Hauke Sebastian, von Appen Alexander, Quidwai Tooba, Ries Jonas, Wombacher Richard
Institute of Pharmacy and Molecular Biotechnology , Ruprecht-Karls-University Heidelberg , Im Neuenheimer Feld 364 , 69120 Heidelberg , Germany . Email:
European Molecular Biology Laboratory , Meyerhofstraße 1 , 69117 Heidelberg , Germany.
Chem Sci. 2017 Jan 1;8(1):559-566. doi: 10.1039/c6sc02088g. Epub 2016 Sep 5.
We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging inside live mammalian cells. These custom-designed, photo-caged Q-rhodamines and fluoresceins are cell-permeable, bright and localize specifically to intracellular targets. We utilized established orthogonal protein labeling strategies to precisely attach the photoactivatable fluorophores to proteins with subsequent activation of fluorescence by irradiation with UV light. That way, diffusive cytosolic proteins, histone proteins as well as filigree mitochondrial networks and focal adhesion proteins were visualized inside living cells. We applied the new photoactivatable probes in inverse fluorescence recovery after photo-bleaching (iFRAP) experiments, gaining real-time access to protein dynamics from live biological settings with resolution in space and time. Finally, we used the caged Q-rhodamine for photo-activated localization microscopy (PALM) on both fixed and live mammalian cells, where the superior molecular brightness and photo-stability directly resulted in improved localization precisions for different protein targets.
我们展示了用于活的哺乳动物细胞内双色光激活和超分辨率成像的新型荧光团共轭物。这些定制设计的、光笼式Q-罗丹明和荧光素具有细胞渗透性,亮度高且能特异性定位于细胞内靶点。我们利用已确立的正交蛋白质标记策略,将光激活荧光团精确地连接到蛋白质上,随后通过紫外光照射激活荧光。通过这种方式,可在活细胞内观察到扩散的胞质蛋白、组蛋白以及精细的线粒体网络和粘着斑蛋白。我们将新型光激活探针应用于光漂白后的反向荧光恢复(iFRAP)实验,能够从活生物环境中实时获取蛋白质动力学信息,具备时空分辨率。最后,我们将笼式Q-罗丹明用于固定和活的哺乳动物细胞的光激活定位显微镜(PALM),其卓越的分子亮度和光稳定性直接提高了不同蛋白质靶点的定位精度。