High-speed volumetric single-molecule imaging using dual-wavelength light sheets and PSF-engineered enhanced biplane...

Area of Science:

• Biophysics
• Optical Microscopy
• Super-resolution Imaging

Background:

• Single-molecule localization microscopy (SMLM) offers nanoscale resolution but faces challenges in 3D imaging speed, density, and efficiency.
• Limitations include background fluorescence, photon inefficiency, and large point-spread function (PSF) footprints.

Purpose of the Study:

• To present SoLiD-3D, a novel single-objective light-sheet microscopy platform engineered for high-speed, high-density 3D SMLM.
• To overcome key limitations of conventional SMLM techniques.

Main Methods:

• Integration of dual-wavelength light-sheet illumination with dual-color, multi-configuration biplane imaging.
• Utilizing PSF engineering for enhanced biplane detection and parallel acquisition.
• Employing dynamically displaced light sheets for volumetric coverage and the Hummus PSF for compact, high-precision 3D localization.

Main Results:

• Achieved high-speed single- and dual-target dual-color imaging, doubling localization density without compromising photon efficiency.
• Demonstrated continuous volumetric imaging with improved axial localization over extended depth ranges.
• Showcased the benefits of the Hummus PSF for high-precision 3D localization with a reduced spatial footprint.

Conclusions:

• SoLiD-3D effectively mitigates the trade-off between axial range and localization precision in 3D SMLM.
• The platform offers significantly improved speed and performance compared to conventional 3D SMLM approaches for whole-cell imaging.