Many biomacromolecules are known to cluster in microdomains with specific
subcellular localization. In the case of enzymes, this clustering greatly defines their biological
functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a
role in detoxification and pro-drug activation. Although nitroreductase activity has been detected
in mammalian cells, the subcellular localization of this activity remains incompletely
characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools
of nitroreductase activity within mitochondria. This probe is activated sequentially by
nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination
with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image
the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>
