Combining 3D single molecule localization strategies for reproducible bioimaging
Combining 3D single molecule localization strategies for reproducible bioimaging
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DOI：
10.1038/s41467-019-09901-8
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发表时间：
2019-04-30
影响因子：
16.6
通讯作者：
Leveque-Fort, Sandrine
中科院分区：
文献类型：
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作者：
Cabriel, Clement;Bourg, Nicolas;Leveque-Fort, Sandrine
在这里，我们提出了一个3D定位为基础的超分辨率技术提供了一个缓慢变化的定位精度超过1 μ m的范围内，精度下降到15 nm。轴向定位是通过点扩散函数（PSF）成形和超临界角荧光（SAF），产生绝对的轴向信息的组合。使用双视图方案，轴向检测与横向检测解耦并且独立地优化以在成像范围上提供弱各向异性3D分辨率。这种方法可以很容易地实现在大多数自制的点扩散函数成形设置，并提供无漂移，倾斜不敏感和消色差的结果。它对这些不可避免的实验偏差的不敏感性特别适用于3D超分辨率显微镜，正如我们通过成像细胞骨架，活细菌膜和轴突周期性亚膜支架所证明的那样。我们进一步说明了在几个pm的范围内，通过直接合并在不同深度的多个采集的生物成像技术的兴趣。
Here, we present a 3D localization-based super-resolution technique providing a slowly varying localization precision over a 1 mu m range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently to provide a weakly anisotropic 3D resolution over the imaging range. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tilt-insensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several-pm range by direct merging of multiple acquisitions at different depths.