Images of micrometer-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains' shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been limited. Researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under vitrified, hydrated conditions. The first method is optimized for probe-free applications: Domains are directly distinguished from the surrounding membrane by their thickness. This technique quantitatively and accurately measures area fractions of domains, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to direct and quantitative imaging of submicron membrane domains in vitrified, hydrated vesicles.

脂质双层中微米级域的图像提供了无模型证据的金标准，以了解域的形状，大小和分布。直接和定量评估较小（纳米级和亚微米级）液体区域的相应技术受到限制。研究人员通常试图将膜蛋白的活性与它们所驻留的结构域的属性相关联。这样做取决于膜结构域的鉴定和表征。尽管可以通过间接方法探查膜结构域的某些特征，但这些方法通常受到以下限制：必须在需要多个假设或参数的模型中分析数据。这里，我们通过开发和测试两种在仿生膜中识别亚微米域的方法来应对这一挑战。两种方法都利用了在玻璃化，水合条件下的三元膜的冷冻电子断层图。第一种方法针对无探针应用进行了优化：域的厚度直接与周围的膜区分开。这项技术与已知相图非常吻合，定量，准确地测量了区域的面积分数。第二种方法经过优化，可用于通过高分辨率冷冻电子断层扫描和衍射极限光学显微镜将单个标记物用于膜成像的应用。对于这种方法，我们测试了一组探针，发现三聚体mCherry标签表现最佳，并指定了开发未来高性能，两用探针。这些发展已导致对玻璃化水化囊泡中亚微米膜结构域的直接和定量成像。