Transient binding facilitates super-resolution imaging of functional amyloid fibrils on living bacteria.

Curli, which are the major proteinaceous components of the Escherichia coli biofilm extracellular matrix, help protect cells against environmental stressors, including dehydration and antibiotics. Composed of the amyloid proteins CsgA and CsgB, curli self-assemble as these protomers are secreted into the extracellular space. The mechanisms of curli assembly and their functional roles within the extracellular matrix are incompletely understood. High-resolution imaging tools compatible with live-cell conditions provide a critical means to investigate the assembly and function of curli in their native context. In this study, we use super-resolution imaging to visualize curli fibrils on living bacterial cells. Transient amyloid binding of the fluorogenic dye Nile blue facilitates two complementary super-resolution fluorescence microscopy approaches: single-molecule imaging via points accumulation for imaging in nanoscale topography and super-resolution optical fluctuation imaging via pixel-wise autocorrelation. Additionally, imaging fluorescence correlation spectroscopy was used to measure the characteristic relaxation times associated with Nile blue binding to CsgA fibrils. Together, these approaches offer a framework for imaging-based biophysical characterization of curli structures on living cells.