We present protein-PAINT – the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes.

中文翻译：

---

具有高度光稳定的可再生信号的活细胞荧光显微镜的蛋白质标记

我们介绍了蛋白质PAINT –活细胞蛋白质标记PAINT（纳米级地形中成像的点累加）一般原理的实现。我们的方法利用细胞可渗透的荧光染料与遗传编码的蛋白质标签的特异性结合。我们设计了三种细菌脂质运载蛋白Blc突变体，它们与荧光染料具有不同的亲和力，并在结合后荧光强度显着增加。这允许在几秒到几分钟的时间范围内快速标记和冲洗细胞内靶标。我们证明了与光谱相似的荧光蛋白相比，荧光信号的光稳定性更高一个数量级。