Abstract

Fluorescence microscopy is the method of choice for studying intracellular dynamics. However, its success depends on the availability of specific and stable markers. A prominent example of markers that are rapidly gaining interest are nanobodies (Nbs, ~ 15 kDa), which can be functionalized with bright and photostable organic fluorophores. Due to their relatively small size and high specificity, Nbs offer great potential for high-quality long-term subcellular imaging, but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells. We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability. Being a laser-based technology, it is readily compatible with light microscopy and the typical cell recipients used for that. Spurred by these promising initial results, we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells. We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm, actin-bundling protein Fascin, and the histone H2A/H2B heterodimers. With an efficiency of more than 80% labeled cells and minimal toxicity (∼ 2%), photoporation proved to be an excellent intracellular delivery method for Nbs. Time-lapse microscopy revealed that cell division rate and migration remained unaffected, confirming excellent cell viability and functionality. We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells, laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.

中文翻译：

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通过激光诱导光穿孔传递标记纳米抗体的长期活细胞显微镜

摘要

荧光显微镜是研究细胞内动力学的首选方法。然而，它的成功取决于特定和稳定标记的可用性。快速引起人们兴趣的一个突出例子是纳米抗体 (Nbs, ~ 15 kDa)，它可以用明亮且光稳定的有机荧光团进行功能​​化。由于其相对较小的尺寸和较高的特异性，Nbs 为高质量的长期亚细胞成像提供了巨大的潜力，但它们无法自发地穿过活细胞的质膜。我们最近发现，激光诱导的光穿孔非常适合向活细胞提供外在标记，而不会影响其生存能力。作为一种基于激光的技术，它很容易与光学显微镜和用于此的典型细胞受体兼容。在这些有希望的初步结果的刺激下，我们在这里首次证明了活细胞中带有标记纳米抗体的特定亚细胞结构的成功长期成像。我们使用靶向细胞质中可访问的 GFP/YFP 蛋白构建体的 Nb、肌动蛋白捆绑蛋白 Fascin 和组蛋白 H2A/H2B 异源二聚体来说明这一点。光穿孔被证明是一种极好的 Nbs 细胞内递送方法，其标记细胞的效率超过 80%，毒性最小（~2%）。延时显微镜显示细胞分裂率和迁移不受影响，证实了出色的细胞活力和功能。我们得出结论，激光诱导的光穿孔标记的 Nbs 可以很容易地传递到活细胞中，