Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics

Nitric oxide ([Image: see text]) is a free radical with a wide range of biological effects, but practically impossible to visualize in single cells. Here we report the development of novel multicoloured fluorescent quenching-based [Image: see text] probes by fusing a bacteria-derived [Image: see text]-binding domain close to distinct fluorescent protein variants. These genetically encoded [Image: see text] probes, referred to as geNOps, provide a selective, specific and real-time read-out of cellular [Image: see text] dynamics and, hence, open a new era of [Image: see text] bioimaging. The combination of geNOps with a Ca(2+) sensor allowed us to visualize [Image: see text] and Ca(2+) signals simultaneously in single endothelial cells. Moreover, targeting of the [Image: see text] probes was used to detect [Image: see text] signals within mitochondria. The geNOps are useful new tools to further investigate and understand the complex patterns of [Image: see text] signalling on the single (sub)cellular level.