Rational development of Nile red derivatives with significantly improved specificity and photostability for advanced fluorescence imaging of lipid droplets

Since the first report of Nile Red as a fluorescent probe for lipid droplets (LDs) imaging was published in 1985, this fluorescent probe has been widely used for nearly 40 years, and so far, it is still one of the most commonly used probes for LDs imaging. Although Nile Red has achieved continuous success, it has gradually emerged two major limitations (poor LDs specificity and low photostability) which directly limit the study of LDs via advanced fluorescence imaging techniques. In this context, we have developed a new synthetic route to conveniently prepare a series of Nile Red derivatives (NR-1 to NR-15). With these 15 derivatives in hand, the relationships between molecular structures and their properties (LDs specificity, photostability) have been comprehensively investigated. Consequently, we have rationally designed a new Nile Red derivative, NR-11, which exhibits significantly improved LDs specificity and photostability. Utilizing this new LDs probe, we have successfully conducted various advanced fluorescence imaging, e.g. time-lapse three-dimensional (3D) confocal imaging of cells, time-lapse 3D dynamic tracking of a single LD, and two-photon 3D imaging of tissues. These advanced imaging results not only demonstrate the utility of this new fluorescent probe but also provide novel insights into the cell biology study of LDs.