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单分子定位技术可以绕过光学系统的衍射限制, 在生物样品的单粒子追踪和超分辨显微成像中得到了广泛应用. 多通道单分子定位采用多个成像通道, 可以实现对不同目标的同时追踪或多色超分辨成像, 也可以提升单粒子追踪的轴向深度或实现更高的定位精度和密度. 但各通道图像间的差异会影响协同定位或定量分析, 因此图像配准是其图像数据预处理的关键环节; 且由于单分子定位精度高, 其对多通道图像配准精度的要求也很高. 现有技术一般采用基于控制点的配准方法, 且多采用复杂而精密的方式来获取基准物网格图像用于定位得到控制点对, 以实现高精度图像配准, 对样品或实验设备要求高, 难以直接推广. 为此, 本文基于局部非线性变换和误匹配点剔除, 发展了一种可以直接采用随机分布荧光珠样品作为基准物的高精度图像配准方法, 通过在特征匹配和变换模型参数估计的过程中对控制点进行监测和迭代筛选, 以剔除因单分子定位不准确或精度差而导致未精确匹配的控制点对, 从而消除以随机分布荧光珠样品作为基准物时对于控制点准确获取和精确匹配所带来的不良影响, 同时采用基于局部加权平均的二阶多项式拟合进行变换模型参数估计, 以更好地适用于不同通道间存在局部非线性形变的情形. 结果表明, 采用该方法只需要3次迭代, 就可以将未准确定位和精确匹配的控制点对找到并剔除, 从而实现更准确的变换模型参数估计, 将配准精度提高一个数量级, 在图像局部非线性形变情况严重的正交像散双通道单分子定位成像系统中实现了约6 nm的配准精度.Single-molecule localization technology has been widely used in single-particle tracking and super-resolution imaging of biological samples, as it can bypass the diffraction limit of optical systems. Multi-channel single-molecule localization uses multiple imaging channels to simultaneously track different targets or perform multi-color super-resolution imaging, and can also improve the axial depth of single-particle tracking or achieve higher localization precision and density for super-resolution imaging. However, the difference between images in each channel can affect collaborative localization or quantitative analysis, so image registration is a key step in its image data preprocessing. Moreover, due to the high precision of single-molecule localization, its requirements for multi-channel image registration accuracy are also high. Existing technologies generally use control point-based registration methods and often use complicated and precise methods to obtain fiducial images for locating control point pairs to achieve high-precision image registration, which involves high sample or experimental equipment requirements and is difficult to directly extend to other systems. Therefore, developed in this work, is a high-precision image registration method that can directly use randomly distributed fluorescent beads as fiducial samples based on local nonlinear transformation and elimination of mismatched points. By monitoring and iteratively filtering control points in the process of feature matching and transformation model parameter estimation to eliminate control point pairs that are not accurately matched due to inaccurate or poor precision of single-molecule localization, the adverse effects on accurate acquisition and precise matching of control points when using randomly distributed fluorescent beads as fiducial samples are eliminated. At the same time, a second-order polynomial fitting based on local weighted mean is used for estimating the transformation model parameter to better adapt to the existence of local nonlinear deformation between different channels. The results show that using this method only requires three iterations to find and eliminate control point pairs that are not accurately located and matched, thereby achieving more accurate transformation model parameter and improving the registration accuracy by an order of magnitude, achieving a registration accuracy of about 6 nm in a complex dual-channel single-molecule localization imaging system based on orthogonal astigmatism.
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Keywords:
- single molecule localization/
- multi-channel imaging/
- image registration/
- elimination of mismatched points
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迭代次数 剔除控制点对标号 平均基准配准误差/ nm 0 — 29.3 1 3, 4, 6, 7 11.7 2 1, 2, 3, 4, 5 3.4 3 2, 3, 4, 5 2.9 误差阈值/nm 每次迭代剔除的控制点对数量 每次迭代后的平均配准误差/nm 1 2 3 4 1 2 3 4 10 4 16 10 9 11.7 5.8 5.6 5.4 20 4 9 4 — 11.7 3.5 2.9 — 30 4 5 4 — 11.7 3.4 2.9 — 40 4 3 — — 11.7 10.4 — — 50 4 2 — — 11.7 11.6 — — 误匹配点剔除算法 迭代成功率 迭代成功所需的
平均迭代次数RANSAC 0.84 165 RSCFDI 0.49 217 RASCFDI+
RANSAC2 match0.49 260 线间距/nm 对比度/% b c d 平均 b c d 平均 Control 58.6 59.4 57.2 58.4 82.1 87.2 76.2 81.8 LWM 55.2 58.8 55.8 56.6 77.7 83.3 48.5 69.8 LWM+EMP-FRE 57.2 58.4 59.0 58.2 74.7 79.4 82.1 78.7 -
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