Lu Jun, Xu Lei, Liao Shuyao, Wang Wei, Dong Biqin
Academy for Engineering and Technology, Yiwu Research Institute, Fudan University, Shanghai 200433, China.
Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033, China.
Biomed Opt Express. 2025 Jan 28;16(2):718-735. doi: 10.1364/BOE.545534. eCollection 2025 Feb 1.
Single-molecule localization microscopy (SMLM) is a powerful super-resolution imaging technique that offers resolution far beyond the optical diffraction limit. The commonly used high numerical-aperture (NA) objective lenses in SMLM can only provide a nearly ideal point-spread function (PSF) at the center of the field-of-view (FOV), whereas the off-axis PSF is often distorted due to optical aberrations. Since precision and accuracy of three-dimensional (3D) spatial localization of single molecules heavily depend on the system's PSF, the FOV of 3D SMLM is often restricted to about 50 µm × 50 µm limiting its applications in visualizing intra-/intercellular interactions and high-throughput single-molecule analysis. Here we present a systematic study to show the influence of optical aberrations on large FOV 3D SMLM using unmodified, astigmatic, and double-helix PSFs. Our results show that optical aberrations introduce significant localization errors during image reconstruction and thereby produce unreliable imaging results at the corner of the FOV. To maximize SMLM's FOV, we proposed and verified the potential of using discrete field-dependent PSFs to retain precise and accurate single-molecule localization and compare their reconstruction results using simulated resolution test patterns and biological structures. Moreover, GPU acceleration empowers a discrete PSF calibration model with high localization speed, which can provide real-time SMLM image reconstruction. We envision these results will further guide the development of strategies that can provide real-time and reliable image reconstruction in large FOV 3D SMLM.
单分子定位显微镜（SMLM）是一种强大的超分辨率成像技术，其分辨率远超光学衍射极限。SMLM中常用的高数值孔径（NA）物镜仅能在视场（FOV）中心提供近乎理想的点扩散函数（PSF），而离轴PSF常因光学像差而失真。由于单分子三维（3D）空间定位的精度和准确性在很大程度上取决于系统的PSF，3D SMLM的FOV通常限制在约50 µm×50 µm，这限制了其在可视化细胞内/细胞间相互作用和高通量单分子分析中的应用。在此，我们进行了一项系统研究，以展示光学像差对使用未修正、像散和双螺旋PSF的大FOV 3D SMLM的影响。我们的结果表明，光学像差在图像重建过程中会引入显著的定位误差，从而在FOV边缘产生不可靠的成像结果。为了最大化SMLM的FOV，我们提出并验证了使用离散场相关PSF来保持精确和准确的单分子定位的潜力，并使用模拟分辨率测试图案和生物结构比较了它们的重建结果。此外，GPU加速使离散PSF校准模型具有高定位速度，能够提供实时SMLM图像重建。我们设想这些结果将进一步指导相关策略的开发，这些策略能够在大FOV 3D SMLM中提供实时且可靠的图像重建。
