Subcellular chemical mapping using correlated cryogenic electron and mass spectrometry imaging

Electron cryomicroscopy (cryo-EM) allows high spatial resolution visualization of biological specimens; however, it is challenging to chemically identify densities observed in cryo-EM. To overcome this, we combined cryo-EM with chemical imaging using focused ion beam secondary ion mass spectrometry (FIB-SIMS) for integrated spatiochemical analysis of untagged specimens. We show that our correlative workflow permits subcellular localization of molecules inside bacterial cells and is compatible with cryogenic light microscopy and FIB-milled lamellae of eukaryotic specimens. To highlight biological insights enabled by the workflow, we studied the uptake of bisphenol-AF, a widespread chemical pollutant, by environmental bacteria, revealing the storage of these chemicals within cytosolic phase-separated aggregates in pollutant-exposed cells, where they cannot be removed by the bacterial efflux machinery despite its robust upregulation. Cryo-EM-FIB-SIMS therefore represents an effective approach to map elemental and molecular signatures in near-native biological samples.