Single‐Molecule Imaging and Spectroscopy Enables Quantification of Location‐Dependent Light–Matter Interactions on Nanoantennas

Individual dye molecules coupled to a plasmonic nanoantenna have been established as a versatile foundation for single-molecule studies. Crucial parameters such as fluorescence intensity enhancement, spectral properties, and emission patterns sensitively depend on the exact position of the dye molecule relative to the antenna and its hot spots. Knowledge about the binding location is therefore of paramount interest, however, it is highly challenging to obtain. We present a comprehensive approach based on correlative microspectroscopy of the optical properties of single fluorophores that transiently bind to gold nanocones using the DNA-PAINT method. These 3D nanoantennas offer independently tunable in- and out-of-plane plasmon resonances with strong electric field enhancements at the tip apex and the nanocone base. We exploit site-specific deformations of the point spread function in a high-throughput approach as a means to correlate the position of the fluorophores on the nanoantenna surface to previously inaccessible parameters, investigating location-specific binding probability, mode-dependent spectral reshaping, and location-resolved fluorescence enhancement factors. Our approach provides unprecedented multimodal quantification by correlating spatial and spectral information to open new avenues in fundamental studies of light-matter interactions and applications like biosensing.