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首页> 美国卫生研究院文献>Nucleus >Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes
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Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes

机译：使用Hoechst和DAPI荧光探针的染色质分布的单分子定位显微镜

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摘要

Several approaches have been described to fluorescently label and image DNA and chromatin in situ on the single-molecule level. These superresolution microscopy techniques are based on detecting optically isolated, fluorescently tagged anti-histone antibodies, fluorescently labeled DNA precursor analogs, or fluorescent dyes bound to DNA. Presently they suffer from various drawbacks such as low labeling efficiency or interference with DNA structure. In this report, we demonstrate that DNA minor groove binding dyes, such as Hoechst 33258, Hoechst 33342, and DAPI, can be effectively employed in single molecule localization microscopy (SMLM) with high optical and structural resolution. Upon illumination with low intensity 405 nm light, a small subpopulation of these molecules stochastically undergoes photoconversion from the original blue-emitting form to a green-emitting form. Using a 491 nm laser excitation, fluorescence of these green-emitting, optically isolated molecules was registered until “bleached”. This procedure facilitated substantially the optical isolation and localization of large numbers of individual dye molecules bound to DNA in situ, in nuclei of fixed mammalian cells, or in mitotic chromosomes, and enabled the reconstruction of high-quality DNA density maps. We anticipate that this approach will provide new insights into DNA replication, DNA repair, gene transcription, and other nuclear processes.
机译：已经描述了几种在单分子水平上对DNA和染色质进行荧光标记和成像的方法。这些超分辨率显微镜技术基于检测光学分离的，荧光标记的抗组蛋白抗体，荧光标记的DNA前体类似物或与DNA结合的荧光染料。目前，它们具有各种缺点，例如标记效率低或干扰DNA结构。在此报告中，我们证明了DNA小沟结合染料，例如Hoechst 33258，Hoechst 33342和DAPI，可以有效地用于具有高光学和结构分辨率的单分子定位显微镜（SMLM）。在用低强度405 nm的光照射时，这些分子的一小部分随机地从原始的发蓝光形式转换为发绿光形式。使用491 nm激光激发，记录这些发出绿色光的光学隔离分子的荧光，直到“漂白”为止。该程序实质上促进了光学分离和原位结合在固定哺乳动物细胞核中或有丝分裂染色体中的大量与DNA结合的单个染料分子，并能够重建高质量的DNA密度图。我们预计这种方法将为DNA复制，DNA修复，基因转录和其他核过程提供新的见解。

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