Fast widefield scan provides tunable and uniform illumination optimizing super-resolution microscopy on large fields

• Published in: Nature communications 2021-05, Vol.12 (1), p.3077-3077, Article 3077
• Main Authors: Mau, Adrien, Friedl, Karoline, Leterrier, Christophe, Bourg, Nicolas, Lévêque-Fort, Sandrine
• Format: Article
• Language: English
• Subjects: 639/624/1107/328/2236; 639/624/1107/328/2238; 639/624/1107/328/2239; Algorithms; Animals; Biological activity; Blinking; Chlorocebus aethiops; Clathrin; Cluster analysis; Clustering; Coated pits; COS Cells; Field of view; Fluorescence; Fluorescence microscopy; Humanities and Social Sciences; Illumination; Imaging techniques; Lasers; Life Sciences; Light; Lighting; Localization; Microscopes; Microscopy; Microscopy, Fluorescence - instrumentation; Microscopy, Fluorescence - methods; Microtubules; multidisciplinary; Neurobiology; Neurons and Cognition; Optical Imaging - instrumentation; Optical Imaging - methods; Optical sectioning; Reproducibility of Results; Science; Science (multidisciplinary); Sectioning; Single Molecule Imaging - instrumentation; Single Molecule Imaging - methods; Spectrin
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-23405-4

Non-uniform illumination limits quantitative analyses of fluorescence imaging techniques. In particular, single molecule localization microscopy (SMLM) relies on high irradiances, but conventional Gaussian-shaped laser illumination restricts the usable field of view to around 40 µm × 40 µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile illumination technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection fluorescence (TIRF). For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power over fields-of-view up to 200 µm × 200 µm. We demonstrate that ASTER improves clustering analysis and nanoscopic size measurements by imaging nanorulers, microtubules and clathrin-coated pits in COS-7 cells, and β2-spectrin in neurons. ASTER’s sharp and quantitative illumination paves the way for high-throughput quantification of biological structures and processes in classical and super-resolution fluorescence microscopies. Uniform illumination is a prerequisite for quantitative analyses in both classical fluorescence microscopy and single molecule localisation microscopy. Here, the authors introduce ASTER, an illumination technique that generates uniform illumination over large and adaptable fields of view, compatible with epifluorescence, HiLo and TIRF illumination schemes.