Orientation of the chromophore dipoles in the TOTO-DNA system

Background: Flow cytometry has been applied successfully to the sizing of m edium to large-sized DNA molecules, thanks to the excellent staining proper ties of cyanine chromophores such as TOTO (homodimer of thiazole orange) (P etty et al.: Anal Chem 67:1755-1761, 1995). The hydrodynamic flow, used to focus the sample molecules in a small laser-illuminated volume, is also res ponsible for their alignment, thereby allowing the determination of the TOT O-dipole orientation with respect to the DNA axis (Agronskaia et al.: Appl Opt 38:714-719, 1999). Methods: We present model calculations of the fluorescence yield of TOTO-st ained DNA measured in a flow-cytometric setup with high numerical aperture. The models consider different orientations of the chromophore dipoles. Results: Comparison of measurement and calculation suggests that the absorp tion dipoles of the TOTO molecule make a mean angle of 61 degrees with the helix axis of the DNA molecule. This mean angle can be the consequence of t wo binding modes. Conclusions: Our results indicate that any model with a significant contrib ution of perpendicularly-oriented chromophores fails to reproduce the exper imental results. Cytometry 37:230-237, 1999, (C) 1999 Wiley-Liss, Inc.