We studied polarized fluorescence in biological coenzyme NADH in water-methanol solutions upon two-photon excitation with femtosecond laser pulses at 720 nm. The polarized fluorescence decay was recorded by a time correlated single photon counting (TCSPC) system. Fluorescence decay times, rotational diffusion time, fluorescence anisotropy, and the ratio of two pre-exponential factors have been determined and studied as a function of methanol concentration. The results obtained were interpreted on the basis of a model of NADH denaturation processes in solutions and can be used for modeling of NADH binding with various dehydrogenases in living cells.
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