Comparing the Accuracy of Reconstructed Image Size in Super-Resolution Imaging of Fluorophore-Labeled Gold Nanorods Using Different Fit Models
Comparing the Accuracy of Reconstructed Image Size in Super-Resolution Imaging of Fluorophore-Labeled Gold Nanorods Using Different Fit Models
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DOI：
10.1021/acs.jpcc.5b04993
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发表时间：
2015-08-20
影响因子：
3.7
通讯作者：
Willets, Katherine A.
中科院分区：
文献类型：
--
作者：
Blythe, Karole L.;Titus, Eric J.;Willets, Katherine A.
我们使用三重态介导的超分辨率荧光成像技术来定位单个荧光标记的双链DNA（dsDNA）结合到金纳米棒表面的位置。在每个衍射极限点内，我们必须考虑两个不同的发射源：来自荧光标记的随机荧光和金纳米棒的稳定背景发光。为了从荧光标记中分离出贡献，我们减去平均金纳米棒发光贡献，用二维高斯或偶极发射模型建模。然后将来自标记的dsDNA的荧光与二维高斯拟合，以重建纳米棒表面上每个单独发射体的位置。使用任一发光模型得到的重建图像与下面的纳米棒的形状和取向一致，并且基于荧光标记的定位在纳米棒的表面上显示出类似的明显的dsDNA结合异质性。使用偶极发射模型用于发光允许在应用拟合阈值之后保留来自荧光标记的更多发射事件，并且产生包含更多质心点的更稳健的重建图像，所述质心点显示dsDNA的表观位置。不幸的是，重建的纳米棒图像的尺寸小于预期，尽管使用了更准确的金发光模型，这表明这种耦合染料-纳米棒系统的光物理学比使用孤立的荧光团时更复杂。
We use a triplet-state-mediated super-resolution fluorescence imaging technique to localize the position of individual fluorescently labeled double-stranded DNA (dsDNA) bound to the surface of gold nanorods. Within each diffraction-limited spot, we must account for two different emission sources: the stochastic fluorescence from the fluorescent labels and the steady background luminescence of the gold nanorod. To isolate the contribution from the fluorescent label, we subtract the average gold nanorod luminescence contribution, modeled with either a two-dimensional Gaussian or a dipolar emission model. The fluorescence from the labeled dsDNA is then fit with a two-dimensional Gaussian to reconstruct the positions of each individual emitter on the nanorod surface. The resulting reconstructed images, using either luminescence model, agree with the shape and orientation of the underlying nanorod, and show similar apparent dsDNA binding heterogeneity across the surface of the nanorod based on the localization of the fluorescent labels. Using the dipolar emission model for the luminescence allows for the retention of more emission events from the fluorescent label, after applying a fitting threshold, and yields a more robust reconstructed image containing more centroid points that show the apparent locations of the dsDNA. Unfortunately, the sizes of the reconstructed nanorod images were smaller than expected, despite the use of the more accurate model for the gold luminescence, suggesting that the photophysics of this coupled dye-nanorod system are more complicated than when using the isolated fluorophores.