An alternative to MINFLUX that enables nanometer resolution in a confocal microscope

• Published in: Light, science & applications science & applications, 2022-06, Vol.11 (1), p.199-9, Article 199
• Main Authors: Masullo, Luciano A., Szalai, Alan M., Lopez, Lucía F., Pilo-Pais, Mauricio, Acuna, Guillermo P., Stefani, Fernando D.
• Format: Article
• Language: English
• Subjects: 639/624/1107/328/2238; 639/624/1107/510; Fluorophores; Lasers; Localization; Microscopes; Microwaves; Optical and Electronic Materials; Optical Devices; Optics; Photonics; Physics; Physics and Astronomy; RF and Optical Engineering; Scanning; Spatial discrimination
• ISSN: 2047-7538; 2095-5545; 2047-7538
• DOI: 10.1038/s41377-022-00896-4

Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale and beyond ensemble averaging. Examples include single-molecule tracking and super-resolution imaging by single-molecule localization microscopy. Among the numerous localization methods available, MINFLUX outstands for achieving a ~10-fold improvement in resolution over wide-field camera-based approaches, reaching the molecular scale at moderate photon counts. Widespread application of MINFLUX and related methods has been hindered by the technical complexity of the setups. Here, we present RASTMIN, a single-molecule localization method based on raster scanning a light pattern comprising a minimum of intensity. RASTMIN delivers ~1–2 nm localization precision with usual fluorophores and is easily implementable on a standard confocal microscope with few modifications. We demonstrate the performance of RASTMIN in localization of single molecules and super-resolution imaging of DNA origami structures. Localization of single fluorescent emitters is key for physicochemical and biophysical measurements at the nanoscale. Recently, the method called MINFLUX has achieved a ~10-fold improvement in spatial resolution over previously developed techniques, reaching molecular resolution with moderate photon counts. However, the technical complexity of this technique has hindered its widespread application. The new technique called RASTMIN provides equivalent resolution to MINFLUX while it can be implemented in standard scanning (confocal) microscopes.