Procedures for the quantification of whole-tissue immunofluorescence images obtained at single-cell resolution during murine tubular organ development.
Procedures for the quantification of whole-tissue immunofluorescence images obtained at single-cell resolution during murine tubular organ development.
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DOI：
10.1371/journal.pone.0135343
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Adachi T
中科院分区：
文献类型：
--
作者：
Hirashima T;Adachi T
单细胞分辨率的全组织量化已成为进一步定量了解器官发育过程中形态发生的必然途径。最近光学组织清除和显微镜技术的进步极大地提高了该方法的可行性。然而，这种方法导致成功的全组织量化所需的一系列程序还远远没有开发出来。为了提供适当的程序，我们在这里展示了整个过程中每个关键步骤的提示，包括免疫荧光固定、光学透明和数字图像处理，并以发育中的小鼠附睾、肾脏和肺等内脏为例。通过对固定液和清除方法的比较，我们找到了实现特定免疫标记靶点更清晰的深层组织成像的最佳条件，并解释了哪些方法可以获得生动的体积成像。此外，我们还展示了光学清洗后的三维数字图像处理为整个组织分析提供了客观的定量数据，重点是附睾管中有丝分裂细胞的空间分布。本文所示的全组织量化方法有助于系统地测量器官发育中的细胞过程，促进在单细胞水平上对形态发生的进一步理解。
Whole-tissue quantification at single-cell resolution has become an inevitable approach for further quantitative understanding of morphogenesis in organ development. The feasibility of the approach has been dramatically increased by recent technological improvements in optical tissue clearing and microscopy. However, the series of procedures required for this approach to lead to successful whole-tissue quantification is far from developed. To provide the appropriate procedure, we here show tips for each critical step of the entire process, including fixation for immunofluorescence, optical clearing, and digital image processing, using developing murine internal organs such as epididymis, kidney, and lung as an example. Through comparison of fixative solutions and of clearing methods, we found optimal conditions to achieve clearer deep-tissue imaging of specific immunolabeled targets and explain what methods result in vivid volume imaging. In addition, we demonstrated that three-dimensional digital image processing after optical clearing produces objective quantitative data for the whole-tissue analysis, focusing on the spatial distribution of mitotic cells in the epididymal tubule. The procedure for the whole-tissue quantification shown in this article should contribute to systematic measurements of cellular processes in developing organs, accelerating the further understanding of morphogenesis at the single cell level.