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.
Quantitative analyses in classical fluorescence microscopy and Single Molecule Localization Microscopy (SMLM) require uniform illumination over the field of view; ideally coupled with optical sectioning techniques such as Total Internal Reflection Fluorescence (TIRF) to remove out of focus background. In SMLM, high irradiances (several kW/cm²) are crucial to drive the densely labeled sample into the single molecule regime, and conventional gaussianshaped lasers will typically restrain the usable field of view to around 40 µm x 40 µm. Here we present Adaptable Scanning for Tunable Excitation Regions (ASTER), a novel and versatile illumination technique that generates uniform illumination over adaptable fields of view and is compatible with illumination schemes from epifluorescence to speckle-free TIRF. For SMLM, ASTER delivers homogeneous blinking kinetics at reasonable laser power, providing constant precision and higher throughput over fields of view 25 times larger than typical (up to 200 µm x 200 µm). This allows improved clustering analysis and uniform size measurements on sub-100 nm objects, as we demonstrate by imaging nanorulers, microtubules and clathrin-coated pits in COS cells, as well as periodic β2spectrin along the axons of neurons. ASTER's sharp, quantitative TIRF and SMLM images up to 200 µm x 200 µm in size pave the way for high-throughput quantification of cellular structures and processes.
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