Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy.
Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy.
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DOI：
10.1364/optica.2.000985
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发表时间：
2015-11-20
期刊：
影响因子：
10.4
通讯作者：
Moerner WE
中科院分区：
文献类型：
--
作者：
von Diezmann A;Lee MY;Lew MD;Moerner WE
单个荧光分子的局域化使得能够以亚衍射精度成像分子结构和动力学，并且可以利用点扩展函数(PSF)工程扩展到三维。然而，在3D单分子显微镜的视野中定位的纳米级精度还没有得到严格的检查。通过使用填充了荧光染料的规则间隔的亚衍射孔，我们揭示了与场相关的高达50-100 nm的像差，并表明它们可以在扩展的3D焦体积上被校正到小于25 nm。我们演示了这种技术在两种工程PSF中的适用性，即双螺旋PSF和散光PSF。我们预计这些结果将广泛适用于要求高精度的3D单分子跟踪和超分辨率方法。
The localization of single fluorescent molecules enables the imaging of molecular structure and dynamics with subdiffraction precision and can be extended to three dimensions using point spread function (PSF) engineering. However, the nanoscale accuracy of localization throughout a 3D single-molecule microscope’s field of view has not yet been rigorously examined. By using regularly spaced subdiffraction apertures filled with fluorescent dyes, we reveal field-dependent aberrations as large as 50–100 nm and show that they can be corrected to less than 25 nm over an extended 3D focal volume. We demonstrate the applicability of this technique for two engineered PSFs, the double-helix PSF and the astigmatic PSF. We expect these results to be broadly applicable to 3D single-molecule tracking and superresolution methods demanding high accuracy.