The ability to monitor NADH in vivo remained limited until the development of two-photon microscopy techniques, which utilize the two-photon cross section of chromophores to enable excitation at double the wavelength increasing tissue penetration depth. Additionally, methods to couple two-photon microscopy with time resolved fluorescence lifetime imaging microscopy (FLIM) have been developed and used to probe the microenvironment of tissue chromophores. In the current work by Yaseen et al the first use of two-photon FLIM to detect NADH fluorescence in cerebral tissues in vivo is demonstrated. The authors demonstrated detection of four species of NADH using two-photon FLIM, which define the different microenvironments within the tissue where NADH resides. A multi-component fitting algorithm is developed and utilized to model the measured NADH in vivo fluorescence lifetime. The microenvironmental difference between the components was tested by generating brief periods of anoxia, where the four NADH fluorescence components were found to respond differently likely indicating different enzymatic formulations. Thus, the ability to measure individual components of the NADH fluorescence lifetime may enable detection of the specific molecular pathways involved in oxidative metabolism in future studies.
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