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Tissue microarrays (TMAs) are commonly used for the rapid analysis of large numbers of tissue samples, often in morphological assessments but increasingly in spectroscopic analysis, where specific molecular markers are targeted via immunostaining. Here we report the use of an automated high-throughput system based on desorption electrospray ionization (DESI) mass spectrometry (MS) for the rapid generation and online analysis of high-density (6144 samples/array) TMAs, at rates better than 1 sample/second. Direct open-air analysis of tissu