Fluorescent Flavoprotein Heterodimers: Combining Photostability with Singlet Oxygen Generation

Flavoproteins that combine stable fluorescence emission with the ability to photosensitize reactive oxygen species (ROS) have great potential for advanced imaging techniques, such as correlative light and electron microscopy (CLEM). However, ROS photosensitization results in substantial fluorescence photobleaching in flavoproteins, limiting their application as genetically encoded microscopy tags. To address this issue, we have engineered two novel flavoprotein heterodimers from monomers with complementary properties, and show that a good compromise between photostability and singlet oxygen photosensitization can be achieved. The potential of the heterodimers as tags for CLEM is also investigated and discussed. Since the size of the tandem heterodimers is still small, similar to that of a single green fluorescent protein, these novel tags are a useful addition to the flavoprotein toolbox for a growing number of advanced imaging techniques.