Quantitative Imaging of Intracellular Density with Ratiometric Stimulated Raman Scattering Microscopy
Quantitative Imaging of Intracellular Density with Ratiometric Stimulated Raman Scattering Microscopy
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DOI：
10.1021/acs.jpcb.2c04355
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发表时间：
2022-09-22
影响因子：
3.3
通讯作者：
Fu, Dan
中科院分区：
文献类型：
--
作者：
Figueroa, Benjamin;Xu, Fiona Xi;Fu, Dan
哺乳动物细胞中的细胞大小和密度受到严格控制。它们影响广泛的生理功能，包括渗透调节、组织动态平衡和生长调节。与大小相比，给定细胞类型的密度变化通常要小得多，这意味着细胞类型的特定密度在细胞功能中发挥着重要作用。然而，关于细胞密度如何影响细胞功能或它是如何被调控的，人们知之甚少。目前用于测量细胞内细胞密度的工具仅限于悬浮细胞或生长在2D基质上的细胞，这两种工具都不能概括完整组织中单个细胞的生理学。虽然光学测量有可能非侵入性地原位测量细胞密度，但多细胞系统中的光散射阻碍了直接量化。在这里，我们介绍了一种基于比率受激拉曼散射显微镜(RSRS)的细胞内密度成像技术。它使用细胞内大分子的固有振动信息来量化干质量密度。此外，还使用水作为内部标准，以校正像差和光散射效应。我们演示了细胞内密度的实时测量，并表明密度在不同的细胞类型中受到严格控制，可以用于区分细胞类型和细胞状态。我们进一步展示了对渗透压挑战的密度变化的动态成像，以及三维肿瘤球体的细胞内密度成像。我们的技术有可能成像完整组织中的细胞内密度，并了解密度调节及其在组织动态平衡中的作用。
Cell size and density are tightly controlled in mammalian cells. They impact a wide range of physiological functions, including osmoregulation, tissue homeostasis, and growth regulation. Compared to size, density variation for a given cell type is typically much smaller, implying that cell-type specific density plays an important role in cell function. However, little is known about how cell density affects cell function or how it is regulated. Current tools for intracellular cell density measurements are limited to either suspended cells or cells grown on 2D substrates, neither of which recapitulate the physiology of single cells in intact tissue. While optical measurements have the potential to noninvasively measure cell density in situ, light scattering in multicellular systems prevents direct quantification. Here, we introduce an intracellular density imaging technique based on ratiometric stimulated Raman scattering microscopy (rSRS). It uses intrinsic vibrational information from intracellular macromolecules to quantify dry mass density. Moreover, water is used as an internal standard to correct for aberration and light scattering effects. We demonstrate real-time measurement of intracellular density and show that density is tightly regulated across different cell types and can be used to differentiate cell types as well as cell states. We further demonstrate dynamic imaging of density change in response to osmotic challenge as well as intracellular density imaging of a 3D tumor spheroid. Our technique has the potential for imaging intracellular density in intact tissue and understanding density regulation and its role in tissue homeostasis.