Effects of Tuning Fluorophore Density, Identity, and Spacing on Reconstructed Images in Super-Resolution Imaging of Fluorophore-Labeled Gold Nanorods

A triplet-state mediated super-resolution imaging technique is used to optically investigate the locations of single fluorescently labeled double-stranded DNA (dsDNA) ligands bound to gold nanorods (AuNRs). Previous work on this system has shown significant apparent heterogeneity in dsDNA ligand binding across the AuNR surface, but the reconstructed images have yielded dimensions that are smaller than the underlying AuNR substrate. Here, we tune the fluorophore density and identity to determine if the properties of the fluorophore impact the reconstructed images. While lowering the fluorophore density on the AuNR surface reduces the probability of simultaneous emission events and fluorophore self-quenching, we do not observe an improvement in the reconstructed image size and ultimately suffer from under-sampling of the surface. As alternative strategies, the identity of the fluorescent label, and thus the photophysics of the system, was also changed and again, the size of the reconstructed images remained smaller than expected in nearly all cases. Lastly, we increased the dsDNA linker length to decrease any interactions between the fluorophore and the gold surface and found that while the increased fluorophore-nanoparticle spacing impacted the photophysics of the fluorophore, the reconstructed image sizes remained consistently smaller than expected. Thus, we conclude that the photophysics of the fluorescent tag is not the primary origin of the incorrect size of the super-resolution images. However, in all cases, we continue to observe significant heterogeneity in the reconstructed images, further supporting our hypothesis that ligand binding from solution is nonuniform across the AuNR surfaces.