Automatic three‐dimensional segmentation of mouse embryonic stem cell nuclei by utilising multiple channels of confocal fluorescence images
Automatic three‐dimensional segmentation of mouse embryonic stem cell nuclei by utilising multiple channels of confocal fluorescence images
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DOI：
10.1111/jmi.12949
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发表时间：
2020-07
影响因子：
2
通讯作者：
Yuan-Hsiang Chang;H. Yokota;K. Abe;Ming-Dar Tasi;S. Chu
中科院分区：
文献类型：
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作者：
Yuan-Hsiang Chang;H. Yokota;K. Abe;Ming-Dar Tasi;S. Chu
来自携带报告基因组蛋白H2B-mCherry和Mvh-Venus的小鼠胚胎干细胞（ESC）的延时共聚焦荧光显微镜图像已用于监测活ESC的细胞/分化特征的动态变化。准确的细胞核分割需要在单细胞分辨率下分析ESC动力学和分化。几种方法使用核轮廓上的凹陷来分割重叠的细胞核。我们提出的方法不仅评估凹，而且每个2D核区域的大小和形状，以确定是否满足任何海峡，挤出，凸度和大直径标准，以分割区域内的重叠核。然后，我们使用3D分割方法来重建简单的，凸的，和合理大小的3D细胞核沿着图像堆叠方向使用的半径和中心的每个分割区域在各自的显微镜图像。为了避免核边界上的假凹面，H2B-mCherry报告基因的荧光图像用于定位细胞核，Venus荧光图像用于确定细胞集落范围。我们使用了一系列的图像预处理程序，以消除噪声外，内细胞集落，并在各自的核，并平滑核边界的基础上殖民地范围。我们提出了动态数据结构来记录每个分割的核区域和固体的3D共聚焦图像集（卷）。实验结果表明，所提出的图像预处理方法保留了显微图像中小鼠胚胎干细胞核的区域，分割方法有效地分割出了大小和形状合理的细胞核。共焦显微镜图像的集合（体积）中的所有3D细胞核可以由用于3D重建的动态数据结构访问。可以跟踪延时共聚焦显微镜图像中的3D细胞核，以计算连续体积中的细胞运动和增殖，从而了解ESC分化特征的动态。
Time‐lapse confocal fluorescence microscopy images from mouse embryonic stem cells (ESCs) carrying reporter genes, histone H2B‐mCherry and Mvh‐Venus, have been used to monitor dynamic changes in cellular/differentiation characteristics of live ESCs. Accurate cell nucleus segmentation is required to analyse the ESC dynamics and differentiation at a single cell resolution. Several methods used concavities on nucleus contours to segment overlapping cell nuclei. Our proposed method evaluates not only the concavities but also the size and shape of every 2D nucleus region to determine if any of the strait, extrusion, convexity and large diameter criteria is satisfied to segment overlapping nuclei inside the region. We then use a 3D segmentation method to reconstruct simple, convex, and reasonably sized 3D nuclei along the image stacking direction using the radius and centre of every segmented region in respective microscopy images. To avoid false concavities on nucleus boundaries, fluorescence images of the H2B‐mCherry reporter are used for localisation of cell nuclei and Venus fluorescence images are used for determining the cell colony ranges. We use a series of image preprocessing procedures to remove noise outside and inside cell colonies, and in respective nuclei, and to smooth nucleus boundaries based on the colony ranges. We propose dynamic data structures to record every segmented nucleus region and solid in sets (volumes) of 3D confocal images. The experimental results show that the proposed image preprocessing method preserves the areas of mouse ESC nuclei on microscopy images and that the segmentation method effectively segment out every nucleus with a reasonable size and shape. All 3D nuclei in a set (volume) of confocal microscopy images can be accessed by the dynamic data structures for 3D reconstruction. The 3D nuclei in time‐lapse confocal microscopy images can be tracked to calculate cell movement and proliferation in consecutive volumes for understanding the dynamics of the differentiation characteristics about ESCs.