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MALDI imaging provides molecular information from tissue and to detect biomarkers. Lateral resolution (“pixel size”) of MALDI images determines the level of morphological correlation between molecular and histological image. Therefore, increasing the image resolution at no loss in signal intensity is paramount for a method that is meant to provide clinically relevant results. Lateral resolution is limited by sample preparation protocols, laser properties and instrument sensitivity. In addition, high acquisition (multiple pixesl/second) speed is required to keep measurement times for high resolution images in reasonable limits. We developed a novel laser design in a new axial MALDI-TOF instrument, which enables 20μm image resolution for proteins at 1 kHz laser repetition rate. Using classical Nd:YAG-solid state lasers with Gaussian energy profiles, we observed a dramatic loss in signal intensity from MALDI images - even at focus diameters exceeding 100 μm. Smartbeam solid state lasers employ a structured, non-Gaussian beam profile instead that counteracts this limitation efficiently. Until recently, this effect was limited to larger (~80μm) focus sizes, but the redesigned smartbeam II optics used in the present study allow beam profile modulation down to 10μm while maintaining an efficient readout, which is a prerequisite for high definition MALDI imaging. We introduce rat testis tissue as an excellent model system to demonstrate lateral resolution in a MALDI imaging experiment. A single tissue cross-section shows numerous seminiferous tubules of ~200μm diameter. Different stages of spermatid development are present simultaneously, but can only be visualized in a MALDI image if the resolution is sufficient to separate tubule wall and spermatocytes based on distinct molecular signals (~20 ′ 20 μm pixel size). In addition, we demonstrate the importance of performing post-acquisition histological staining of the same section and co-registering digital photographs at microscopic resolution (from a Zeiss MIRAX scanner) for the interpretation of MALDI images.