Lipid phase separation in cellular membranes is thought to play an important role in many biological functions. This has prompted the development of synthetic membranes to study lipid–lipid interactions in vitro, alongside optical microscopy techniques aimed at directly visualizing phase partitioning. In this context, there is a need to overcome the limitations of fluorescence microscopy, where added fluorophores can significantly perturb lipid packing. Raman-based optical imaging is a promising analytical tool for label-free chemically specific microscopy of lipid bilayers. In this work, we demonstrate the application of hyperspectral coherent Raman scattering microscopy combined with a quantitative unsupervised data analysis methodology developed in-house to visualize lipid partitioning in single planar membrane bilayers exhibiting liquid-ordered and liquid-disordered domains. Two home-built instruments were utilized, featuring coherent anti-Stokes Raman scattering and stimulated Raman scattering modalities. Ternary mixtures of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol were used to form phase-separated domains. We show that domains are consistently resolved, both chemically and spatially, in a completely label-free manner. Quantitative Raman susceptibility spectra of the domains are provided alongside their spatially resolved concentration maps.

中文翻译：

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高光谱相干拉曼散射的单双层脂质域的定量无标记成像。

人们认为细胞膜中的脂质相分离在许多生物学功能中起着重要作用。这促进了合成膜的研究，以研究体外的脂质-脂质相互作用以及旨在直接可视化相分配的光学显微镜技术。在这种情况下，需要克服荧光显微镜的局限性，在荧光显微镜中添加的荧光团会显着干扰脂质堆积。基于拉曼的光学成像是一种有前途的分析工具，可用于脂质双分子层的无标记化学特异性显微镜检查。在这项工作中，我们证明了高光谱相干拉曼散射显微镜结合内部开发的定量无监督数据分析方法的应用，以可视化显示具有液体有序和液体无序域的单个平面膜双层中的脂质分配。利用了两种家用仪器，其特征是相干的反斯托克斯拉曼散射和受激拉曼散射模态。二醇油酰磷脂酰胆碱，鞘磷脂和胆固醇的三元混合物用于形成相分离的域。我们显示，域是在化学和空间上以完全无标签的方式一致解析的。随域的空间分辨浓度图一起提供了域的定量拉曼敏感性光谱。