Aberration-accounting calibration for 3D single-molecule localization microscopy

We propose a straightforward sample-based technique to calibrate the axial detection in 3D single-molecule localization microscopy. Using microspheres coated with fluorescent molecules, the calibration curves of point spread function-shaping or intensity-based measurements can be obtained over the imaging depth range. This experimental method takes into account the effect of the spherical aberration without requiring computational correction. We demonstrate its efficiency for astigmatic imaging in a 1.2 μm range above the coverslip.