Near-infrared co-illumination of fluorescent proteins reduces photobleaching and phototoxicity

Here we present a method to reduce the photobleaching of fluorescent proteins and the associated phototoxicity. It exploits a photophysical process known as reverse intersystem crossing, which we induce by near-infrared co-illumination during fluorophore excitation. This dual illumination method reduces photobleaching effects 1.5-9.2-fold, can be easily implemented on commercial microscopes and is effective in eukaryotic and prokaryotic cells with a wide range of fluorescent proteins. A dual illumination method reduces photobleaching for green and yellow fluorescent proteins.