Fast scanned widefield scheme provides tunable and uniform illumination for optimized SMLM on large fields of view

Abstract

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/cm{superscript 2}) are crucial to drive the densely labeled sample into the single molecule regime, and conventional gaussian-shaped lasers will typically restrain the usable field of view to around 40 {micro}m x 40 {micro}m. 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. 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 {beta}2-spectrin along the axons of neurons. ASTERs sharp, quantitative TIRF and SMLM images up to 200 {micro}m x 200 {micro}m in size pave the way for high-throughput quantification of cellular structures and processes.