Bimolecular fluorescence complementation (BiFC) is an effective tool for visualizing protein–protein interactions (PPIs). However, a BiFC system with long wavelength and high fluorescence intensity is yet to be developed for in vivo imaging. In this study, we constructed a tandem near-infrared BiFC (tBiFC) system by splitting a near-infrared phytochrome, IFP2.0. This system allows the identification and visualization of PPIs in live cells and living mice. The photophysical properties of the complementary fluorescence of the tBiFC system were similar to those of its parent protein IFP2.0, but the intensity was twice that of a single-copy IFP2.0-based BiFC system. Compared with previously reported near infrared BiFC systems—iRFP-BiFC and IFP1.4-BiFC—the signal intensity of the tBiFC system increased by ~1.48- and ~400-fold for weak PPIs in living cells, and ~1.51- and ~8-fold for strong PPIs, respectively. When applied to imaging PPIs in live mice, the complementary fluorescence intensity of the tBiFC system was also significantly higher than that of the other near-infrared BiFC systems. The use of this bright phytochrome in a tandem arrangement constitutes a powerful tool for imaging PPIs in the near infrared region.

双分子荧光互补（BiFC）是可视化蛋白质-蛋白质相互作用（PPI）的有效工具。但是，附设系统的长波长和高荧光强度却为待开发在体内成像。在这项研究中，我们通过拆分近红外植物色素IFP2.0构建了串联近红外BiFC（tBiFC）系统。该系统可以识别和可视化活细胞和活小鼠中的PPI。tBiFC系统的互补荧光的光物理性质与其亲本IFP2.0相似，但强度是基于单拷贝IFP2.0的BiFC系统的强度的两倍。与以前报道的近红外BiFC系统（iRFP-BiFC和​​IFP1.4-BiFC）相比，活细胞中较弱的PPI的tBiFC系统的信号强度分别提高了约1.48和400倍，以及约1.51和8 -分别表示强PPI。当应用于活小鼠的PPI成像时，tBiFC系统的互补荧光强度也显着高于其他近红外BiFC系统。串联使用这种明亮的植物色素构成了在近红外区域成像PPI的强大工具。