High-throughput multi-color reliable SMLM nanoscopy

Beyond all super-resolution microscopy methods, single molecule localization microscopy (SMLM) offers best 3D spatial resolution, up to several nanometers. Despite its super-resolutive property, SMLM has still a low throughput. In this context, SMLM offers a mono-color limited field of view (FOV). SMLM suffers indeed from fluorophore photoswitching characteristics, which depends on the wavelength, and significant chromatic aberration at the nanoscale. Multicolor SMLM imaging is classically achieved by sequential acquisitions at different excitation wavelengths, but lengthy acquisition times, drifts between the different color recordings, and cross excitation by different lasers may hamper quantitative reconstruction. In this talk, we will present two strategies to increase the FOV up to 200 × 200 µm² while improving the number of colors up to 3: Adaptable Scanning for Tunable Excitation Regions (ASTER) and spectral-demixing respectively. The association of ASTER and spectral demixing leads to increase the throughput by a factor 50 compared to standard nanoscopy approach, in the same acquisition time. We here present a reliable characterization of those methods for homogeneity, localisation precision, crosstalk, labelling density influence. By using complementary samples, we could obtain a rigorous estimation of the performances in various settings: DNA origamis, gold standard samples for SMLM, up to real biological applications as hippocampal neurons or extracellular vesicles characterization.