Institute of Applied Physics, Karlsruhe Institute of Technology, 76049 Karlsruhe, Germany.
Institute of Nanotechnology, Karlsruhe Institute of Technology, 76021 Karlsruhe, Germany.
Methods Appl Fluoresc. 2022 Jul 28;10(4). doi: 10.1088/2050-6120/ac7d3f.
Optical fluorescence microscopy plays a pivotal role in the exploration of biological structure and dynamics, especially on live specimens. Progress in the field relies, on the one hand, on technical advances in imaging and data processing and, on the other hand, on progress in fluorescent marker technologies. Among these, genetically encodable fluorescent proteins (FPs) are invaluable tools, as they allow facile labeling of live cells, tissues or organisms, as these produce the FP markers all by themselves after introduction of a suitable gene. Here we cover FP markers from the GFP family of proteins as well as tetrapyrrole-binding proteins, which further complement the FP toolbox in important ways. A broad range of FP variants have been endowed, by using protein engineering, with photophysical properties that are essential for specific fluorescence microscopy techniques, notably those offering nanoscale image resolution. We briefly introduce various advanced imaging methods and show how they utilize the distinct properties of the FP markers in exciting imaging applications, with the aim to guide researchers toward the design of powerful imaging experiments that are optimally suited to address their biological questions.
光学荧光显微镜在探索生物结构和动态方面起着关键作用，特别是在活体标本上。该领域的进展一方面依赖于成像和数据处理方面的技术进步，另一方面依赖于荧光标记技术的进步。在这些技术中，基因编码的荧光蛋白（FPs）是非常有价值的工具，因为它们可以轻松标记活细胞、组织或生物体，在引入合适的基因后，这些细胞、组织或生物体自身就会产生 FP 标记物。在这里，我们将介绍 GFP 蛋白家族和四吡咯结合蛋白的 FP 标记物，它们以重要的方式进一步补充了 FP 工具包。通过使用蛋白质工程，已经赋予了广泛的 FP 变体具有光物理特性，这些特性对于特定的荧光显微镜技术至关重要，特别是那些提供纳米级图像分辨率的技术。我们简要介绍了各种先进的成像方法，并展示了它们如何利用 FP 标记物的独特特性在令人兴奋的成像应用中发挥作用，旨在引导研究人员设计出强大的成像实验，这些实验最适合解决他们的生物学问题。
