Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking.
Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking.
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DOI：
10.1021/acs.chemrev.6b00629
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发表时间：
2017-06-14
期刊：
影响因子：
62.1
通讯作者：
Moerner WE
中科院分区：
文献类型：
--
作者：
von Diezmann L;Shechtman Y;Moerner WE
单分子超分辨率荧光显微镜和单粒子跟踪是两种成像模式，分别在几十纳米的空间尺度上照亮细胞和材料的特性，或者在毫秒范围内提供有关纳米级粒子运动的动态信息。这些方法通常使用宽视场显微镜和二维相机检测器来定位分子，其精度比衍射极限高得多。鉴于每个单分子标记的总光子有限，这两种方式都需要仔细的数学分析和图像处理。通过扩展到三维（3D）单分子定位，可以获得更多关于所研究系统的信息：如果没有这种能力，在轴向方向上延伸的结构或运动的可视化很容易被错过或混淆，从而损害科学理解。已经设计了各种用于获得3D超分辨率图像和3D跟踪信息的方法，每种方法都有自己的优点和缺点。这些技术包括多焦平面成像、点扩散函数工程和干涉检测。这些方法可以基于它们提供具有有限光子的单分子发射体的准确和精确的位置信息的能力进行比较。为了成功地应用和进一步发展这些方法，必须考虑许多实际问题，包括光学像差的影响，成像系统中的场依赖性，荧光团标记密度和不同颜色通道之间的配准。3D超分辨率成像和跟踪的选择的例子被描述为从各种生物背景和各种方法的说明，展示了理解复杂系统的3D本地化的力量。
Single-molecule super-resolution fluorescence microscopy and single-particle tracking are two imaging modalities that illuminate the properties of cells and materials on spatial scales down to tens of nanometers, or with dynamical information about nanoscale particle motion in the millisecond range, respectively. These methods generally use wide-field microscopes and two-dimensional camera detectors to localize molecules to much higher precision than the diffraction limit. Given the limited total photons available from each single-molecule label, both modalities require careful mathematical analysis and image processing. Much more information can be obtained about the system under study by extending to three-dimensional (3D) single-molecule localization: without this capability, visualization of structures or motions extending in the axial direction can easily be missed or confused, compromising scientific understanding. A variety of methods for obtaining both 3D super-resolution images and 3D tracking information have been devised, each with their own strengths and weaknesses. These include imaging of multiple focal planes, point-spread-function engineering, and interferometric detection. These methods may be compared based on their ability to provide accurate and precise position information of single-molecule emitters with limited photons. To successfully apply and further develop these methods, it is essential to consider many practical concerns, including the effects of optical aberrations, field-dependence in the imaging system, fluorophore labeling density, and registration between different color channels. Selected examples of 3D super-resolution imaging and tracking are described for illustration from a variety of biological contexts and with a variety of methods, demonstrating the power of 3D localization for understanding complex systems.