Fluorescence lifetime imaging microscopy (FLIM) is an imaging technique that characterizes fluorophores based on the average time between excitation and emission and provides useful information about the state of fluorophores. Our fast and label-free approach targets NADH and NADPH, two endogenous autofluorescent molecules that are abundant in cells, together termed NAD(P)H. NADH, which is more abundant in cells than NAD(P)H, is a key metabolite linked to glycolysis and the electron transport chain. When bound to a protein, its fluorescence lifetime increases from about 0.3-0.8 ns to about 2 to 6 ns. Metabolic shifts in cells can happen very quickly (<1 s) so most photon-counting FLIM techniques such as TCSPC are too slow to capture fast metabolic dynamics. To overcome this limitation, our lab has developed a fast FLIM system capable of video rate (>20 fps) imaging of NAD(P)H. So far we have used fast FLIM to investigate rapid processes such as cell apoptosis and neural dynamics.
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- Sorrells, J. E., Iyer, R. R., Yang, L., Bower, A. J., Spillman, D. R., Chaney, E. J., Tu, H., Boppart, S. A. "Real-time pixelwise phasor analysis for video-rate two-photon fluorescence lifetime imaging microscopy." Biomedical Optics Express 12.7 (2021): 4003-4019. https://doi.org/10.1364/BOE.424533
- Bower, A. J., Sorrells, J. E., Li, J., Marjanovic, M., Barkalifa, R., & Boppart, S. A. "Tracking metabolic dynamics of apoptosis with high-speed two-photon fluorescence lifetime imaging microscopy." Biomedical Optics Express 10.12 (2019): 6408-6421. https://doi.org/10.1364/BOE.10.006408
- Bower, A.J., Li J., Chaney, E.J., Marjanovic, M., Spillman Jr., D.R., Boppart, S.A. "High-speed imaging of transient metabolic dynamics using two-photon fluorescence lifetime imaging microscopy." Optica 5.10 (2018):1290-1296. https://doi.org/10.1364/OPTICA.5.001290