Abstract
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.
Highlights
Beyond the genomic era, systematic observation and quantification at single-cell resolution has become a powerful approach to investigate which and how cellular processes, such as cell division or active cellular constriction, underlie morphogenesis during the embryonic development [1,2,3]
We first show images obtained via whole-tissue fluorescence labeling of F-actin and E-cadherin, both of which are generally expressed in epithelial cells, in the developing epididymis (E16.5), kidney (E13.5), and lung (E13.5) (Fig 1)
These two proteins are known to be key factors for the generation of cellular physical force associated with maintaining epithelial integrity during tissue morphogenesis, the exact distribution of these proteins in whole internal organs remains elusive because the analysis has been mainly performed via slice sectioning [18,19,20,21]
Summary
Systematic observation and quantification at single-cell resolution has become a powerful approach to investigate which and how cellular processes, such as cell division or active cellular constriction, underlie morphogenesis during the embryonic development [1,2,3]. Employing such an approach in morphogenesis research has progressed the understanding of biological processes bridging different scales between the cell and tissue levels. Fluorescence labeling has been widely used for the detection of these cellular processes. Quantification of Whole-Tissue Immunofluorescence Images data collection and analysis, decision to publish, or preparation of the manuscript
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