Multicolored nanometre-resolution mapping of single protein-ligand binding complexes using far-field photostable optical nanoscopy (PHOTON).

Mapping of individual ligand molecules and their binding sites in single protein-ligand complexes at nanometer resolution in real-time would enable probing their structures and functions in vitro and in vivo. In this study, we have developed far-field photostable optical nanoscopy (PHOTON) for mapping single ligand molecules (biotin) and their binding sites in individual protein-ligand complexes (streptavidin-biotin) with 1.2 nm spatial resolution and 100 ms temporal resolution. PHOTON includes one standard far-field optical microscope with a halogen-lamp illuminator; single-molecule-nanoparticle-optical-biosensors (SMNOBS) with exceptionally high quantum-yield (QY) of Rayleigh scattering and photostability (non-photobleaching, non-photoblinking) as imaging probes; and Multispectral Imaging System (MSIS) for spectral isolation of individual SMNOBS with 1 nm wavelength resolution. Intrinsic size- and shape- dependent localized-surface-plasmon-resonance (LSPR) spectra of single SMNOBS provide multiple-spectral (color) nanoprobes for sub-diffraction imaging, offering feasibility of probing of binding structures and functions of single protein-ligand complexes at nm (potentially achieving Ångstrom) resolution in real-time.