For more than a century, hematoxylin and eosin (H&E) staining has been the de facto standard for histological studies. Consequently, the legacy of histological knowledge is largely based on H&E staining. Due to the recent advent of multi-photon excitation microscopy, the observation of live tissue is increasingly being used in many research fields. Adoption of this technique has been further accelerated by the development of genetically encoded biosensors for ions and signaling molecules. However, H&E-based histology has not yet begun to fully utilize in vivo imaging due to the lack of proper morphological markers. Here, we report a genetically encoded fluorescent marker, NuCyM (Nucleus, Cytosol, and Membrane), which is designed to recapitulate H&E staining patterns in vivo. We generated a transgenic mouse line ubiquitously expressing NuCyM by using a ROSA26 bacterial artificial chromosome (BAC) clone. NuCyM evenly marked the plasma membrane, cytoplasm and nucleus in most tissues, yielding H&E staining-like images. In the NuCyM-expressing cells, cell division of a single cell was clearly observed as five basic phases during M phase by three-dimensional imaging. We next crossed NuCyM mice with transgenic mice expressing an ERK biosensor based on the principle of Förster resonance energy transfer (FRET). Using NuCyM, ERK activity in each cell could be extracted from the FRET images. To further accelerate the image analysis, we employed machine learning-based segmentation methods, and thereby automatically quantitated ERK activity in each cell. In conclusion, NuCyM is a versatile cell morphological marker that enables us to grasp histological information as with H&E staining.Key words: in vivo imaging, histology, machine learning, molecular activity.

中文翻译：

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一种用于在单细胞水平上分析分子活性的新型形态标记。

一个多世纪以来，苏木精和曙红（H＆E）染色已成为组织学研究的事实上的标准。因此，组织学知识的遗留很大程度上基于H＆E染色。由于最近出现了多光子激发显微镜，所以在许多研究领域中越来越多地使用活组织的观察。通过开发用于离子和信号分子的基因编码生物传感器，进一步加快了该技术的采用。但是，由于缺乏适当的形态学标记，基于H＆E的组织学尚未开始充分利用体内成像。在这里，我们报告了一种遗传编码的荧光标记NuCyM（核蛋白，胞质溶胶和膜），其旨在概述体内H＆E染色模式。我们通过使用ROSA26细菌人工染色体（BAC）克隆生成了普遍表达NuCyM的转基因小鼠品系。NuCyM均匀地标记了大多数组织的质膜，细胞质和细胞核，从而产生了类似于H＆E染色的图像。在表达NuCyM的细胞中，通过三维成像清楚地观察到单个细胞的细胞分裂为M期期间的五个基本期。接下来，我们根据Förster共振能量转移（FRET）的原理，将NuCyM小鼠与表达ERK生物传感器的转基因小鼠杂交。使用NuCyM，可以从FRET图像中提取每个细胞中的ERK活性。为了进一步加速图像分析，我们采用了基于机器学习的分割方法，从而自动量化了每个单元格中的ERK活性。综上所述，