Fluorescence lifetime imaging of nuclear DNA: Effect of fluorescence resonance energy transfer

Background: DNA fluorescence dyes have been used to study DNA dynamics, chr omatin structure, and cell cycle analysis. However, most microscopic fluore scence studies of DNA use only steady-state measurements and do not take ad vantage of the additional information content of the time-resolved fluoresc ence In this paper, we combine fluorescence imaging of DNA with time-resolv ed measurements to examine the proximity of donors and accepters bound to c hromatin. Methods: We used frequency-domain fluorescence life-time imaging microscopy to study the spatial distribution of DNA-bound donors and accepters in fix ed 3T3 nuclei. Over 50 cell nuclei were imaged in the presence of an AT-spe cific donor, Hoechst 33258 (Ho), and a GC-specific acceptor, 7-aminoactinom ycin D (7-AAD). Results: The intensity images of Ho alone showed a spatially irregular dist ribution due to the various concentrations of DNA or AT-rich DNA throughout the nuclei. The lifetime imaging of the He-stained nuclei was typically fl at. Addition of 7-AAD decreased the fluorescence intensity and lifetime of the He-stained DNA. The spatially dependent phase and modulation values of Ho in the presence of 7-AAD showed that the no decay becomes nonexponential , as is expected for a resonance energy transfer (RET) with multiple accept ers located over a range of distances. In approximately 40 nuclei, the inte nsity and lifetime decrease was spatially homogeneous. In approximately 10 nuclei, addition of 7-AAD resulted in a spatially nonhomogeneous decrease i n intensity and lifetime. The RET efficiency was higher in G(2)/M than in G (0/1) phase cells. Conclusions: Because RET efficiency depends on the average distance between Ho and 7-AAD, data suggest that the heterogeneity of lifetimes and spatial variation of the RET efficiency are caused by the presence of highly conde nsed regions of DNA in nuclei. (C) 2000 Wiley-Liss, Inc.