Characterisation of the effects of optical aberrations in single molecule techniques.
Characterisation of the effects of optical aberrations in single molecule techniques.
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DOI：
10.1364/boe.7.001755
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发表时间：
2016-05-01
影响因子：
3.4
通讯作者：
Lo Schiavo V
中科院分区：
文献类型：
--
作者：
Coles BC;Webb SE;Schwartz N;Rolfe DJ;Martin-Fernandez M;Lo Schiavo V
光学像差降低了荧光显微镜的图像质量，包括单分子技术。这些依赖于后处理来定位图像序列中的单个分子。利用模拟数据，我们展示了光学像差对定位成功、精度和精度的影响。当像差强度大于1.0 rad RMS时，峰值强度和成功定位的比例大大降低，而每个定位的精度减半。使用雷暴包时，在像差强度仅为~0.6 rad RMS时，假阳性定位的数量超过真阳性定位数量的10%，而使用径向对称包时，其像差强度大于1.0 rad RMS。存在彗差时，在像差强度~0.6 rad RMS下，定位误差达到100 nm。本文还考虑了噪声和像散对轴向分辨率的影响。了解像差的影响是至关重要的，当决定是否增加自适应光学到单分子显微镜可以显著增加从图像序列获得的信息。
Optical aberrations degrade image quality in fluorescence microscopy, including for single-molecule based techniques. These depend on post-processing to localize individual molecules in an image series. Using simulated data, we show the impact of optical aberrations on localization success, accuracy and precision. The peak intensity and the proportion of successful localizations strongly reduces when the aberration strength is greater than 1.0 rad RMS, while the precision of each of those localisations is halved. The number of false-positive localisations exceeded 10% of the number of true-positive localisations at an aberration strength of only ~0.6 rad RMS when using the ThunderSTORM package, but at greater than 1.0 rad RMS with the Radial Symmetry package. In the presence of coma, the localization error reaches 100 nm at ~0.6 rad RMS of aberration strength. The impact of noise and of astigmatism for axial resolution are also considered. Understanding the effect of aberrations is crucial when deciding whether the addition of adaptive optics to a single-molecule microscope could significantly increase the information obtainable from an image series.