Comparing the Accuracy of Reconstructed Image Size in Super-Resolution Imaging of Fluorophore-Labeled Gold Nanorods Using Different Fit Models

We use a triplet-state-mediated super-resolution fluorescence imaging technique to localize the position of individual fluorescently labeled double-stranded DNA (dsDNA) bound to the surface of gold nanorods. Within each diffraction-limited spot, we must account for two different emission sources: the stochastic fluorescence from the fluorescent labels and the steady background luminescence of the gold nanorod. To isolate the contribution from the fluorescent label, we subtract the average gold nanorod luminescence contribution, modeled with either a two-dimensional Gaussian or a dipolar emission model. The fluorescence from the labeled dsDNA is then fit with a two-dimensional Gaussian to reconstruct the positions of each individual emitter on the nanorod surface. The resulting reconstructed images, using either luminescence model, agree with the shape and orientation of the underlying nanorod, and show similar apparent dsDNA binding heterogeneity across the surface of the nanorod based on the localization of the fluorescent labels. Using the dipolar emission model for the luminescence allows for the retention of more emission events from the fluorescent label, after applying a fitting threshold, and yields a more robust reconstructed image containing more centroid points that show the apparent locations of the dsDNA. Unfortunately, the sizes of the reconstructed nanorod images were smaller than expected, despite the use of the more accurate model for the gold luminescence, suggesting that the photophysics of this coupled dye–nanorod system are more complicated than when using the isolated fluorophores.