Single-molecule-based super-resolution images in the presence of multiple fluorophores.
Single-molecule-based super-resolution images in the presence of multiple fluorophores.
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DOI：
10.1021/nl203560r
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发表时间：
2011-11-09
期刊：
影响因子：
10.8
通讯作者：
Selvin PR
中科院分区：
文献类型：
--
作者：
Simonson PD;Rothenberg E;Selvin PR
存在几种超分辨率技术，但大多数需要多个激光器，使用大或弱发射荧光团，或涉及化学操作。在这里，我们展示了一种简单的技术，在固定样品上超过标准衍射极限5- 15倍，但允许用户从拥挤的荧光团中定位单个荧光团。它只依赖于明亮的有机荧光团和灵敏的相机，这两者都是商业上可用的。超分辨率是通过减去连续图像来找到在从一帧过渡到下一帧时光漂白（暂时或永久）、光激活或结合到感兴趣结构的荧光团来实现的。然后，这些荧光团可以通过高斯拟合与选择性帧平均来定位，以实现比衍射极限更好的精度。信噪比随着附近荧光团数量的平方根而降低，产生通常< 30 nm的平均单分子定位误差。令人惊讶的是，当有大约20个荧光团围绕感兴趣的荧光团时，通常可以提取信号。显示的例子包括微管（体外和固定细胞）和染色体DNA。
Several super-resolution techniques exist, yet most require multiple lasers, use either large or weakly emitting fluorophores, or involve chemical manipulation. Here we show a simple technique that exceeds the standard diffraction limit by 5–15x on fixed samples, yet allows the user to localize individual fluorophores from among groups of crowded fluorophores. It relies only on bright, organic fluorophores and a sensitive camera, both of which are commercially available. Super-resolution is achieved by subtracting sequential images to find the fluorophores that photobleach (temporarily or permanently), photo-activate, or bind to the structure of interest in transitioning from one frame to the next. These fluorophores can then be localized via Gaussian fitting with selective frame averaging to achieve accuracies much better than the diffraction limit. The signal-to-noise ratio decreases with the square root of the number of nearby fluorophores, producing average single-molecule localization errors that are typically < 30 nm. Surprisingly, one can often extract signal when there are approximately 20 fluorophores surrounding the fluorophore of interest. Examples shown include microtubules (in vitro and in fixed cells) and chromosomal DNA.
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影响因子：
3.4
作者：
Thompson, RE;Larson, DR;Webb, WW
通讯作者：
Webb, WW
影响因子：
2.9
作者：
Flors, Cristina;Ravarani, Charles N. J.;Dryden, David. T. F.
通讯作者：
Dryden, David. T. F.
影响因子：
56.9
作者：
Bates, Mark;Huang, Bo;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
DOI：
10.1073/pnas.0401638101
发表时间：
2004-04-27
影响因子：
11.1
作者：
Gordon, MP;Ha, T;Selvin, PR
通讯作者：
Selvin, PR
影响因子：
56.9
作者：
Schermelleh, Lothar;Carlton, Peter M.;Sedat, John W.
通讯作者：
Sedat, John W.