Fig 1: Sphingolipid ExM visualizes intracellular pathogens and their interactions with mitochondrial proteins.a–c Cells were infected with Simkania negevensis for 96 h, fed with α-NH2-ω-N3-C6-ceramide, fixed, permeabilized and stained with DBCO-Alexa Fluor 488 (green), and then imaged. The images show different cells before expansion (a), after 4x expansion (b), and 10x expansion (c) recorded by confocal microscopy. d–f Cells were infected with Chlamydia trachomatis for 24 h, fed with α-NH2-ω-N3-C6-ceramide, fixed, permeabilized and stained with DBCO-Alexa Fluor 488 (green). Different cells were imaged before expansion (d), after 4x expansion (e), and 10x expansion (f) by confocal microscopy. In the unexpanded (d) and 4x expanded image (e) chlamydial HSP60 was immunolabeled with ATTO647N secondary antibody (magenta). g The mitochondrial marker protein Prx3 was stained by immunolabeling with an ATTO 647N secondary antibody (magenta). The confocal fluorescence image of 10x expanded samples revealed a close contact between Chlamydia and mitochondria at the inclusion membrane. h SIM images of 10x expanded samples uncover that some Prx3 molecule are inserted into the bacterial membrane. The data were obtained from n = 3 independent experiments. Scale bars, 5 µm (unexpanded images a, d), 10 µm (4x and 10x expanded images b, c, e, f, g, h), and 2 µm (magnified views in images g, h).