光激活定位显微镜中多闪烁伪影的校正。

Correction of multiple-blinking artifacts in photoactivated localization microscopy.

作者信息

Jensen Louis G, Hoh Tjun Yee, Williamson David J, Griffié Juliette, Sage Daniel, Rubin-Delanchy Patrick, Owen Dylan M

机构信息

Department of Mathematics, Aarhus University, Aarhus, Denmark.

Institute for Statistical Science, School of Mathematics, University of Bristol, Bristol, UK.

出版信息

Nat Methods. 2022 May;19(5):594-602. doi: 10.1038/s41592-022-01463-w. Epub 2022 May 11.

Abstract

Photoactivated localization microscopy (PALM) produces an array of localization coordinates by means of photoactivatable fluorescent proteins. However, observations are subject to fluorophore multiple blinking and each protein is included in the dataset an unknown number of times at different positions, due to localization error. This causes artificial clustering to be observed in the data. We present a 'model-based correction' (MBC) workflow using calibration-free estimation of blinking dynamics and model-based clustering to produce a corrected set of localization coordinates representing the true underlying fluorophore locations with enhanced localization precision, outperforming the state of the art. The corrected data can be reliably tested for spatial randomness or analyzed by other clustering approaches, and descriptors such as the absolute number of fluorophores per cluster are now quantifiable, which we validate with simulated data and experimental data with known ground truth. Using MBC, we confirm that the adapter protein, the linker for activation of T cells, is clustered at the T cell immunological synapse.

摘要

光激活定位显微镜(PALM)通过光激活荧光蛋白产生一系列定位坐标。然而,由于定位误差,观测结果会受到荧光团多次闪烁的影响,并且每个蛋白质在数据集中会在不同位置被包含未知次数。这导致在数据中观察到人为聚类。我们提出了一种“基于模型的校正”(MBC)工作流程,使用无校准的闪烁动力学估计和基于模型的聚类来生成一组校正后的定位坐标,这些坐标代表具有更高定位精度的真实潜在荧光团位置,性能优于现有技术。校正后的数据可以可靠地进行空间随机性测试或通过其他聚类方法进行分析,并且现在每个聚类中荧光团的绝对数量等描述符是可量化的,我们用模拟数据和具有已知真实情况的实验数据进行了验证。使用MBC,我们证实衔接蛋白(T细胞激活连接蛋白)在T细胞免疫突触处聚集。

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