Spectrally Resolved Localization Microscopy with Ultrahigh-Throughput

Abstract Single-molecule localization microscopy (SMLM) has become a strong technique in the toolbox of chemists, biologists, physicists, and engineers in recent years for its unique ability to resolve characteristic features quickly and accurately in complex environments at the nanoscopic level. Multicolor super-resolution imaging has seen the greatest advancement among SMLM techniques, drastically improving the differentiation ability of nanostructures beyond the diffraction limit and increasing the resolution with which previously unresolvable structures are studied. However, current multicolor SMLM methodologies present low spatial resolution and throughput and require complex optical systems. Here, we overcome these drawbacks by developing an ultrahigh-throughput SMLM methodology that allows for ultrahigh throughput multicolor imaging at the nanoscopic level using a color glass filter. Our methodology can readily distinguish fluorophores of close spectral emission and achieves sub-10 nm localization and sub-5nm spectral precisions.