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 on 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.

中文翻译：

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用于超分辨率成像和单粒子跟踪的单分子的三维定位

单分子超分辨率荧光显微镜和单粒子跟踪是两种成像方式，分别以几十纳米的空间尺度或具有毫秒级范围内的纳米尺度运动的动态信息照亮了细胞和材料的特性。这些方法通常使用宽视场显微镜和二维照相机检测器将分子定位到比衍射极限高得多的精度。鉴于每个单分子标记可用的总光子有限，两种模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，可以获得有关正在研究的系统的更多信息：如果没有此功能，沿轴向方向延伸的结构或运动的可视化很容易被遗漏或混淆，从而损害了科学的理解。已经设计了用于获得3D超分辨率图像和3D跟踪信息的多种方法，每种方法都有其自身的优点和缺点。这些包括多个焦平面的成像，点扩展功能工程和干涉检测。可以根据它们在具有有限光子的单分子发射器上提供准确和精确的位置信息的能力来比较这些方法。为了成功应用和进一步开发这些方法，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，荧光团标记密度以及不同颜色通道之间的配准。