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Sample and substrate preparation for exploring living neurons in culture with quantitative-phase imaging
Methods ( IF 4.8 ) Pub Date : 2018-03-01 , DOI: 10.1016/j.ymeth.2018.02.001
Sébastien A. Lévesque , Jean-Michel Mugnes , Erik Bélanger , Pierre Marquet

Quantitative-phase imaging (QPI) has recently emerged as a powerful new quantitative microscopy technique suitable for the noninvasive exploration of the structure and dynamics of transparent specimens, including living cells in culture. Indeed, the quantitative-phase signal (QPS), induced by transparent living cells, can be detected with a nanometric axial sensitivity, and contains a wealth of information about both cell morphology and content. However, QPS is also sensitive to various sources of experimental noise. In this chapter, we emphasize how to properly and specifically measure the cell-mediated QPS in a wet-lab environment, when measuring with a digital holographic microscope (DHM). First, we present the substrate-requisite characteristics for properly achieving such cell-mediated QPS measurements at single-cell level. Then, we describe how quantitative-phase digital holographic microscopy (QP-DHM) can be used to numerically process holograms and subsequently reshape wavefronts in association with post-processing algorithms, thereby allowing for highly stable QPS obtainable over extended periods of time. Such stable QPS is a prerequisite for exploring the dynamics of specific cellular processes. We also describe experimental procedures that make it possible to extract important biophysical cellular parameters from QPS including absolute cell volume, transmembrane water permeability, and the movements of water in and out of the cell. To illustrate how QP-DHM can reveal the dynamics of specific cellular processes, we show how the monitoring of transmembrane water movements can be used to resolve the neuronal network dynamics at single-cell level. This is possible because QPS can measure the activity of electroneutral cotransports, including NKCC1 and KCC2, during a neuronal firing mediated by glutamate, the main excitatory neurotransmitter in the brain. Finally, we added a supplemental section, with more technical details, for readers who are interested in troubleshooting live-cell QP-DHM.

中文翻译:

用定量相位成像探索培养物中活神经元的样品和底物制备

定量相位成像 (QPI) 最近已成为一种强大的新型定量显微镜技术,适用于透明标本(包括培养中的活细胞)的结构和动力学的无创探索。事实上,由透明活细胞诱导的定量相位信号 (QPS) 可以以纳米轴向灵敏度进行检测,并包含有关细胞形态和含量的丰富信息。然而,QPS 对各种实验噪声源也很敏感。在本章中,我们强调在使用数字全息显微镜 (DHM) 进行测量时,如何在湿实验室环境中正确且具体地测量细胞介导的 QPS。首先,我们提出了在单细胞水平上正确实现这种细胞介导的 QPS 测量所需的底物特征。然后,我们描述了如何使用定量相位数字全息显微镜 (QP-DHM) 对全息图进行数值处理,并随后与后处理算法相关联地重塑波前,从而允许在长时间内获得高度稳定的 QPS。这种稳定的 QPS 是探索特定细胞过程动力学的先决条件。我们还描述了可以从 QPS 中提取重要生物物理细胞参数的实验程序,包括绝对细胞体积、跨膜水渗透率以及水进出细胞的运动。为了说明 QP-DHM 如何揭示特定细胞过程的动力学,我们展示了如何使用跨膜水运动的监测来解决单细胞水平的神经元网络动力学。这是可能的,因为 QPS 可以测量在由谷氨酸(大脑中的主要兴奋性神经递质)介导的神经元放电期间,包括 NKCC1 和 KCC2 在内的电中性协同转运的活动。最后,我们为对活细胞 QP-DHM 故障排除感兴趣的读者添加了一个补充部分,其中包含更多技术细节。
更新日期:2018-03-01
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