Nile Red Fluorescence: Where’s the Twist?

Nile Red is a fluorescent dye used extensively in bioimaging due to its strong solvatochromism. The photophysics underpinning Nile Red’s fluorescence has been disputed for decades, with some studies claiming that the dye fluoresces from two excited states, and/or the main emissive state is twisted and intramolecular charge-transfer (ICT) in character, as opposed to planar ICT (PICT). To resolve these long-standing questions, a combined experimental and theoretical study was used to unravel the mechanism of Nile Red’s fluorescence. Time-resolved fluorescence measurements indicated Nile Red emission occurs from a single excited state. Theoretical calculations revealed no evidence for a low-lying TICT state, with the S1 minimum corresponding to a PICT state. Ultrafast pump-probe spectroscopic data contained no signatures associated with an additional excited state involved in the fluorescent decay of Nile Red. Collectively, these data in polar and non-polar solvents refute dual fluorescence in Nile Red and definitively demonstrate that emission occurs from a PICT state.