Mirror-enhanced 4Pi-SMLM with one objective enables isotropic nanoscale imaging.

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• Dual-objective 4Pi-SMLM provides isotropic nanoscale resolution but is complex.
• Instrumental complexity and alignment hinder widespread adoption of 4Pi-SMLM.

Purpose of the Study:

• Introduce mirror-enhanced 4Pi-SMLM (me4Pi-SMLM) as a simplified, single-objective alternative.
• Enhance axial resolution and reduce system complexity compared to existing methods.

Main Methods:

• Developed a single-objective 4Pi-SMLM configuration using mirror-based retroreflection.
• Generated phase-tunable interference fringes for improved resolution.

Main Results:

• Achieved approximately fivefold improvement in axial resolution for astigmatism-based methods.
• Reached near-isotropic localization precision of 2-3 nm in biological samples.
• Demonstrated sub-15 nm isotropic resolution in brain slices, enabling advanced imaging techniques.

Conclusions:

• me4Pi-SMLM offers performance comparable to conventional 4Pi-SMLM with significantly reduced complexity.
• The system facilitates high-fidelity multicolor imaging, whole-cell reconstruction, and live-cell imaging.
• Seamless integration into existing 3D-SMLM systems enhances performance cost-effectively.