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 fluorescence probes specific for AT and GC base pairs, respectively. YOYO-1 (Yo) was used as a dye that displays distinct fluorescence lifetimes when bound 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 information content of the time-resolved fluorescence. Because a single nucleus 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 intensity-based analysis of Ho- or 7-AAD-stained images indicates increased number and larger size of the DNA condensation centers in the G2/M-phases compared to G0/1-phases. The lifetime-based study of Yo-stained images suggests spatial separation of the AT- or GC-rich DNA regions in the G2/M-phase. Texture analysis of fluorescence intensity and lifetime images was used to quantitatively study the spatial change of condensation and separation of AT- and GC-rich DNA during the cell cycle.
Murata
S
S
Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland at Baltimore, School of Medicine, Baltimore, Maryland 21201, USA.
Herman
P
P
Lakowicz
J R
JR
D016475:3T3 Cells; D020856:AT Rich Sequence; D000818:Animals; D001583:Benzoxazoles; D006690:Bisbenzimidazole; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D003609:Dactinomycin / Q000031:analogs & derivatives*; D005456:Fluorescent Dyes; D020862:GC Rich Sequence; D019044:Image Cytometry; D051379:Mice; D008856:Microscopy, Fluorescence; D011806:Quinolinium Compounds
D001583:Benzoxazoles; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D004247:DNA; D006690:Bisbenzimidazole