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Background  Imaging mass spectrometry (imaging MS) is an enabling technology for spatial metabolomics of tissue sections with rapidly growing areas of applications in biology and medicine. However, imaging MS data is polluted with off-sample ions caused by sample preparation, particularly by the MALDI (matrix-assisted laser desorption/ionization) matrix application. Off-sample ion images confound and hinder statistical analysis, metabolite identification and downstream analysis with no automated solutions available.    Results  We developed an artificial intelligence approach to recognize off-sample ion images. First, we created a high-quality gold standard of 23,238 expert-tagged ion images from 87 public datasets from the METASPACE knowledge base. Next, we developed several machine and deep learning methods for recognizing off-sample ion images. The following methods were able to reproduce expert judgements with a high agreement: residual deep learning ( F 1-score 0.97), semi-automated spatio-molecular biclustering ( F 1-score 0.96), and molecular co-localization ( F 1-score 0.90). In a test-case study, we investigated off-sample images corresponding to the most common MALDI matrix (2,5-dihydroxybenzoic acid, DHB) and characterized properties of matrix clusters.    Conclusions  Overall, our work illustrates how artificial intelligence approaches enabled by open-access data, web technologies, and machine and deep learning open novel avenues to address long-standing challenges in imaging MS.

OffsampleAI: artificial intelligence approach to recognize off-sample mass spectrometry images