Heberle Frederick A, Welsch Doug, Scott Haden L, Waxham M Neal
Department of Chemistry, University of Tennessee, Knoxville, Tennessee.
Department of Neurobiology and Anatomy, University of Texas Health Science Center, Houston, Texas.
Biophys Rep (N Y). 2022 Dec 10;3(1):100090. doi: 10.1016/j.bpr.2022.100090. eCollection 2023 Mar 8.
Cryogenic electron microscopy (cryo-EM) is among the most powerful tools available for interrogating nanoscale structure of biological materials. We recently showed that cryo-EM can be used to measure the bilayer thickness of lipid vesicles and biological membranes with subangstrom precision, resulting in the direct visualization of nanoscopic domains of different thickness in multicomponent lipid mixtures and giant plasma membrane vesicles. Despite the great potential of cryo-EM for revealing the lateral organization of biomembranes, a large parameter space of experimental conditions remains to be optimized. Here, we systematically investigate the influence of instrument parameters and image postprocessing steps on the ability to accurately measure bilayer thickness and discriminate regions of different thickness within unilamellar liposomes. This unique application of cryo-EM places particular demands on image acquisition optimization and analysis due to the facts that 1) each vesicle is a different size with different curvature, 2) the domains in each vesicle can be heterogenous in size, and 3) the random orientation of vesicles amplifies the variability of domain size in projected images. We also demonstrate a spatial autocorrelation analysis to extract additional information about lateral heterogeneity.
低温电子显微镜（cryo-EM）是用于探究生物材料纳米级结构的最强大工具之一。我们最近表明，cryo-EM可用于以亚埃精度测量脂质囊泡和生物膜的双层厚度，从而直接观察多组分脂质混合物和巨型质膜囊泡中不同厚度的纳米级区域。尽管cryo-EM在揭示生物膜横向组织方面具有巨大潜力，但仍有很大的实验条件参数空间有待优化。在此，我们系统地研究了仪器参数和图像后处理步骤对准确测量单层脂质体双层厚度以及区分不同厚度区域能力的影响。由于以下事实，cryo-EM的这种独特应用对图像采集优化和分析提出了特殊要求：1）每个囊泡大小不同且曲率不同；2）每个囊泡中的区域大小可能存在异质性；3）囊泡的随机取向放大了投影图像中区域大小的变异性。我们还展示了一种空间自相关分析，以提取有关横向异质性的更多信息。
