Uy Jeanne L, Asbury Charles L, Petersen Timothy W, van den Engh Ger
Cell Sorting Facility, Department of Pathology, University of Washington, Seattle, Washington, USA.
Cytometry A. 2004 Sep;61(1):18-25. doi: 10.1002/cyto.a.20059.
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.
偏振光源（如流式细胞术中使用的激光）诱导的荧光通常是偏振且各向异性的。此外，大多数光学检测系统对偏振方向敏感。这两个因素会影响荧光强度测量的准确性。只有当偏振方向和偏振程度的所有细节都已知时，才能比较两个光源的强度。在先前的一项研究中，我们观察到荧光偏振可能会因染料 - 染料相互作用而改变。本报告进一步研究染料浓度在荧光偏振各向异性中的作用。
我们测量了用常用DNA染料染色的样品的偏振分布。为了确定荧光团接近程度的作用，我们比较了DNA染料溴化乙锭（EB）、噻唑橙（TO）和恶唑黄（YO）的单体形式和二聚体形式。
在所有采样的染料中，高染料浓度下的荧光偏振低于低浓度下的荧光偏振。相同种类的单体染料比二聚体染料表现出更高的偏振度。
DNA染料荧光的偏振与掺入DNA螺旋的密度有关。紧密相邻分子之间的能量转移会减弱激发偶极子和发射偶极子之间的联系，从而降低发射的偏振程度。
