Mass spectrometric imaging(MSI)is an emerging tool for biological research.Matrix-assisted laser desroption ioniization(MALDI)imaging is the most widely used MSI technology at present especially for protein imaging.Meanwhile,matrix-free molecular imaging at atmospheric pressure has been developed in the past decade.Molecular imaging of lipids and metabolites in tissues under ambient conditions is readily achieved using desorption electrospray ionization(DESI),but DESI is essentially blind to the presence of proteins.We demonstrated that the use of nanospray desorption electrospray ionization(nanoDESI)in combination with an inverted microscope overcomes this problem and allows us to visualize proteins in tissue samples in a label-free manner at atmospheric pressure with only minimum sample preparation.Later we interfaced a 2D stage with nanoDESI for direct and continuous mass spectrometry scannings on tissue sections.Multiply charged proteins with masses up to 15 kDa were successfully mapped.In a mice brain section,expression of proteins previously found by MALDI imaging,including ubiquitin,β-thymosin,and myelin basic protein,were spatially resolved,and then identified via top-down approaches.This imaging modality was further implemented to MYC-induced lymphomas.We observed an array of truncated proteins in the region where normal thymus cells were infiltrated by tumor cells,in contrast to healthy tissue.It is expected that this new tool will provide important biological insight into cellular processes.