Aberration-free calibration for 3D single molecule localization microscopy

TL;DR

This paper introduces a simple, sample-based calibration method for 3D single molecule localization microscopy that accounts for spherical aberration without computational correction, improving axial detection accuracy.

Contribution

A novel experimental calibration technique for 3D SMLM that directly measures calibration curves across depth ranges, eliminating the need for computational corrections.

Findings

Calibration curves can be obtained for depths from hundreds of nanometers to tens of microns.

The method effectively accounts for spherical aberration.

Calibration improves the accuracy of 3D localization measurements.

Abstract

We propose a straightforward sample-based technique to calibrate the axial detection in 3D single molecule localization microscopy (SMLM). Using microspheres coated with fluorescent molecules, the calibration curves of PSF-shaping- or intensity-based measurements can be obtained for any required depth range from a few hundreds of nanometers to several tens of microns. This experimental method takes into account the effect of the spherical aberration without requiring computational correction.