Department of Chemistry, University of Washington, Seattle, Washington 98195, United States.
J Phys Chem B. 2022 Oct 6;126(39):7595-7603. doi: 10.1021/acs.jpcb.2c04355. Epub 2022 Sep 22.
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 , 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.
哺乳动物细胞中的细胞大小和密度受到严格控制。它们影响着广泛的生理功能，包括渗透调节、组织稳态和生长调节。与大小相比，给定细胞类型的密度变化通常要小得多，这意味着细胞类型特异性密度在细胞功能中起着重要作用。然而，人们对细胞密度如何影响细胞功能以及它是如何被调节的知之甚少。目前用于测量细胞内密度的工具仅限于悬浮细胞或在 2D 基质上生长的细胞，两者都不能重现完整组织中单细胞的生理学。虽然光学测量有潜力非侵入性地测量细胞密度，但多细胞系统中的光散射会阻止直接定量。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。它利用细胞内大分子的固有振动信息来定量干物质密度。此外，还使用水作为内部标准来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以及细胞状态。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对 3D 肿瘤球体的细胞内密度成像。我们的技术有可能对完整组织中的细胞内密度进行成像，并深入了解密度调节及其在组织稳态中的作用。
