Cryogenic Far-Field Fluorescence Nanoscopy: Evaluation with DNA Origami
Cryogenic Far-Field Fluorescence Nanoscopy: Evaluation with DNA Origami
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DOI：
10.1021/acs.jpcb.0c04721
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发表时间：
2020-09-03
影响因子：
3.3
通讯作者：
Fujiyoshi, Satoru
中科院分区：
文献类型：
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作者：
Furubayashi, Taku;Ishida, Keita;Fujiyoshi, Satoru
利用DNA折纸作为1纳米级的精确支架，展示了在1.8K温度下单个荧光团的远场荧光定位纳米显微镜。将红色和近红外荧光团修饰到支架上，荧光团的间距为11 nm或77 nm。我们用远场荧光显微镜在1.8K下对这两个荧光团进行了定位纳米观察。在低温条件下，荧光团被完全固定，其光漂白被显著抑制；因此，横向空间精度(重复性的衡量标准)提高到1 nm。然而，横向空间精度(真实程度的衡量标准)仍然是几十纳米。我们观察到荧光团的质心随着轴向位置的变化而横向移动。由于在低温条件下荧光团的跃迁偶极子的取向是固定的，单个固定偶极子的各向异性发射导致了横向位移。这种由偶极取向效应引起的系统误差可以通过单个荧光团的三维定位来校正，空间精度(横向)为1 nm，(轴)为17 nm。此外，两个荧光团相距11 nm的支架的三维(3D)取向引起的XY误差估计为0.3 nm。结果，DNA折纸上的单个荧光团被定位在设计的位置，横向空间精度被量化为标准误差中的4 nm。
Far-field fluorescence localization nanoscopy of individual fluorophores at a temperature of 1.8 K was demonstrated using DNA origami as a one-nanometer-accurate scaffold. Red and near-infrared fluorophores were modified to the scaffold, and the fluorophores were 11 or 77 nm apart. We performed the localization nanoscopy of these two fluorophores at 1.8 K with a far-field fluorescence microscope. Under the cryogenic conditions, the fluorophores were perfectly immobilized and their photobleaching was drastically suppressed; consequently, the lateral spatial precision (a measure of reproducibility) was increased to 1 nm. However, the lateral spatial accuracy (a measure of trueness) remained tens of nanometers. We observed that the fluorophore centroids were laterally shifted as a function of the axial position. Because the orientation of the transition dipole of the fluorophores was fixed under cryogenic conditions, the anisotropic emission from the single fixed dipole had led to the lateral shift. This systematic error due to the dipole-orientation effect could be corrected by the three-dimensional localization of the individual fluorophores with spatial precisions of (lateral) 1 nm and (axial) 17 nm. In addition, the xy-error arising from the three-dimensional (3D) orientation of the scaffold with the two fluorophores 11 nm apart was estimated to be 0.3 nm. As a result, the individual fluorophores on the DNA origami were localized at the designed position, and the lateral spatial accuracy was quantified to be 4 nm in the standard error.