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Journal of Histochemistry and Cytochemistry, Vol. 49, 1443-1452, November 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

Texture Analysis of Fluorescence Lifetime Images of AT- and GC-rich Regions in Nuclei

Shin-ichi Murataa, Petr Hermana, and Joseph R. Lakowicza
a Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland at Baltimore, School of Medicine, Baltimore, Maryland

Correspondence to: Joseph R. Lakowicz, Center for Fluorescence Microscopy, Dept. of Biochemistry and Molecular Biology, U. of Maryland at Baltimore, Schl. of Medicine, 725 W. Lombard St., Baltimore, MD 21201.

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.

(J Histochem Cytochem 49:1443–1451, 2001)

Key Words: fluorescence lifetime imaging, microscopy, texture analysis, Hoechst 33258, 7-aminoactinomycin D, YOYO-1, cell cycle


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