Temperature dependence for fluorescence of beta-NADH in glycerol/water solution and in trehalose/sucrose glass.

Fluorescence imaging of cells and tissue can be used to evaluate beta-NADH redox and location. At low temperature, beta-NADH fluorescence intensity increases and therefore sensitivity of imaging increases. In this paper, the temperature dependence of fluorescence was evaluated for beta-NADH in glycerol/water solution and in trehalose/sucrose glass. The average fluorescence lifetime for NADH in glycerol/water is 0.66 ns, compared with 5.3 ns in trehalose/ sucrose at 20 degrees C. Emission spectra were recorded from 290 to 12 K. The fluorescence of beta-NADH in glycerol/water increases approximately 16 fold and the emission shifts about 35 nm to the blue as temperature decreases. Much smaller change is seen for fluorescence of beta-NADH in sugar glass. Below 77 K, the beta-NADH spectral features did not change significantly with temperature change, and so no increase in sensitivity is obtained by going to very low temperatures. It is suggested that the sensitivity of beta-NADH fluorescence is related to water relaxation around the excited state molecule. Differences in water in various tissues may contribute to beta-NADH fluorescence changes when cells are altered.