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

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 restricts the usable field view to around 40 µm × µm. We present Adaptable Scanning for Tunable Excitation Regions (ASTER), a versatile technique that generates uniform and adaptable illumination. ASTER is also highly compatible with optical sectioning techniques such as total internal reflection (TIRF). For SMLM, delivers homogeneous blinking kinetics at reasonable power over fields-of-view up 200 demonstrate improves clustering analysis nanoscopic size measurements by nanorulers, microtubules clathrin-coated pits in COS-7 cells, ?2-spectrin neurons. ASTER’s sharp paves way high-throughput quantification biological structures processes classical super-resolution microscopies.