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 concentration insensitive, whereas it is sensitive to the local environment and interactions of fluorophores such as fluorescence resonance energy transfer (RET).


METHODS
Fluorescence microscopy, lifetime imaging, and texture analysis were used to study the spatial distribution of fluorophores bound to nuclear DNA. 3T3-Swiss albino mice fibroblast nuclei were labeled with Hoechst 33258 (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 most of the doubly stained cells (about 80%), the phase and modulation lifetime distributions were spatially homogeneous. In about 20% of the cells, we noticed that lower phase and modulation lifetimes caused by RET were correlated 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 dimensionally condensed regions of DNA.
Murata
S
S
Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland at Baltimore, School of Medicine, Baltimore, Maryland, USA.
Herman
P
P
Lakowicz
J R
JR
D016475:3T3 Cells; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D004735:Energy Transfer; D006207:Half-Life; D019044:Image Cytometry / Q000379:methods*; D008432:Mathematical Computing; D051379:Mice; D013050:Spectrometry, Fluorescence / Q000379:methods
D004247:DNA