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Three dimensional live-cell STED microscopy at increased depth using a water immersion objective.

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Water immersion objectives improve super-resolution microscopy for deep live-cell imaging. This approach corrects aberrations, enhancing axial resolution in stimulated emission depletion (STED) microscopy beyond oil objectives.

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Area of Science:

  • Biophysics
  • Optical Microscopy

Background:

  • Super-resolution microscopy, like stimulated emission depletion (STED), enables nanoscale imaging of living cells.
  • High numerical aperture (NA) oil objectives are common for STED but suffer from spherical aberrations in aqueous media at depth.

Purpose of the Study:

  • To overcome limitations of oil objectives in deep live-cell STED imaging.
  • To enhance axial resolution in z- and 3D-STED microscopy at greater penetration depths.

Main Methods:

  • Utilized a water immersion objective combined with a spatial light modulator for z-STED.
  • Developed a compact system allowing objective switching and real-time STED point spread function (PSF) aberration correction.
  • Theoretically and experimentally investigated the influence of NA on axial STED resolution.

Main Results:

  • Demonstrated superior performance of water immersion objectives compared to oil objectives for deep live-cell STED imaging (5-180 μm).
  • Successfully corrected for spherical aberrations caused by refractive index mismatch.
  • Significantly improved axial resolution in z-STED measurements.

Conclusions:

  • Water immersion objectives offer a viable solution for high-resolution deep live-cell STED microscopy.
  • The developed system provides enhanced axial resolution and aberration correction capabilities.
  • This advancement is crucial for studying cellular dynamics at nanometer scales within intact biological samples.