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

Adrien Mau, Karoline Friedl, Christophe Leterrier, Nicolas Bourg and Sandrine Lévêque-Fort ()
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Adrien Mau: Institut des Sciences Moléculaires d’Orsay, Université Paris-Saclay, CNRS
Karoline Friedl: Abbelight
Christophe Leterrier: Aix-Marseille Université, CNRS, INP UMR7051, NeuroCyto
Nicolas Bourg: Abbelight
Sandrine Lévêque-Fort: Institut des Sciences Moléculaires d’Orsay, Université Paris-Saclay, CNRS

Nature Communications, 2021, vol. 12, issue 1, 1-11

Abstract: Abstract 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.

Date: 2021
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DOI: 10.1038/s41467-021-23405-4

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