Polarization effect on position accuracy of fluorophore localization
Polarization effect on position accuracy of fluorophore localization
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DOI：
10.1364/oe.14.008111
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发表时间：
2006-09-04
期刊：
影响因子：
3.8
通讯作者：
Selvin, Paul R.
中科院分区：
文献类型：
--
作者：
Enderlein, Joerg;Toprak, Erdal;Selvin, Paul R.
以纳米分辨率确定单个荧光分子位置的技术称为 FIONA，已成为多种生物物理应用的重要工具，例如研究运动蛋白的运动机制。位置确定通常通过将二维高斯（x-y 与光子数）拟合到荧光分子的发射强度分布来完成。然而，发射分子的强度分布不仅取决于其在空间中的位置，还取决于其三维方向。在这里，我们对作为分子方向函数的位置确定的可实现精度进行了广泛的数值研究。我们比较了具有不同数值孔径的物镜，结果表明，要获得良好的位置精度，每个 CCD 像素的有效像素尺寸需要为 100 nm 或更小。尽管如此，取向效应仍然会导致大各向异性的位置误差，对于高数值孔径物镜而言高达 10 nm。然而，当使用数值孔径为 1.2 的物镜时，位置精度明显更好（< 2.5 nm）。当然，各向异性较低的探头会降低位置不确定性。 (c) 2006 年美国光学学会。
The technique of determining the position of individual fluorescent molecules with nanometer resolution, called FIONA, has become an important tool for several biophysical applications such as studying motility mechanisms of motor proteins. The position determination is usually done by fitting a 2-D Gaussian (x-y vs. photon number) to the emission intensity distribution of the fluorescent molecule. However, the intensity distribution of an emitting molecule depends not only on its position in space, but also on its three-dimensional orientation. Here, we present an extensive numerical study of the achievable accuracy of position determination as a function of molecule orientation. We compare objectives with different numerical apertures and show that an effective pixel size of 100 nm or less per CCD pixel is required to obtain good positional accuracy. Nonetheless, orientation effects can still cause position errors for large anisotropy, as high as 10 nm for high numerical aperture objectives. However, position accuracy is significantly better (< 2.5 nm) when using objectives with a numerical aperture of 1.2. Of course, probes with lower anisotropy decrease the positional uncertainty. (c) 2006 Optical Society of America.