Nile Red Fluorescence:\nWhere’s the Twist?

Nile Red is a fluorescent dye used extensively in bioimaging\ndue\nto its strong solvatochromism. The photophysics underpinning Nile\nRed’s fluorescence has been disputed for decades, with some\nstudies claiming that the dye fluoresces from two excited states and/or\nthat the main emissive state is twisted and intramolecular charge-transfer\n(ICT) in character as opposed to planar ICT (PICT). To resolve these\nlong-standing questions, a combined experimental and theoretical study\nwas used to unravel the mechanism of Nile Red’s fluorescence.\nTime-resolved fluorescence measurements indicated that Nile Red emission\noccurs from a single excited state. Theoretical calculations revealed\nno evidence for a low-lying TICT state, with the S<sub>1</sub> minimum\ncorresponding to a PICT state. Ultrafast pump–probe spectroscopic\ndata contained no signatures associated with an additional excited\nstate involved in the fluorescence decay of Nile Red. Collectively,\nthese data in polar and nonpolar solvents refute dual fluorescence\nin Nile Red and definitively demonstrate that emission occurs from\na PICT state.