Single Cell Label-Free Probing of Chromatin Dynamics During B Lymphocyte Maturation.
Single Cell Label-Free Probing of Chromatin Dynamics During B Lymphocyte Maturation.
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DOI：
10.3389/fcell.2021.646616
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发表时间：
2021
影响因子：
5.5
通讯作者：
Stone N
中科院分区：
文献类型：
--
作者：
Morrish R;Yim KHW;Pagliara S;Palombo F;Chahwan R;Stone N
在发育生长期间或响应于环境改变的大规模细胞内信号传导主要由细胞核内的染色质协调。染色质结构的化学和构象修饰是调节差异基因表达和最终决定细胞命运的关键步骤。因此，建立细胞核的化学性质可以为单个细胞尺度上的细胞过程的表型表征提供关键标记。拉曼显微镜是一种灵敏的技术，能够探测单细胞化学成分和亚细胞区域，在一个无标记的光学方式。因此，它在临床和基础研究方面都具有巨大的潜力。然而，感知到的拉曼光谱的局限性，如低信号强度和难以将振动信号的改变与随后的生物效应直接联系起来，阻碍了该领域的进展。在这里，我们使用免疫B淋巴细胞的发展作为一个模型，以评估染色质和转录的变化，使用共聚焦拉曼显微镜结合微流控装置和相关的转录组学，从而连接化学和结构特性的变化，生物学的结果。在成熟之前和之后评估活的B淋巴细胞。应用多变量分析来区分每个细胞内的细胞组分。然后鉴定未活化和活化的B淋巴细胞之间的光谱差异，并与常规RNA-seq分析相比评估它们与已知细胞内生物学变化的相关性。我们的数据表明，光谱分析提供了一个强大的工具来研究基因激活，可以补充传统的分子生物学技术，并开辟了一条道路，映射在单个细胞的生化组成的动态。
Large-scale intracellular signaling during developmental growth or in response to environmental alterations are largely orchestrated by chromatin within the cell nuclei. Chemical and conformational modifications of the chromatin architecture are critical steps in the regulation of differential gene expression and ultimately cell fate determination. Therefore, establishing chemical properties of the nucleus could provide key markers for phenotypic characterization of cellular processes on a scale of individual cells. Raman microscopy is a sensitive technique that is capable of probing single cell chemical composition—and sub-cellular regions—in a label-free optical manner. As such, it has great potential in both clinical and basic research. However, perceived limitations of Raman spectroscopy such as low signal intensity and the difficulty in linking alterations in vibrational signals directly with ensuing biological effects have hampered advances in the field. Here we use immune B lymphocyte development as a model to assess chromatin and transcriptional changes using confocal Raman microscopy in combination with microfluidic devices and correlative transcriptomics, thereby linking changes in chemical and structural properties to biological outcomes. Live B lymphocytes were assessed before and after maturation. Multivariate analysis was applied to distinguish cellular components within each cell. The spectral differences between non-activated and activated B lymphocytes were then identified, and their correlation with known intracellular biological changes were assessed in comparison to conventional RNA-seq analysis. Our data shows that spectral analysis provides a powerful tool to study gene activation that can complement conventional molecular biology techniques and opens the way for mapping the dynamics in the biochemical makeup of individual cells.
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