Triplet-state mediated super-resolution imaging of fluorescently-labeled ligands on gold nanorods : a single molecule, single particle approach

Super-resolution imaging was used to study the binding interactions between fluorescently-labeled double-stranded DNA (dsDNA) ligands and gold nanorods (AuNRs). The dsDNA is attached to AuNRs via a thiol linker and the fluorescent label served as a reporter molecule. A triplet-state mediated technique was used to modulate emission from the reporter molecules such that ideally only one was emitting at a time. The steady AuNR luminescence contribution was subtracted from the fluorophore emission, and the result was fit to a model function to extract the centroid position of the reporter molecule, which maps the apparent location of the dsDNA with respect to the AuNR surface. With this technique, we have observed instances where the majority of ligands bind preferentially at the ends of the AuNR, one end of the AuNR has more binding than the other, or the binding is spread evenly over the surface of the AuNR. We hypothesize that the differences in fluorophore localization between functionalized AuNRs was due to apparent binding heterogeneity, providing insight that is not attainable with bulk studies. By mapping the apparent locations of the dsDNA bound to the AuNRs, we were also able to reconstruct the shape and orientation of the underlying AuNR, but the size of the reconstructed image was almost always smaller than expected, when compared to dimensions obtained using electron or atomic force microscopy. To address this mismatch we tested several hypotheses. We used a better model (a dipolar emission model instead of two-dimensional Gaussian) to fit the AuNR luminescence contribution and the improved model led to more robust reconstructed images by allowing more centroid positions to pass the quality-of-fit criterion. We also altered the concentration of the fluorescently-labeled DNA available for binding and tested reporter molecules with different non-fluorescent lifetimes. These strategies aimed to lower the probability of reporter molecules emitting simultaneously, but these experiments did not fix the size mismatch. Nevertheless, under those different experimental conditions, we continually observed apparent binding heterogeneity of dsDNA across the surface of AuNRs based on the super-localization of the reporter molecules, indicating the power of this technique for observing nanoscale heterogeneity.