Joint estimation of point spread function and molecule positions in SMLM informed from multiple planes.

Area of Science:

• Biophysics
• Optical Microscopy
• Nanotechnology

Background:

• Single Molecule Localization Microscopy (SMLM) enables nanoscale imaging of biological structures.
• Systematic errors from optical aberrations, especially in 3D SMLM, often degrade image quality.
• Improving localization precision has been the primary focus, neglecting aberration correction.

Purpose of the Study:

• To develop an SMLM imaging and data processing approach for simultaneous estimation of molecule positions and optical aberrations.
• To minimize systematic errors in SMLM reconstructions without requiring additional experimental calibration.
• To assess the method's reliability, particularly for challenging 'flat' samples with limited image diversity.

Main Methods:

• Jointly estimating molecule positions and optical aberrations within the SMLM data processing pipeline.
• Utilizing auxiliary SMLM data sets from slightly defocused planes to improve ill-posed inverse problem solutions.
• Validating the approach through numerical simulations and experimental imaging of calibration probes and nuclear pore complexes.

Main Results:

• The proposed method effectively corrects for optical aberrations in SMLM reconstructions.
• Systematic errors are minimized without the need for fluorescent bead z-stacks or other calibration steps.
• The approach demonstrates reliability even for samples with low structural diversity.

Conclusions:

• This method offers a simple and integrated solution for enhancing SMLM image quality by addressing optical aberrations.
• It seamlessly integrates into existing SMLM setups without requiring system modifications.
• The technique improves the accuracy of nanoscale biological imaging.