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 that 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.