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

Area of Science:

• Optical microscopy
• Biophysics
• Cell biology

Background:

• Non-uniform illumination in fluorescence microscopy limits quantitative analysis.
• Conventional Gaussian illumination restricts the field of view in single molecule localization microscopy (SMLM) to approximately 40 µm × 40 µm.
• High laser irradiances are crucial for SMLM, but non-uniformity affects quantitative measurements.

Purpose of the Study:

• To introduce Adaptable Scanning for Tunable Excitation Regions (ASTER), a novel illumination technique for fluorescence microscopy.
• To overcome the limitations of non-uniform illumination in quantitative imaging.
• To enhance the capabilities of techniques like SMLM and total internal reflection fluorescence (TIRF).

Main Methods:

• ASTER employs a scanning method to generate uniform and adaptable illumination patterns.
• The technique is compatible with optical sectioning methods like TIRF.
• ASTER was tested for its performance in single molecule localization microscopy (SMLM).

Main Results:

• ASTER achieves homogeneous blinking kinetics in SMLM at moderate laser power.
• It enables uniform illumination over significantly larger fields of view, up to 200 µm × 200 µm.
• Improved clustering analysis and nanoscopic size measurements were demonstrated using ASTER.

Conclusions:

• ASTER provides sharp, quantitative, and uniform illumination for fluorescence microscopy.
• The technique significantly expands the usable field of view for SMLM and other super-resolution methods.
• ASTER facilitates high-throughput quantification of biological structures and processes.