Texture analysis of fluorescence lifetime images of nuclear DNA with effect of fluorescence resonance energy transfer

Background: Fluorescence lifetime imaging microscopy (FLIM) is becoming an important tool in cellular imaging. In FLIM, the image contrast is concentr ation insensitive, whereas it is sensitive to the local environment and int eractions of fluorophores such as fluorescence resonance energy transfer(RE T). Methods: Fluorescence microscopy, lifetime imaging, and texture analysis we re used to study the spatial distribution of fluorophores bound to nuclear DNA. 3T3-Swiss albino mice fibroblast nuclei were labeled with Hoechst 3325 8 (Ho), an AT-specific dye, and 7-aminoactinomycin D (7-AAD), a GC-specific dye. Ho is a RET donor to the 7-AAD acceptor. Results: Texture analysis of 50 alcohol-fixed nuclei quantitatively showed changes of spatial distribution of apparent donor lifetimes. RET increased the spatial heterogeneity in the phase and modulation lifetime images. In m ost of the doubly stained cells (about 80%), the phase and modulation lifet ime distributions were spatially homogeneous. In about 20% of the cells, we noticed that lower phase and modulation lifetimes caused by RET were corre lated with regions of high Ho intensity in the nuclei. Conclusions: The spatial lifetime heterogeneity of Ho in presence of 7-AAD seems to be caused by RET between closely spaced strands in the three dimen sionally condensed regions of DNA. (C) 2001 Wiley-Liss. Inc.