Orientation of the chromophore dipoles in the TOTO-DNA system

✍ Scribed by Juleon M. Schins; Alexandra Agronskaia; Bart G. de Grooth; Jan Greve

• Book ID: 101243746
• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 355 KB
• Volume: 37
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19991101)37:3<230::aid-cyto10>3.0.co;2-#

✦ Synopsis

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 flowcytometric 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°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.