BPS2026 – Molecular-scale dissection of bacterial protein interactions with 3D expansion-PALM

Super-resolution microscopy has revolutionized biological imaging by enabling visualization of cellular structures beyond the diffraction limit. Yet, applying 3D single-molecule localization microscopy bacterial cells, remains highly challenging. In highly confined samples asymmetric point spread functions overlap is predominant, causing significant loss of localizations, thus affecting structure sampling and the final image resolution. Here, we present a novel approach combining photo-activated localization microscopy (PALM) with expansion microscopy (ExM), termed Ex-PALM, to achieve multicolor 3D super-resolution imaging in Escherichia coli. By physically expanding bacterial cells up to fourfold, ExM reduces molecular crowding and restores the ability to localize individual photoactivable fluorescent proteins in 3D. We optimized bacterial expansion protocols, including cell wall digestion and protein anchoring, to ensure isotropic expansion and reproducibility. Additionally, we corrected chromatic aberrations and signal crosstalk, and applied highly selective inclined illumination to maximize single-molecule sensitivity. Together, these advances enable single-molecule localization with ∼5 nm precision in 2D and ∼11 nm in 3D, representing an order-of-magnitude improvement over PALM alone. We applied Ex-PALM to investigate the spatial organization of HisF and HisH enzymes involved in the histidine biosynthetic pathway of E. coli. By tagging each protein with distinct photoactivable fluorescent proteins (PAGFP and PAmCherry1, respectively), we quantified their co-localization in 3D and measured their intermolecular distance with nanometric accuracy, finding an average separation of 19 nm. Ex-PALM thus provides a powerful technique for exploring protein organization and interactions at the molecular scale in bacteria, where traditional super-resolution techniques face severe limitations. Beyond prokaryotes, this approach is broadly applicable to the study of highly crowded systems such as organelles and molecular condensates. By combining labeling specificity with physical expansion, Ex-PALM opens new avenues for dissecting subcellular architecture with unprecedented resolution, offering significant opportunities in microbiology, synthetic biology, and cellular biophysics.