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

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.