Department of Cell and Molecular Biology, Uppsala University, Box 596, 751 24 Uppsala, Sweden.
Nat Commun. 2017 May 3;8:15115. doi: 10.1038/ncomms15115.
Pointwise localization of individual fluorophores is a critical step in super-resolution localization microscopy and single particle tracking. Although the methods are limited by the localization errors of individual fluorophores, the pointwise localization precision has so far been estimated using theoretical best case approximations that disregard, for example, motion blur, defocus effects and variations in fluorescence intensity. Here, we show that pointwise localization precision can be accurately estimated directly from imaging data using the Bayesian posterior density constrained by simple microscope properties. We further demonstrate that the estimated localization precision can be used to improve downstream quantitative analysis, such as estimation of diffusion constants and detection of changes in molecular motion patterns. Finally, the quality of actual point localizations in live cell super-resolution microscopy can be improved beyond the information theoretic lower bound for localization errors in individual images, by modelling the movement of fluorophores and accounting for their pointwise localization uncertainty.
点定位是超分辨率定位显微镜和单粒子跟踪中的关键步骤。尽管这些方法受到单个荧光团定位误差的限制，但迄今为止，点定位精度一直是使用理论最佳情况近似值来估计的，这些近似值忽略了例如运动模糊、离焦效应和荧光强度变化等因素。在这里，我们展示了可以使用受简单显微镜特性约束的贝叶斯后验密度，直接从成像数据中准确估计点定位精度。我们进一步证明，估计的定位精度可用于改进下游的定量分析，例如扩散常数的估计和分子运动模式变化的检测。最后，通过对荧光团的运动进行建模并考虑其点定位不确定性，可以提高活细胞超分辨率显微镜中实际点定位的质量，超出了单个图像中定位误差的信息论下限。
