Live-cell fluorescence nanoscopy is a powerful tool to study cellular biology on a molecular scale, yet its use is held back by the paucity of suitable fluorescent probes. Fluorescent probes based on regular fluorophores usually suffer from a low cell permeability and an unspecific background signal. Here we report a general strategy to transform regular fluorophores into fluorogenic probes with an excellent cell permeability and a low unspecific background signal. Conversion of a carboxyl group found in rhodamines and related fluorophores into an electron-deficient amide does not affect the spectroscopic properties of the fluorophore, but allows us to rationally tune the dynamic equilibrium between two different forms: a fluorescent zwitterion and a non-fluorescent, cell-permeable spirolactam. Furthermore, the equilibrium generally shifts towards the fluorescent form when the probe binds to its cellular targets. The resulting increase in fluorescence can be up to 1,000-fold. Using this simple design principle, we created fluorogenic probes in various colours for different cellular targets for wash-free, multicolour, live-cell nanoscopy.

中文翻译：

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开发用于多色纳米显微镜的细胞可渗透和荧光探针的一般策略。

活细胞荧光纳米显微镜是研究分子生物学细胞生物学的有力工具，但由于缺乏合适的荧光探针，其使用受到了限制。基于常规荧光团的荧光探针通常具有较低的细胞渗透性和非特异性背景信号。在这里，我们报告一种将常规荧光团转化为具有优异细胞渗透性和低非特异性背景信号的荧光探针的一般策略。将若丹明和相关荧光团中存在的羧基转化为电子缺陷型酰胺不会影响荧光团的光谱性质，但可以使我们合理地调整两种不同形式之间的动态平衡：荧光两性离子和非荧光两性，细胞可渗透的螺内酰胺。此外，当探针与其细胞靶标结合时，平衡通常朝着荧光形式转移。导致的荧光增加最多可达1,000倍。使用这种简单的设计原理，我们为不同的细胞靶标创建了多种颜色的荧光探针，用于免洗，多色，活细胞纳米检查。