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There is a need for fast methods of enumeration, identification, and viability evaluation of bacteria in diverse environments. Using multiphoton microscopy, NAD(P)H and FAD autofluorescence intensities and lifetimes have been widely used to study metabolism in a variety of biological specimens, but have traditionally been limited by slow acquisition. Recent work has developed technology to enable faster (~1 Hz) multiphoton fluorescence intensity and lifetime imaging with photon counting resolution with capabilities for dual-channel simultaneous NAD(P)H and FAD autofluorescence imaging. This enables label-free imaging of bacteria in native environments such as on skin and imaging of fast time-lapse responses to antibiotics.
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Janet E. Sorrells, Rishyashring R. Iyer, Lingxiao Yang, Farzana R. Zaki, Eric J. Chaney, Marina Marjanovic, Stephen A. Boppart, "Fast label-free multiphoton fluorescence lifetime imaging microscopy for metabolic characterization of the skin microbiome," Proc. SPIE PC12358, Photonic Diagnosis, Monitoring, Prevention, and Treatment of Infections and Inflammatory Diseases 2023, PC1235802 (17 March 2023); https://doi.org/10.1117/12.2648984