Expansion microscopy (ExM) has become a powerful super-resolution method in cell biology. It is a simple, yet robust approach, which does not require any instrumentation or reagents beyond those present in a standard microscopy facility. In this study, we used kinetoplastid parasites Trypanosoma brucei and Leishmania major, which possess a complex, yet well-defined microtubule-based cytoskeleton, to demonstrate that this method recapitulates faithfully morphology of structures as previously revealed by a combination of sophisticated electron microscopy (EM) approaches. Importantly, we also show that due to the rapidness of image acquisition and three-dimensional reconstruction of cellular volumes ExM is capable of complementing EM approaches by providing more quantitative data. This is demonstrated on examples of less well-appreciated microtubule structures, such as the neck microtubule of T. brucei or the pocket, cytosolic and multivesicular tubule-associated microtubules of L. major. We further demonstrate that ExM enables identifying cell types rare in a population, such as cells in mitosis and cytokinesis. Three-dimensional reconstruction of an entire volume of these cells provided details on the morphology of the mitotic spindle and the cleavage furrow. Finally, we show that established antibody markers of major cytoskeletal structures function well in ExM, which together with the ability to visualize proteins tagged with small epitope tags will facilitate studies of the kinetoplastid cytoskeleton.

膨胀显微镜（ExM）已成为细胞生物学中强大的超分辨率方法。这是一种简单而强大的方法，除了标准显微镜设施中存在的仪器或试剂之外，不需要任何仪器或试剂。在这项研究中，我们使用了动质体寄生虫布氏锥虫和大型利什曼原虫，它具有复杂但明确的基于微管的细胞骨架，以证明该方法忠实地再现了先前通过复杂的电子显微镜（EM）方法组合所揭示的结构形态。重要的是，我们还表明，由于图像采集和细胞体积三维重建的快速性，ExM 能够通过提供更多定量数据来补充 EM 方法。这一点在一些不太被广泛认可的微管结构的例子中得到了证明，例如T. brucei的颈部微管或L. Major的口袋、胞质和多泡小管相关的微管。我们进一步证明 ExM 能够识别群体中罕见的细胞类型，例如有丝分裂和胞质分裂中的细胞。这些细胞整个体积的三维重建提供了有丝分裂纺锤体和卵裂沟形态的详细信息。最后，我们表明，已建立的主要细胞骨架结构的抗体标记在 ExM 中功能良好，再加上可视化带有小表位标签的蛋白质的能力，将有助于对动质体细胞骨架的研究。