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.

Single Molecule Localization Microscopy (SMLM) is a straightforward approach to reach sub-50 nm resolution using techniques such as Stochastic Optical Reconstruction Microscopy (STORM) or DNA-Point Accumulation for Imaging in Nanoscale Topography (PAINT), and to resolve the arrangement of cellular components in their native environment. However, SMLM acquisitions are slow, particularly for multicolor experiments where channels are usually acquired in sequence. In this work, we evaluate two approaches to speed-up multicolor SMLM using a module splitting the fluorescence emission toward two cameras: simultaneous 2-color PAINT (S2C-PAINT) that images spectrally-separated red and far-red imager strands on each camera, and spectral demixing STORM (SD-STORM) that uses spectrally-close far-red fluorophores imaged on both cameras before assigning each localization to a channel by demixing. For each approach, we carefully evaluate the crosstalk between channels using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets. We then devise experiments to assess how crosstalk can potentially affect the detection of biologically-relevant subdiffraction patterns. Finally, we show how these approaches can be combined with astigmatism to obtain three- dimensional data, and how SD-STORM can be extended three-color imaging, making spectral separation and demixing attractive options for robust and versatile multicolor SMLM investigations.

Graphical Abstract3D cell collagen-based model with tunable environment conditions (oxygen concentration, nanodrug exposure or irradiations).

No abstract

No abstract

No abstract

No abstract