Texture analysis of fluorescence lifetime images of AT- and GC-rich regions in nuclei

We used intensity and fluorescence lifetime microscopy (FLIM) of 3T3 nuclei to investigate the existence of AT-rich and GC-rich regions of the nuclear DNA. Hoechst 33258 (Ho) and 7-aminoactinomycin D (7-AAD) were used as fluo rescence probes specific for AT and GC base pairs, respectively. YOYO-1 (Yo ) was used as a dye that displays distinct fluorescence lifetimes when boun d to AT or GC base pairs. We combined fluorescence imaging of Ho and 7-AAD with time-resolved measurements of Yo and took advantage of an additional i nformation content of the time-resolved fluorescence. Because a single nucl eus could not be stained and measured with all three dyes, we used texture analysis to compare the spatial distribution of AT-rich and GC-rich DNA in 100 nuclei in different phases of the cell cycle. The fluorescence intensit y-based analysis of Ho- or 7-AAD-stained images indicates increased number and larger size of the DNA condensation centers in the G(2)/M-phases compar ed to G(0/1)-phases. The lifetime-based study of Yo-stained images suggests spatial separation of the AT- or GC-rich DNA regions in the G(2)/M-phase. Texture analysis of fluorescence intensity and lifetime images was used to quantitatively study the spatial change of condensation and separation of A T- and GC-rich DNA during the cell cycle.