Metabolic characterization of bacteria using multimodal label-free nonlinear optical microscopy

Optical metabolic imaging is performed by quantifying the autofluorescence intensity and lifetime of endogenous metabolic cofactors NAD(P)H and FAD. Bacteria energy metabolism is similar to mammalian energy metabolism, yet the bulk of previous work in optical metabolic imaging has focused on mammalian samples. Here, we combine multiphoton fluorescence lifetime imaging microscopy of NAD(P)H and FAD with coherent anti-Stokes Raman scattering microscopy to examine the optical metabolic properties of bacteria. Nonlinear optical microscopy provides high resolution and label-free contrast, and can thus be used to study bacteria in diverse in vivo environments, unlike most methods for bacteria characterization. Bacteria are examined in a variety of diverse situations, including the dynamics of antibiotic treatment. Furthermore, optical metabolic differences between planktonic bacteria and biofilms were identified. This work presents preliminary success for optical metabolic imaging of bacteria by quantifying capabilities and highlighting useful cases, opening the door for future investigations.