Extended Data Fig. 6: Cryogenic FIB-SIMS of E. coli ∆tolC exposed to 250 µM PFNA show intra-cellular accumulation of PFAS compared to the DMSO control.

During cryogenic FIB-SIMS imaging, the vitrified sample is scanned by a Gallium focused ion beam (FIB) and time-of-flight secondary electron mass spectrometry (ToF-SIMS) is performed on the secondary ions created by the interaction of ion beam and sample at each scanning position. Thus, the chemical composition of the sample can be visualised pixel-by-pixel. a-c. E. coli ∆tolC exposed to 250 µM PFNA. d-f. E. coli ∆tolC exposed to DMSO control. For both cases, a spatial image of a cell resulting from the secondary electrons created during FIB imaging is shown on the left (a,d). Please note that because the specimens were frozen on a holey cryo-EM grid, round holes in the film are additionally observed, in addition to the elongated cells. On the right (b,c,e,f), ToF-SIMS images of the same cell are shown for mass-to-charge ratios 19 and 63, corresponding to F⁻ and PO₂⁻, respectively. The colour bars indicate ion count, that is, regions shown in red are those where the most ions of the particular species were detected. F⁻ signal indicates the presence of PFNA, PO₂⁻ was chosen as a comparison as it occurs in most bacterial cells and hence is a reliable marker for cells in negative SIMS imaging mode. In E. coli ∆tolC cells exposed to PFNA, a strong F⁻ signal is observed within the cell (b), which is not the case for the DMSO treated cells (e), in which a faint background signal can only be observed when adjusting the colour scale by several orders of magnitude.