We present a novel, cost-effective, versatile, and practical frequency-domain fluorescence lifetime imaging/microscopy (FD-FLIM) implementation capable of simultaneous multi-wavelength excitation, simultaneous multispectral detection, and sub-nanosecond to nanosecond fluorescence lifetime estimation. Rigorous validation experiments have demonstrated the capabilities of this novel FD-FLIM implementation to accurately measure fluorescence lifetimes in the range of ~0.4-12 ns. This novel technology has been adopted to implement an FD-FLIM endoscopy system for label-free metabolic imaging of oral lesions based on NADH (375-nm excitation, 484/37-nm emission) and FAD (445-nm excitation, 553/93-nm emission) autofluorescence imaging. Future efforts will focus on developing imaging systems for simultaneous endogenous (UV-VI excitation) and exogenous (NIR excitation) fluorescence lifetime imaging.
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