Measurement precision bounds on aberrated single molecule emission patterns.
Measurement precision bounds on aberrated single molecule emission patterns.
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测量精度限制了畸变的单分子发射模式。
DOI：
10.1101/2023.11.30.569462
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Huang,Fang
中科院分区：
文献类型：
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作者：
Fang,Li;Huang,Fang
单分子定位显微镜（SMLM）通过提供精细的纳米级空间分辨率彻底改变了生物现象的研究。然而，样品和系统缺陷引起的光学像差会扭曲单分子发射图案（即 PSF），导致 SMLM 的精度和分辨率降低，特别是在三维 (3D) 应用中。尽管已采用各种分析方法和仪器方法来减轻这些像差，但仍然缺乏对不同类型的常见像差如何影响单分子实验及其图像形成的全面分析。在本研究中，我们通过对存在像差的情况下位置和波前畸变测量的理论精度极限进行定量研究来解决这一差距。利用 Fisher 信息和 Cramér-Rao 下限 (CRLB)，我们定量分析和比较了不同像差类型（包括折射率失配像差）对双平面和像散 3D 模态以及 2D SMLM 成像中的定位精度的影响。此外，我们研究了从像差单分子发射模式中可实现的波前估计精度，这是通过厚样品在 SMLM 中成功实现自适应光学的关键一步。该分析为SMLM在全细胞、组织和大视场中的发展和应用奠定了定量基础，深入了解单分子成像中不同像差类型的行为，从而为开发高效像差校正策略和提高3D SMLM的精度和可靠性提供理论指导。
Single-molecule localization microscopy (SMLM) has revolutionized the study of biological phenomena by providing exquisite nanoscale spatial resolution. However, optical aberrations induced by sample and system imperfections distort the single-molecule emission patterns (i.e. PSFs), leading to reduced precision and resolution of SMLM, particularly in three-dimensional (3D) applications. While various methods, both analytical and instrumental, have been employed to mitigate these aberrations, a comprehensive analysis of how different types of commonly encountered aberrations affect single-molecule experiments and their image formation remains missing. In this study, we addressed this gap by conducting a quantitative study of the theoretical precision limit for position and wavefront distortion measurements in the presence of aberrations. Leveraging Fisher information and Cramér-Rao lower bound (CRLB), we quantitively analyzed and compared the effects of different aberration types, including index mismatch aberrations, on localization precision in both biplane and astigmatism 3D modalities as well as 2D SMLM imaging. Furthermore, we studied the achievable wavefront estimation precision from aberrated single-molecule emission patterns, a pivot step for successful adaptive optics in SMLM through thick specimens. This analysis lays a quantitative foundation for the development and application of SMLM in whole-cells, tissues and with a large field of view, providing in-depth insights into the behavior of different aberration types in single-molecule imaging and thus generating theoretical guidelines for developing highly efficient aberration correction strategies and enhancing the precision and reliability of 3D SMLM.