Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg, Germany.
Optical Microscopy Facility, Max Planck Institute for Medical Research, Heidelberg, Germany.
Nat Chem. 2020 Feb;12(2):165-172. doi: 10.1038/s41557-019-0371-1. Epub 2019 Dec 2.
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.
活细胞荧光纳米显微镜是研究细胞生物学的一种强大工具，但由于缺乏合适的荧光探针，其应用受到限制。基于常规荧光团的荧光探针通常存在细胞通透性差和非特异性背景信号的问题。在这里，我们报告了一种将常规荧光团转化为具有优异细胞通透性和低非特异性背景信号的荧光探针的通用策略。将在罗丹明和相关荧光团中发现的羧基转化为缺电子酰胺不会影响荧光团的光谱性质，但使我们能够合理地调节两种不同形式之间的动态平衡：荧光内盐和非荧光、可穿透细胞的螺内酯。此外，当探针与细胞靶标结合时，平衡通常向荧光形式移动。荧光强度的增加可达 1000 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各种颜色的荧光探针，用于无冲洗、多色、活细胞纳米显微镜。
