The polarization of fluorescence of DNA stains depends on the incorporation density of the dye molecules
Article first published online: 29 JUN 2004
Copyright © 2004 Wiley-Liss, Inc.
Cytometry Part A
Volume 61A, Issue 1, pages 18–25, September 2004
How to Cite
Uy, J. L., Asbury, C. L., Petersen, T. W. and van den Engh, G. (2004), The polarization of fluorescence of DNA stains depends on the incorporation density of the dye molecules. Cytometry, 61A: 18–25. doi: 10.1002/cyto.a.20059
- Issue published online: 17 AUG 2004
- Article first published online: 29 JUN 2004
- Manuscript Accepted: 15 MAR 2004
- Manuscript Revised: 3 MAR 2004
- Manuscript Received: 22 OCT 2003
- NSF Science and Technology Center. Grant Number: BIR 9214821
- Genome Training Grant NIH. Grant Number: T32 GM00035-05
- fluorescence polarization;
- energy transfer;
- fluorescence depolarization;
- ethidium bromide (EB);
- thiazole orange (TO);
- oxazole yellow (YO);
- ethidium homodimer (EBEB)
The fluorescence induced by polarized light sources, such as the lasers that are used in flow cytometry, is often polarized and anisotropic. In addition, most optical detector systems are sensitive to the direction of polarization. These two factors influence the accuracy of fluorescence intensity measurements. The intensity of two light sources can be compared only if all details of the direction and degree of polarization are known. In a previous study, we observed that fluorescence polarization might be modified by dye–dye interactions. This report further investigates the role of dye density in fluorescence polarization anisotropy.
We measured the polarization distribution of samples stained with commonly used DNA dyes. To determine the role of fluorophore proximity, we compared the monomeric and a dimeric form of the DNA dyes ethidium bromide (EB), thiazole orange (TO), and oxazole yellow (YO).
In all dyes sampled, fluorescence polarization is less at high dye concentrations than at low concentrations. The monomeric dyes exhibit a higher degree of polarization than the dimeric dyes of the same species.
The polarization of fluorescence from DNA dyes is related to the density of incorporation into the DNA helix. Energy transfer between molecules that are in close proximity loosens the linkage between the excitation and emission dipoles, thereby reducing the degree of polarization of the emission. © 2004 Wiley-Liss, Inc.