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首页> 美国卫生研究院文献>Biophysical Journal >Double-beam autocompensation for fluorescence polarization measurements in flow cytometry.
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Double-beam autocompensation for fluorescence polarization measurements in flow cytometry.

机译：双光束自动补偿用于流式细胞仪中的荧光偏振测量。

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The degree of depolarization of fluorescent light emitted from an organic dye, which is used as molecular probe, is a powerful tool in probing the microenvironment. By fluorescence depolarization the macromolecular structure can be investigated as well as the the mobility of the marker molecule itself or of the complex formed by the probe. Additional information such as energy transfer rates, donor-acceptor distances, and orientations are also measurable. These data are of particular interest if they can be measured from whole cells. Using flow cytometry, we can analyze a large number of cells with high statistical significance in a short period of time. We describe a newly developed double-beam epi-illumination arrangement for fluorescence polarization measurements that uses an autocompensation technique. This new technique permits the various depolarizing effects within the optical as well as the electronic components of the system to be continually compensated for on a cell by cell basis. Simultaneous measurements of other cell parameters for cell cycle analysis by total fluorescence intensity remains possible. The sensitivity of the system to measure polarization was determined as +/- 0.006 p (0 less than or equal to p less than or equal to 0.5 in isotropic media), which amounts to +/- 1.2% of the maximum p value. Polarization data for latex microspheres plotted in the histogram mode were measured with a standard deviation of 0.006, which proved the high resolution and the high performance of the system.
机译：从有机染料发出的荧光的去偏振度（用作分子探针）是探测微环境的有力工具。通过荧光去极化，可以研究大分子结构以及标记分子本身或由探针形成的复合物的迁移率。还可以测量其他信息，例如能量传输速率，施主-受主距离和方向。如果可以从整个细胞中进行测量，这些数据将特别有意义。使用流式细胞仪，我们可以在短时间内分析大量具有统计学意义的细胞。我们描述了一种新开发的采用自动补偿技术的荧光偏振测量双光束落射照明装置。这项新技术允许逐个电池不断地补偿系统的光学和电子组件内的各种去极化效果。通过总荧光强度同时测量其他细胞参数以进行细胞周期分析仍然是可能的。确定系统测量极化的灵敏度为+/- 0.006 p（在各向同性介质中，0小于或等于p小于或等于0.5），等于最大p值的+/- 1.2％。以直方图模式绘制的乳胶微球的极化数据以0.006的标准偏差进行了测量，证明了该系统的高分辨率和高性能。

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