4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.
4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.
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DOI：
10.1371/journal.pone.0020645
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Soeller C
中科院分区：
文献类型：
--
作者：
Baddeley D;Crossman D;Rossberger S;Cheyne JE;Montgomery JM;Jayasinghe ID;Cremer C;Cannell MB;Soeller C
荧光生物样品的光学超分辨成像正迅速成为研究蛋白质在分子水平上分布的重要工具。因此，重要的是开发实用的超分辨率方法，允许捕捉生物系统的全部三维性质，并且还可以在同一样本中可视化多个蛋白质物种。我们发现，结合使用传统的近红外染料，如Alexa 647、Alexa 680和Alexa 750，都是由671 nm的半导体激光激发的，能够实现复杂生物样品的3D多色超分辨率成像。光学厚的样品，包括人体组织切片、大鼠心肌细胞和密集生长的神经元培养物，横向分辨率为∼15 nm(Std)。Dev.)同时将标记串扰降低到1%。使用散光，轴向分辨率为∼65 nm(标准Dev.)是例行公事的成就。能够区分的标记物种的数量取决于单分子事件的平均光子数。利用∼2000的Alexa680的典型光子产生量，最多5个标记物原则上可以用2%的串扰来解决。我们的方法完全基于传统的、商业上可用的标记的使用，并且只需要一个激光器。它提供了一种在纳米尺度上研究生物样本的非常直接的方法，并应有助于将实用的4D超分辨率显微镜作为许多实验室的常规研究工具。
Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample. We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk. Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.
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影响因子：
16.2
作者：
Dani, Adish;Huang, Bo;Bergan, Joseph;Dulac, Catherine;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
影响因子：
56.9
作者：
Bates, Mark;Huang, Bo;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
DOI：
10.1073/pnas.0900245106
发表时间：
2009-03-03
影响因子：
11.1
作者：
Pavani, Sri Rama Prasanna;Thompson, Michael A.;Moerner, W. E.
通讯作者：
Moerner, W. E.
影响因子：
3.5
作者：
Cheyne, Juliette E.;Montgomery, Johanna M.
通讯作者：
Montgomery, Johanna M.
影响因子：
10.8
作者：
Evans, AM;Cannell, MB
通讯作者：
Cannell, MB