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3D high-density localization microscopy using hybrid astigmatic/ biplane imaging and sparse image reconstruction

机译:使用混合像散/双平面成像和稀疏图像重建的3D高密度定位显微镜

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摘要

Localization microscopy achieves nanoscale spatial resolution by iterative localization of sparsely activated molecules, which generally leads to a long acquisition time. By implementing advanced algorithms to treat overlapping point spread functions (PSFs), imaging of densely activated molecules can improve the limited temporal resolution, as has been well demonstrated in two-dimensional imaging. However, three-dimensional (3D) localization of high-density data remains challenging since PSFs are far more similar along the axial dimension than the lateral dimensions. Here, we present a new, high-density 3D imaging system and algorithm. The hybrid system is implemented by combining astigmatic and biplane imaging. The proposed 3D reconstruction algorithm is extended from our state-of-the art 2D high-density localization algorithm. Using mutual coherence analysis of model PSFs, we validated that the hybrid system is more suitable than astigmatic or biplane imaging alone for 3D localization of high-density data. The efficacy of the proposed method was confirmed via simulation and real data of microtubules. Furthermore, we also successfully demonstrated fluorescent-protein-based live cell 3D localization microscopy with a temporal resolution of just 3 seconds, capturing fast dynamics of the endoplasmic recticulum.
机译:定位显微镜通过稀疏激活分子的迭代定位来实现纳米级的空间分辨率,这通常会导致较长的采集时间。通过实施先进的算法来处理重叠点扩散函数(PSF),密集激活分子的成像可以改善有限的时间分辨率,这在二维成像中已得到充分证明。但是,高密度数据的三维(3D)定位仍然具有挑战性,因为PSF沿轴向尺寸比侧向尺寸更相似。在这里,我们提出了一种新的高密度3D成像系统和算法。通过组合像散和双平面成像来实现混合系统。所提出的3D重建算法是从我们最新的2D高密度定位算法扩展而来的。使用模型PSF的互相关分析,我们验证了混合系统比单独的像散或双平面成像更适合用于高密度数据的3D定位。通过仿真和微管的真实数据证实了该方法的有效性。此外,我们还成功地证明了基于荧光蛋白的活细胞3D定位显微镜,其时间分辨率仅为3秒,捕获了内质网的快速动态。

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