Viscosity sensitive fluorophores termed 'molecular rotors' represent a convenient and quantitative tool for measuring intracellular viscosity via Fluorescence Lifetime Imaging Microscopy (FLIM). We compare the FLIM performance of two BODIPY-based molecular rotors bound to HaloTag protein expressed in different subcellular locations. While both rotors are able to penetrate live cells and specifically label the desired intracellular location, we found that the rotor with a longer HaloTag protein recognition motif was significantly affected by photo-induced damage when bound to the HaloTag protein, while the other dye showed no changes upon irradiation. Molecular dynamics modelling indicates that the irradiation-induced electron transfer between the BODIPY moiety and the HaloTag protein is a plausible explanation for these photostability issues. Our results demonstrate that binding to the targeted protein may significantly alter the photophysical behaviour of a fluorescent probe and therefore its thorough characterisation in the protein bound form is essential prior to any in vitro and in cellulo applications.

中文翻译：

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接头长度影响细胞微粘度蛋白质靶向传感器的光稳定性。

称为“分子转子”的粘度敏感荧光团代表了一种方便的定量工具，用于通过荧光寿命成像显微镜 (FLIM) 测量细胞内粘度。我们比较了两种基于 BODIPY 的分子转子的 FLIM 性能，这些转子与在不同亚细胞位置表达的 HaloTag 蛋白结合。虽然两个转子都能够穿透活细胞并专门标记所需的细胞内位置，但我们发现具有较长 HaloTag 蛋白识别基序的转子在与 HaloTag 蛋白结合时受到光诱导损伤的显着影响，而另一种染料则没有辐照后的变化。分子动力学模型表明，辐照诱导的 BODIPY 部分和 HaloTag 蛋白之间的电子转移是对这些光稳定性问题的合理解释。我们的研究结果表明，与目标蛋白质的结合可能会显着改变荧光探针的光物理行为，因此在任何体外和纤维素应用之前，它在蛋白质结合形式中的彻底表征是必不可少的。