在三维单分子定位显微镜中以纳米级精度校正场相关像差。
Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy.
作者信息
von Diezmann Alex, Lee Maurice Y, Lew Matthew D, Moerner W E
机构信息
Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Department of Chemistry, Stanford University, Stanford, California 94305, USA; Biophysics Program, Stanford University, Stanford, California 94305, USA.
出版信息
Optica. 2015 Nov 20;2(11):985-993. doi: 10.1364/OPTICA.2.000985. Epub 2015 Nov 19.
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.
单个荧光分子的定位能够以亚衍射精度对分子结构和动力学进行成像，并且可以利用点扩散函数（PSF）工程扩展到三维。然而，在整个三维单分子显微镜视野范围内定位的纳米级精度尚未得到严格检验。通过使用填充有荧光染料的规则间隔亚衍射孔径，我们揭示了高达50 - 100纳米的场依赖像差，并表明在扩展的三维焦体积内可以将其校正到小于25纳米。我们证明了该技术对两种工程化PSF（双螺旋PSF和像散PSF）的适用性。我们期望这些结果能广泛应用于要求高精度的三维单分子跟踪和超分辨率方法。
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