Understanding the three-dimensional world from two-dimensional immunofluorescent adjacent sections.
Understanding the three-dimensional world from two-dimensional immunofluorescent adjacent sections.
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DOI：
10.4103/2153-3539.158052
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发表时间：
2015-01-01
影响因子：
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通讯作者：
Manova-Todorova, Katia
中科院分区：
文献类型：
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作者：
Fujisawa, Sho;Yarilin, Dmitry;Manova-Todorova, Katia
在三维（3D）层面上可视化组织结构对于理解正常和病理现象至关重要。然而，对厚切片和整装样本进行染色和成像可能具有挑战性。几十年来，研究人员对大型组织进行连续切片，并费力地重建3D体积。从切片到对齐的自动化技术的进步，现在极大地加快了这一过程。此外，免疫荧光染色方法允许同时检测多种抗原并进行立体分析。目的是将多通道免疫荧光染色和自动化技术应用于连续切片的3D重建以进行立体分析。石蜡包埋的样本采用手动切片，但以自动化方式进行处理、染色、成像和对齐。对重建的堆叠图像进行3D定量分析。通过将自动化免疫荧光染色与重建相邻切片的可靠方法相结合，我们不仅能够详细地可视化样本的几何结构，还能看到其3D环境中多种感兴趣的蛋白质和分子的存在及相互作用。技术和软件算法的进步显著加快了连续切片的3D重建。自动化的多抗原免疫荧光染色将极大地拓宽可通过这种方法回答的科学问题的范围和复杂性。
Visualizing tissue structures in three-dimensions (3D) is crucial to understanding normal and pathological phenomena. However, staining and imaging of thick sections and whole mount samples can be challenging. For decades, researchers have serially sectioned large tissues and painstakingly reconstructed the 3D volume. Advances in automation, from sectioning to alignment, now greatly accelerate the process. In addition, immunofluorescent staining methods allow multiple antigens to be simultaneously detected and analyzed volumetrically. The objective was to incorporate multi-channel immunofluorescent staining and automation in 3D reconstruction of serial sections for volumetric analysis. Paraffin-embedded samples were sectioned manually but were processed, stained, imaged and aligned in an automated fashion. Reconstructed stacks were quantitatively analyzed in 3D. By combining automated immunofluorescent staining and tried-and-true methods of reconstructing adjacent sections, we were able to visualize, in detail, not only the geometric structures of the sample but also the presence and interactions of multiple proteins and molecules of interest within their 3D environment. Advances in technology and software algorithms have significantly expedited the 3D reconstruction of serial sections. Automated, multi-antigen immunofluorescent staining will significantly broaden the range and complexity of scientific questions that can be answered with this methodology.