Background:
Dna fluorescence dyes have been used to study dna dynamics, chromatin structure, and cell cycle analysis. however, most microscopic fluorescence studies of dna use only steady-state measurements and do not take advantage of the additional information content of the time-resolved fluorescence. in this paper, we combine fluorescence imaging of dna with time-resolved measurements to examine the proximity of donors and acceptors bound to chromatin.
Methods:
We used frequency-domain fluorescence lifetime imaging microscopy to study the spatial distribution of dna-bound donors and acceptors in fixed 3t3 nuclei. over 50 cell nuclei were imaged in the presence of an at-specific donor, hoechst 33258 (ho), and a gc-specific acceptor, 7-aminoactinomycin d (7-aad).
Results:
The intensity images of ho alone showed a spatially irregular distribution due to the various concentrations of dna or at-rich dna throughout the nuclei. the lifetime imaging of the ho-stained nuclei was typically flat. addition of 7-aad decreased the fluorescence intensity and lifetime of the ho-stained dna. the spatially dependent phase and modulation values of ho in the presence of 7-aad showed that the ho decay becomes nonexponential, as is expected for a resonance energy transfer (ret) with multiple acceptors located over a range of distances. in approximately 40 nuclei, the intensity and lifetime decrease was spatially homogeneous. in approximately 10 nuclei, addition of 7-aad resulted in a spatially nonhomogeneous decrease in 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 condensed regions of dna in nuclei.