This dissertation elucidates the instrumentation and application of a hybrid ambient ionization source, laser electrospray mass spectrometry (LEMS), for the direct analysis and molecular imaging of biological tissue without matrix deposition. In LEMS, laser pulses from a Ti:Sapphire laser amplifier (60 fs, 800 nm, and 1 mJ) interact with surface analytes and transfer them from the condensed phase into the gas phase without the requirement of either exogenous matrix or endogenous water in the

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... ple. The laser vaporized analytes are captured and ionized by an electrospray source, and finally detected by a mass analyzer. It was found that a turn-key, robust femtosecond fiber laser with longer wavelength, longer duration, and lower pulse energy at 1042 nm, 425 fs, and 50 µJ, respectively, provided comparable results with the Ti:Sapphire laser. Vaporization of intact, dried or aqueous cytochrome c and lysozyme samples was demonstrated by the fiber laser. A charge states distribution at lower charge states indicating folded conformation of proteins and the hemoglobin α subunit-heme complex from whole blood was observed. Endogenous anthocyanins, sugars, and other metabolites were detected and revealed the anticipated metabolite profile for the flower petal and leaf samples by the fiber laser. Phospholipids, especially phosphatidylcholine, were identified from a fresh mouse brain section sample. These lipid features were suppressed in both the fiber laser and Ti:Sapphire LEMS measurement in the presence of optimal cutting temperature compounds which are commonly used in animal tissue cryosectioning. This dissertation also details the design of an automated mass spectrometry imaging source based on the Ti:Sapphire LEMS. The laser, translation stage, and mass analyzer are synchronized and controlled using a customized user interface to enable step-by-step scanning of the area of interest on a given tissue sample. The imaging source is coupled with a high resolution accurate mass quadrupole time-of-flight (QTOF) mass analy [...]