Orientation of the chromophore dipoles in the TOTO-DNA system.

Background: Flow cytometry has been applied successfully to the sizing of medium to large-sized DNA molecules, thanks to the excellent staining properties of cyanine chromophores such as TOTO (homodimer of thiazole orange) (Petty 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 responsible for their alignment, thereby allowing the determination of the TOTO-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-stained 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 absorption 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 two binding modes.

Conclusions: Our results indicate that any model with a significant contribution of perpendicularly-oriented chromophores fails to reproduce the experimental results.