Subnanometre single-molecule localization, registration and distance measurements
Subnanometre single-molecule localization, registration and distance measurements
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DOI：
10.1038/nature09163
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发表时间：
2010-07-29
期刊：
影响因子：
64.8
通讯作者：
Chu, Steven
中科院分区：
文献类型：
--
作者：
Pertsinidis, Alexandros;Zhang, Yunxiang;Chu, Steven
光学显微镜在纳米距离的测量方面取得了显着的进展。如果衍射将点物体的图像模糊成艾里斑，其均方根（rms）大小为 s = 0.44 lambda/2NA（对于波长为 lambda = 600 nm 的光，类似于 90 nm，物镜的数值孔径为 NA = 1.49），则将使用的远场显微镜的分辨率限制为 d = 2.4s，近似于 200 nm，另外关于样本的知识可以发挥很大的作用。例如，如果已知光源是两个空间分辨的荧光分子，则它们之间的距离由两个荧光图像中心的间隔给出(1)。在高分辨率微波和光学光谱中，有许多以小于线宽的 10(-6) 精度确定线中心的例子。相比之下，在生物应用中，最亮的单个荧光发射器可以以接近 100 的信噪比进行检测，从而将质心定位精度限制为 r.m.s 的 s(loc) >= 1% (>= 1 nm)。显微镜点扩散函数 (PSF)(2) 的大小 s。此外，共定位两个或多个单个发射器的误差明显更差，仍然大于 PSF 尺寸 (3-8) 的 5-10% (5-10 nm)。在这里，我们报告了在生理缓冲条件下使用传统远场荧光成像测量不同颜色荧光分子之间的分离时，距离分辨率为 s(reg) = 0.50 nm (1 sigma)，绝对精度为 s(distance) = 0.77 nm (1 sigma)。相同单分子样本集合的平均值的统计不确定性仅受收集的光子总数的限制，s(loc) 约为 0.3 nm，相当于光学 PSF 大小的 3 x 10(-3) 倍。我们的方法还可用于提高许多亚波长、远场成像方法的分辨率，例如基于空间中随机开启的分子共定位的方法(6-8)。分辨率的提高将允许在单分子水平上破译生物相关环境中大型多亚基生物复合物的结构。
Remarkable progress in optical microscopy has been made in the measurement of nanometre distances. If diffraction blurs the image of a point object into an Airy disk with a root-mean-squared (r.m.s.) size of s = 0.44 lambda/2NA (similar to 90 nm for light with a wavelength of lambda = 600 nm and an objective lens with a numerical aperture of NA = 1.49), limiting the resolution of the far-field microscope in use to d = 2.4s approximate to 200 nm, additional knowledge about the specimen can be used to great advantage. For example, if the source is known to be two spatially resolved fluorescent molecules, the distance between them is given by the separation of the centres of the two fluorescence images(1). In high-resolution microwave and optical spectroscopy, there are numerous examples where the line centre is determined with a precision of less than 10(-6) of the linewidth. In contrast, in biological applications the brightest single fluorescent emitters can be detected with a signal-to-noise ratio of similar to 100, limiting the centroid localization precision to s(loc) >= 1% (>= 1 nm) of the r.m.s. size, s, of the microscope point spread function (PSF)(2). Moreover, the error in co-localizing two or more single emitters is notably worse, remaining greater than 5-10% (5-10 nm) of the PSF size(3-8). Here we report a distance resolution of s(reg) = 0.50 nm (1 sigma) and an absolute accuracy of s(distance) = 0.77 nm (1 sigma) in a measurement of the separation between differently coloured fluorescent molecules using conventional far-field fluorescence imaging in physiological buffer conditions. The statistical uncertainty in the mean for an ensemble of identical single-molecule samples is limited only by the total number of collected photons, to s(loc) approximate to 0.3 nm, which is similar to 3 x 10(-3) times the size of the optical PSF. Our method may also be used to improve the resolution of many subwavelength, far-field imaging methods such as those based on co-localization of molecules that are stochastically switched on in space(6-8). The improved resolution will allow the structure of large, multisubunit biological complexes in biologically relevant environments to be deciphered at the single-molecule level.