaty, N.; Tao, W. A.; Pan, Z. Z.; Cooks, R. G. Chem. Commun. 2007, 61–63. (6) Meetani, M. A.; Shin, Y. S.; Zhang, S. F.; Mayer, R.; Basile, F. J. Mass Spectrom. 2007, 42, 1186–1193. (7) Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1712–1716. (8) Sampson, J. S.; Hawkridge, A. 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