Bimolecular fluorescence complementation (BiFC): A 5-year update and future perspectives

Over the past decade, bimolecular fluorescence complementation (BiFC) has emerged as a key technique to visualize protein-protein interactions in a variety of model organisms. The BiFC assay is based on reconstitution of an intact fluorescent protein when two complementary non-fluorescent fragments are brought together by a pair of interacting proteins. While the originally reported BiFC method has enabled the study of many protein-protein interactions, increasing demands to visualize protein-protein interactions under various physiological conditions have not only prompted a series of recent BiFC technology improvements, but also stimulated interest in developing completely new approaches. Here we review current BiFC technology, focusing on the development and improvement of BiFC systems, the understanding of split sites in fluorescent proteins, and enhancements in the signal-to-noise ratio. In addition, we provide perspectives on possible future improvements of the technique.