在单细胞水平上分析分子活动的一种新的形态标记。

IF 2 4区 生物学 Q4 CELL BIOLOGY
Cell structure and function Pub Date : 2018-08-10 Epub Date: 2018-06-29 DOI:10.1247/csf.18013
Ayako Imanishi, Tomokazu Murata, Masaya Sato, Kazuhiro Hotta, Itaru Imayoshi, Michiyuki Matsuda, Kenta Terai
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引用次数: 15

摘要

一个多世纪以来,苏木精和伊红(H&E)染色一直是组织学研究的事实上的标准。因此,组织学知识的遗产主要是基于H&E染色。由于近年来多光子激发显微镜的出现,活体组织的观察越来越多地应用于许多研究领域。基因编码的离子和信号分子生物传感器的发展进一步加速了这项技术的采用。然而,由于缺乏合适的形态学标记,基于h&e的组织学尚未开始充分利用体内成像。在这里,我们报告了一种基因编码的荧光标记,NuCyM(细胞核,细胞质和膜),它被设计用来概括体内的H&E染色模式。利用ROSA26细菌人工染色体(BAC)克隆获得了一株表达NuCyM的转基因小鼠。在大多数组织中,NuCyM均匀地标记在质膜、细胞质和细胞核上,形成H&E染色样图像。在表达nucim的细胞中,通过三维成像可以清楚地观察到单个细胞在M期分裂为5个基本相。接下来,我们将NuCyM小鼠与基于Förster共振能量转移(FRET)原理表达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,从而自动量化每个细胞中的ERK活性。总之,NuCyM是一种多功能的细胞形态标记,使我们能够像H&E染色一样掌握组织学信息。关键词:体内成像,组织学,机器学习,分子活性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A Novel Morphological Marker for the Analysis of Molecular Activities at the Single-cell Level.

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.

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来源期刊
Cell structure and function
Cell structure and function 生物-细胞生物学
CiteScore
2.50
自引率
0.00%
发文量
6
审稿时长
>12 weeks
期刊介绍: Cell Structure and Function is a fully peer-reviewed, fully Open Access journal. As the official English-language journal of the Japan Society for Cell Biology, it is published continuously online and biannually in print. Cell Structure and Function publishes important, original contributions in all areas of molecular and cell biology. The journal welcomes the submission of manuscripts on research areas such as the cell nucleus, chromosomes, and gene expression; the cytoskeleton and cell motility; cell adhesion and the extracellular matrix; cell growth, differentiation and death; signal transduction; the protein life cycle; membrane traffic; and organelles.
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