Exploring bacteria–surface interactions with a fluorescent membrane tension probe

Understanding how bacteria interact with surfaces is critical for advancing applications in biofilm and biofouling prevention, biomaterial development, or biosensing. However, the biophysical mechanisms underlying these interactions remain poorly characterized, and novel microscopy strategies are needed to specifically address the biointerface. In this study, we employ fluorescence lifetime imaging microscopy (FLIM) with the tension reporter Flipper-TR to investigate membrane tension in live bacteria interacting with various surfaces. We show that Flipper-TR stains both Gram-positive and Gram-negative bacterial membranes, exhibiting fluorescence lifetimes shorter than those in eukaryotic cells, with slight variations between bacterial types and likely reflecting differences in membrane composition. Flipper-TR displays lifetime variations along the vertical axis of bacterial cells, suggesting spatial differences in membrane tension influenced by cell wall architecture. Our results further demonstrate that Flipper-TR is responsive to the nature of bacterial interactions with surfaces. By comparing bacterial immobilization on surfaces with different coatings, we show that Flipper-TR can sensitively distinguish differences in membrane tension arising from distinct adhesion mechanisms. Additionally, Flipper-TR detects changes in membrane tension when bacteria are exposed to engineered nanostructured substrates. Overall, this work expands the toolbox to study the mechanical aspects of bacterial-material interactions and contributes to providing design rules for novel materials that influence bacterial behavior.