Correction of depth-dependent aberrations in 3D single-molecule localization and super-resolution microscopy

Area of Science:

• Biophysics
• Optical Microscopy
• Super-resolution Imaging

Background:

• 3D single-molecule localization microscopy (SMLM) enables super-resolution imaging in three dimensions.
• Spherical aberrations, caused by refractive index mismatch, significantly degrade axial (z) localization accuracy, limiting imaging depth.

Purpose of the Study:

• To develop and validate a method for accurate, depth-dependent z-calibration in SMLM.
• To overcome the limitations imposed by spherical aberrations on imaging depth.

Main Methods:

• Measuring the point spread function (PSF) at the coverslip surface.
• Calculating the microscope pupil function via phase retrieval.
• Computing depth-dependent PSFs with simulated spherical aberrations.

Main Results:

• The developed method provides accurate z-calibrations across a wide range of imaging depths.
• Experimental validation demonstrated maintained z-localization accuracy even at depths up to 2.5 μm within a mammalian cell nucleus.
• High-quality super-resolution images were acquired at greater depths than previously possible.

Conclusions:

• This depth-calibration technique significantly enhances the reliability of 3D SMLM in biological samples.
• It overcomes a major hurdle for deep-tissue or thick-sample super-resolution imaging.
• Enables accurate 3D super-resolution imaging deeper within cells and tissues.