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28.See supplementary material at for additional experimental descriptions and figures.[Supplementary Material]

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A vacuum compatible microfluidic reactor, SALVI (System for Analysis at the Liquid Vacuum Interface), was employed for chemical imaging of live biofilms using time-of-flight secondary ion mass spectrometry (ToF-SIMS). Depth profiling by sputtering materials in sequential layers resulted in live biofilmspatial chemical mapping. Two-dimensional (2D) images were reconstructed to report the first three-dimensionalimages of hydrated biofilm elucidating spatial and chemical heterogeneity. 2D image principal component analysis was conducted among biofilms at different locations in the microchannel. Our approach directly visualizedspatial and chemical heterogeneity within the living biofilm by dynamic liquid ToF-SIMS.


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