The nuclear exit of messenger RNA (mRNA) molecules through the nuclear pore complex (NPC) is an essential step in the translation process of all proteins. The current limitations of conventional fluorescence and electron microscopy have prevented elucidation of how mRNA exports through the NPCs of live cells. In the recent years, various single-molecule fluorescence (SMF) microscopy techniques have been developed to improve the temporal and spatial resolutions of live-cell imaging allowing a more comprehensive understanding of the dynamics of mRNA export through native NPCs. In this review, we firstly evaluate the necessity of single-molecule live-cell microscopy in the study of mRNA nuclear export. Then, we highlight the application of single-point edge-excitation sub-diffraction (SPEED) microscopy that combines high-speed SMF microscopy and a 2D-to-3D transformation algorithm in the studies of nuclear transport kinetics and route for mRNAs. Finally, we summarize the new features of mRNA nuclear export found with SPEED microscopy as well as the reliability and accuracy of SPEED microscopy in mapping the 3D spatial locations of transport routes adopted by proteins and mRNAs through the NPCs.

中文翻译：

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通过 SPEED 显微镜研究 mRNA 分子的核输出

信使 RNA (mRNA) 分子通过核孔复合体 (NPC) 离开核是所有蛋白质翻译过程中必不可少的步骤。目前常规荧光和电子显微镜的局限性阻碍了对 mRNA 如何通过活细胞的 NPC 输出的阐明。近年来，已经开发出各种单分子荧光 (SMF) 显微镜技术来提高活细胞成像的时间和空间分辨率，从而更全面地了解通过天然 NPC 输出 mRNA 的动态。在这篇综述中，我们首先评估了单分子活细胞显微镜在 mRNA 核输出研究中的必要性。然后，我们重点介绍了结合高速 SMF 显微镜和 2D 到 3D 转换算法的单点边缘激发亚衍射 (SPEED) 显微镜在核转运动力学和 mRNA 路线研究中的应用。最后，我们总结了 SPEED 显微镜发现的 mRNA 核输出的新特征，以及 SPEED 显微镜在绘制蛋白质和 mRNA 通过 NPC 采用的运输路线的 3D 空间位置的可靠性和准确性。