Two-photon excitation as a new approach to investigate the metal-enhanced fluorescence

Fluorescence-based detection has become indispensable in biosensing and bioimaging owing to its high sensitivity, rapid response, and non-invasive detection nature. Metal-enhanced fluorescence (MEF) refers to the phenomenon that fluorophores near metallic nanostructures exhibit enhanced emission along with a shortened fluorescence lifetime, and thus MEF has the potential to enhance the sensitivity of fluorescence-based detection and imaging photostability simultaneously. The excitation and emission (quantum yield) enhancements are difficult to measure directly from experiment, because both excitation and emission wavelengths overlap with the broad plasmon resonance spectrum of the metallic nanostructure. We introduced a new approach enabling direct experimental determination of excitation and emission contributions to the fluorescence enhancement and quantifying the fluorescence quantum yield enhancement via two-photon excitation, in which circumventing spectral overlap with the plasmon resonance. This method enables the attribution of enhancement origins, providing a new perspective to the underlying mechanism of MEF.