Real-time, high-resolution metabolic characterization of live bacteria using label-free optical metabolic imaging

Label-free optical imaging provides non-invasive, high-speed, high-resolution metabolic characterization of live bacteria with single-cell resolution. Here, we demonstrate the ability of label-free multiphoton autofluorescence microscopy to characterize the fast (between 0 and 30 min) metabolic changes in bacteria in response to antibiotic treatments and observe the cell-to-cell metabolic heterogeneity of planktonic bacteria and biofilms. Results indicate that bacteria exhibit a distinct measurable response to bactericidal treatments within seconds. Furthermore, S. aureus biofilms exhibit metabolic heterogeneity, with local pockets of high metabolic activity. Bacteria in biofilms exhibit altered metabolic profiles compared to planktonic bacteria for all four species examined: S. aureus, P. aeruginosa, M. catarrhalis, and S. pneumoniae. These results shed light on the spatial and temporal metabolic heterogeneity of bacteria and the quantification possibilities using label-free nonlinear optical microscopy.