Expansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known, whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to ~4.2-times physically expanded nuclei corresponding to a maximal resolution of ~50–60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution, the chromatin structures were observed at a resolution of ~25–35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM, we observed that the preservation of the chromatin ultrastructure after the expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei, we localized successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunolabelling. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation), their ultrastructural arrangement was impaired.

膨胀显微镜 (ExM) 是一种物理放大具有保存超微结构的标本的方法。它有可能探索超出光衍射极限的结构特征。该程序已成功用于不同的动物物种，从分离的大分子复合物到细胞再到组织切片。植物来源样品的扩增仍处于起步阶段，染色质超微结构是否会因物理扩增而改变尚不清楚。在这项研究中，我们扩展了分离的大麦细胞核，并比较了 ExM 是否可以提供与超分辨率显微镜相当的染色质结构视图。测试了不同的固定和变性/消化条件以维持染色质超微结构。我们达到了~4。当通过野场 (WF) 显微镜成像时，2 倍物理膨胀的原子核对应于 ~50–60 nm 的最大分辨率。通过应用结构化照明显微镜（SIM，超分辨率）使 WF 分辨率加倍，以~25-35 nm 的分辨率观察染色质结构。WF 显微镜显示保留的核形状和核仁。此外，我们能够检测到染色质结构域，在未扩展的细胞核中是不可见的。然而，通过应用 SIM，我们观察到扩增后染色质超微结构的保存不完整，并且大多数测试条件未能保持超微结构。然而，使用膨胀的原子核，我们通过荧光原位杂交 (FISH) 成功定位了着丝粒重复序列，并通过间接免疫标记定位了着丝粒特异性组蛋白 H3 变体 CENH3。然而，尽管这些重复和蛋白质位于细胞核内的正确位置（表明 Rabl 方向），但它们的超微结构排列受损。