Mirror-enhanced super-resolution microscopy.
Mirror-enhanced super-resolution microscopy.
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镜面增强超分辨率显微镜
DOI：
10.1038/lsa.2016.134
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Jin D
中科院分区：
文献类型：
--
作者：
Yang X;Xie H;Alonas E;Liu Y;Chen X;Santangelo PJ;Ren Q;Xi P;Jin D
超越衍射极限的轴向激发约束对于下一代超分辨率显微镜的发展至关重要。受激发射耗尽（STED）纳米显微镜使用环形光束耗尽提供横向超分辨率，但其轴向分辨率仍超过500 nm。全内反射荧光显微镜广泛用于单分子定位，但其检测分子的能力仅限于距离细胞附着表面约100 nm的倏逝场内。我们发现，在共焦激发过程中的点扩散函数（PSF）的轴向厚度可以很容易地提高到110 nm，通过更换显微镜载玻片与镜子。局部电磁场的干涉将共焦PSF轴向限制在110 nm的光斑，这使得所有激光扫描显微镜都能够实现轴向超分辨率。轴向切片可以通过波长调制或通过控制反射镜和样品之间的间隔来获得。在没有额外复杂性的情况下，镜辅助激发限制增强了STED的轴向分辨率6倍和横向分辨率2倍，它们共同实现了19 nm的分辨率，以分辨核孔复合物的内缘，并区分120 nm病毒细丝的内容。在不增加激光功率的情况下，使用镜面增强STED来增加横向分辨率和减小轴向截面的厚度的能力对于生物样本成像是非常重要的，生物样本不能耐受高激光功率。
Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of~ 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.