Correlation of Live Imaging and Fixed-Expansion Microscopy to study intracellular pathogens

Expansion microscopy (ExM) enables high–resolution fluorescence imaging by physically enlarging biological samples, but its application to intracellular bacteria remains challenging. Here, we adapted the Ten–fold Robust Expansion Microscopy protocol to study host–pathogen interactions. Expansion of Burkholderia thailandensis-infected epithelial cells revealed previously unresolved membrane architectures associated with bacterial cell–to–cell dissemination. While host cells expanded approximately 9–fold, bacterial cells displayed incomplete and heterogeneous expansion. We found that fixation conditions strongly influence bacterial expansion, while presence of peptidoglycan partially limits expansion. To connect the membrane ultrastructures revealed by expansion with infection dynamics, we developed Live–to–Fixed Expansion microscopy (LiFEx), a workflow that correlates dynamic events observed by live–cell imaging with high–resolution cellular structures resolved by ExM. By correlating T6SS–5 assembly with subsequent high-resolution membrane architecture, LiFEx revealed membrane alterations developing within minutes following T6SS–5 assembly. Together, our approach enabled the investigation of dynamic host–pathogen interactions across complementary spatial and temporal scales.