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

Marijn Siemons1, Bas M C Cloin1, Desiree M Salas1

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Adaptive optics with oil immersion objectives significantly enhance deep 3D single-molecule localization microscopy (SMLM) imaging in watery samples, overcoming optical aberrations for nanometric resolution.

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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.