Orthogonal Probing of Single-Molecule Heterogeneity by Correlative Fluorescence and Force Microscopy,ACS Nano

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Orthogonal Probing of Single-Molecule Heterogeneity by Correlative Fluorescence and Force Microscopy
ACS Nano ( IF 15.8 ) Pub Date : 2018-01-05 00:00:00 , DOI: 10.1021/acsnano.7b05405
Wout Frederickx ₁ , Susana Rocha ₁ , Yasuhiko Fujita ₁ , Koen Kennes ₁ , Herlinde De Keersmaecker ₁ , Steven De Feyter ₁ , Hiroshi Uji-i _{1,

2} , Willem Vanderlinden _{1,

3}

Affiliation

Correlative imaging by fluorescence and force microscopy is an emerging technology to acquire orthogonal information at the nanoscale. Whereas atomic force microscopy excels at resolving the envelope structure of nanoscale specimens, fluorescence microscopy can detect specific molecular labels, which enables the unambiguous recognition of molecules in a complex assembly. Whereas correlative imaging at the micrometer scale has been established, it remains challenging to push the technology to the single-molecule level. Here, we used an integrated setup to systematically evaluate the factors that influence the quality of correlative fluorescence and force microscopy. Optimized data processing to ensure accurate drift correction and high localization precision results in image registration accuracies of ∼25 nm on organic fluorophores, which represents a 2-fold improvement over the state of the art in correlative fluorescence and force microscopy. Furthermore, we could extend the Atto532 fluorophore bleaching time ∼2-fold, by chemical modification of the supporting mica surface. In turn, this enables probing the composition of macromolecular complexes by stepwise photobleaching with high confidence. We demonstrate the performance of our method by resolving the stoichiometry of molecular subpopulations in a heterogeneous EcoRV–DNA nucleoprotein ensemble.

中文翻译：

用相关荧光和力显微镜对单分子异质性进行正交探测

通过荧光和力显微镜的相关成像是一种新兴的技术，可以在纳米级获取正交信息。原子力显微镜擅长解决纳米级标本的包膜结构，而荧光显微镜可以检测特定的分子标记，从而能够明确识别复杂装配体中的分子。尽管已经建立了微米级的相关成像，但将技术推向单分子水平仍然具有挑战性。在这里，我们使用一个集成的设置来系统地评估影响相关荧光和力显微镜质量的因素。优化的数据处理可确保准确的漂移校正和较高的定位精度，从而在有机荧光团上产生约25 nm的图像配准精度，与相关的荧光和力显微镜相比，它比现有技术提高了2倍。此外，通过对支持云母表面进行化学修饰，我们可以将Atto532荧光团的漂白时间延长约2倍。继而，这使得能够通过高置信度通过逐步光漂白来探测大分子复合物的组成。我们通过解析异质EcoRV–DNA核蛋白集合中分子亚群的化学计量来证明我们方法的性能。这使得能够通过高可信度逐步光漂白来探测大分子复合物的组成。我们通过解析异质EcoRV–DNA核蛋白集合中分子亚群的化学计量来证明我们方法的性能。这使得能够通过高可信度逐步光漂白来探测大分子复合物的组成。我们通过解析异质EcoRV–DNA核蛋白集合中分子亚群的化学计量来证明我们方法的性能。

更新日期：2018-01-05

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