Precise nanometer localization analysis for individual fluorescent probes
Precise nanometer localization analysis for individual fluorescent probes
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DOI：
10.1016/s0006-3495(02)75618-x
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发表时间：
2002-05-01
影响因子：
3.4
通讯作者：
Webb, WW
中科院分区：
文献类型：
--
作者：
Thompson, RE;Larson, DR;Webb, WW
计算各个荧光颗粒和分子的图像的质心允许在光学显微镜中定位和跟踪到比显微镜分辨率大大约一个数量级的精度。限制这些技术的精度的因素进行检查，并推导出一个简单的方程，描述了在广泛的条件下的定位精度。此外，从最小二乘拟合理论的本地化算法的动机构造和测试的图像堆栈的30 nm的荧光珠和计算机生成的图像（蒙特卡洛模拟）。仿真和实际图像的结果表明，该算法与推导的精度方程吻合良好。一个简单的方程来描述定位精度的可用性，帮助调查人员在评估实验装置的质量和引导注意力的因素，限制进一步改进。对于散粒噪声有限的情况，定位精度缩放为斑点中光子数量的平方根倒数，并且对于背景噪声有限的情况，定位精度缩放为光子数量的倒数。最佳图像放大率取决于光子和背景噪声的预期数量，但是，对于大多数感兴趣的情况，像素大小应该大约等于点扩散函数的标准偏差。
Calculation of the centroid of the images of individual fluorescent particles and molecules allows localization and tracking in light microscopes to a precision about an order of magnitude greater than the microscope resolution. The factors that limit the precision of these techniques are examined and a simple equation derived that describes the precision of localization over a wide range of conditions. In addition, a localization algorithm motivated from least-squares fitting theory is constructed and tested both on image stacks of 30-nm fluorescent beads and on computer-generated images (Monte Carlo simulations). Results from the algorithm show good agreement with the derived precision equation for both the simulations and actual images. The availability of a simple equation to describe localization precision helps investigators both in assessing the quality of an experimental apparatus and in directing attention to the factors that limit further improvement. The precision of localization scales as the inverse square root of the number of photons in the spot for the shot noise limited case and as the inverse of the number of photons for the background noise limited case. The optimal image magnification depends on the expected number of photons and background noise, but, for most cases of interest, the pixel size should be about equal to the standard deviation of the point spread function.