The polarization of fluorescence of DNA stains depends on the incorporation density of the dye molecules.
BACKGROUND
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.


METHODS
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).


RESULTS
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.


CONCLUSIONS
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.
Uy
Jeanne L
JL
Cell Sorting Facility, Department of Pathology, University of Washington, Seattle, Washington, USA.
Asbury
Charles L
CL
Petersen
Timothy W
TW
van den Engh
Ger
G
D000818:Animals; D052160:Benzothiazoles; D001583:Benzoxazoles / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D007364:Intercalating Agents / Q000737:chemistry*; D051379:Mice; D011804:Quinolines / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry
D052160:Benzothiazoles; D001583:Benzoxazoles; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D011804:Quinolines; D013844:Thiazoles; C097015:oxazole yellow; C051000:thiazole orange; D004247:DNA