Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, D-06466 Seeland, Germany.
Centre of the Region Hana for Biotechnological and Agricultural Research, Institute of Experimental Botany of the Czech Academy of Sciences, 77900 Olomouc, Czech Republic.
Int J Mol Sci. 2021 Feb 14;22(4):1903. doi: 10.3390/ijms22041903.
The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.
荧光显微镜对于理解细胞和亚细胞结构和功能的重要性是不可否认的。然而，分辨率受到光衍射的限制（横向约 200-250nm，轴向约 500-700nm）。与此同时，超分辨率显微镜技术，如结构光照明显微镜（SIM），越来越多地被应用于克服这种限制。然而，由于需要特定的样品制备和稳定的染料染色，基于受激发射损耗（STED）的超分辨率显微镜技术的分辨率虽然能够达到横向约 50nm 和轴向约 130nm，但在植物细胞研究中尚未得到广泛应用。单分子定位显微镜（SMLM）包括光激活定位显微镜（PALM）虽然这项纳米技术甚至可以检测单个分子，但尚未得到广泛应用。在这项研究中，我们通过传统的宽场和共聚焦显微镜以及 SIM、STED 和 SMLM 等亚衍射方法比较了大麦中期染色体中的蛋白质成像。染色体通过 DAPI（4'，6-二脒基-2-苯基吲哚）进行标记，DAPI 是一种 DNA 特异性染料，并用针对拓扑异构酶 IIα（Topo II）的抗体进行标记，Topo II 是一种对于正确染色质凝聚很重要的蛋白质。与衍射受限方法相比，三种不同的超分辨率成像技术的组合通过实现更高的分辨率，为植物染色体结构提供了更多的深入了解。
