Optimal 3D single-molecule localization for superresolution microscopy with aberrations and engineered point spread functions
Optimal 3D single-molecule localization for superresolution microscopy with aberrations and engineered point spread functions
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DOI：
10.1073/pnas.1109011108
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发表时间：
2012-01-17
影响因子：
11.1
通讯作者：
Piestun, Rafael
中科院分区：
文献类型：
--
作者：
Quirin, Sean;Pavani, Sri Rama Prasanna;Piestun, Rafael
光激活定位显微术是一种基于单分子定位的远场超分辨成像技术，具有亚衍射极限的精度。这些技术的首字母缩写如PALM（光激活定位显微镜）或STORM（随机光学重建显微镜），通过仅允许稀疏的随机分子组在任何给定时间发光并随后以极高的精度定位每个分子来实现超分辨率。最近，这种技术已经扩展到三维，为探索细胞的结构和功能开辟了前所未有的可能性。有趣的是，通过额外的光学器件对三维（3D）点扩散函数（PSF）进行适当的工程设计，已被证明可以在理论上提高3D位置估计和最终的分辨率。在本文中，一个最佳的3D单分子定位估计提出了一个一般的框架，噪声，畸变和/或工程PSF成像。为了找到每个分子的位置，实现了相位检索使能的最大似然估计器。该估计被证明是有效的，这意味着它达到了基本的Cramer-Rao下限的x，y和z定位精度。使用特定工程PSF显微镜的相位恢复启用最大似然估计器的实验应用展示了无与伦比的低光子计数3D宽场单分子定位性能。
Photo-activation localization microscopy is a far-field superresolution imaging technique based on the localization of single molecules with subdiffraction limit precision. Known under acronyms such as PALM (photo-activated localization microscopy) or STORM (stochastic optical reconstruction microscopy), these techniques achieve superresolution by allowing only a sparse, random set of molecules to emit light at any given time and subsequently localizing each molecule with great precision. Recently, such techniques have been extended to three dimensions, opening up unprecedented possibilities to explore the structure and function of cells. Interestingly, proper engineering of the three-dimensional (3D) point spread function (PSF) through additional optics has been demonstrated to theoretically improve 3D position estimation and ultimately resolution. In this paper, an optimal 3D single-molecule localization estimator is presented in a general framework for noisy, aberrated and/or engineered PSF imaging. To find the position of each molecule, a phase-retrieval enabled maximum-likelihood estimator is implemented. This estimator is shown to be efficient, meaning it reaches the fundamental Cramer-Rao lower bound of x, y, and z localization precision. Experimental application of the phase-retrieval enabled maximum-likelihood estimator using a particular engineered PSF microscope demonstrates unmatched low-photon-count 3D wide-field single-molecule localization performance.