Comparing strategies for deep astigmatism-based single-molecule localization microscopy

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• Single-molecule localization microscopy (SMLM) achieves nanometric resolution in fluorescence microscopy.
• Optical aberrations limit SMLM depth penetration in aqueous samples, especially with high numerical aperture (NA) oil immersion (OI) objectives.
• Adaptive optics (AO) and water immersion (WI) objectives are potential solutions for deep SMLM.

Purpose of the Study:

• To compare the effectiveness of WI and OI objectives, with and without AO, for SMLM at various depths.
• To evaluate astigmatism-based 3D localization encoding using a cylindrical lens versus a deformable mirror (DM).
• To determine the optimal configuration for deep 3D SMLM.

Main Methods:

• Comparative SMLM imaging in aqueous samples at different depths.
• Utilized oil immersion (OI) and water immersion (WI) objectives.
• Employed adaptive optics (AO) with a deformable mirror (DM) and a cylindrical lens for aberration correction and z-encoding.

Main Results:

• OI objectives combined with AO significantly improved localization precision compared to WI-based imaging.
• Deep (>10 µm) 3D localization was successfully achieved using OI with AO.
• Both cylindrical lens and DM enabled astigmatism-based z-encoding, with AO offering superior performance.

Conclusions:

• Adaptive optics is crucial for achieving high-precision, deep 3D SMLM in aqueous environments.
• Oil immersion objectives coupled with AO provide superior performance for deep SMLM over water immersion objectives.
• AO-based aberration correction and z-encoding are essential for advancing SMLM applications in biological imaging.