What advantages do fluorescent proteins have over organic dyes as FRET probes?

Fluorescent proteins (FPs), offer distinct advantages in live cell FRET imaging compared to dyes and quantum dots (QDs). Firstly, FPs are genetically encoded, simplifying the design of FRET sensors by directly fusing FPs to sensing domains through genetic engineering. Unlike dyes and QDs, which require antibodies for labeling sensing domains, FPs allow for the creation of a wider range of sensors. Additionally, FRET sensors based on FPs can be easily introduced into both in vitro and in vivo cellular environments through transfection or virus infection methods. Conversely, introducing dye-based FRET biosensors into cells is more difficult. FPs also demonstrate superior stability in living cells compared to dyes and QDs, remaining stable for extended periods due to the intrinsic stability of FPs. For example, the widely used EGFP has a half-life exceeding 24 hours in cells. Additionally, stable cell lines expressing FRET biosensors can be readily established under antibiotic pressure, which not only enhances cell viability during FRET imaging but also facilitates high-throughput drug screening assays. Lastly, FPs provide high cellular specificity by utilizing tissue-specific promoters and can achieve high subcellular specificity through the incorporation of subcellular targeting sequences. This enables FRET probes to selectively detect activity in desired cell types or subcellular regions.