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We developed a new localization algorithm for interferometric single-molecule localization microscopy (iPALM) that achieves the theoretical 3D resolution limit. This method improves 4Pi-SMS imaging by fitting experimental point spread functions to achieve nanometer resolution.

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Area of Science:

Super-resolution microscopy
Biophysics
Optical imaging

Background:

Interferometric single-molecule localization microscopy (iPALM, 4Pi-SMS) offers nanometer isotropic 3D resolution.
Current iPALM analysis struggles to reach theoretical limits due to suboptimal localization algorithms.

Purpose of the Study:

To develop an improved localization algorithm for iPALM/4Pi-SMS.
To achieve the theoretical resolution limit (CRLB) in 3D super-resolution imaging.

Main Methods:

Developed a method to fit experimentally derived point spread function (PSF) models to the interference 4Pi-PSF.
Decoupled the interference phase term from the 3D PSF position.
Used a spline-interpolated experimental PSF model and global fitting of phase images.

Main Results:

The new method reliably infers the interference period without requiring astigmatism.
Simulated data demonstrated achievement of the Cramér-Rao lower bound (CRLB) for 3D resolution.
Reduced microscope complexity by removing the need for astigmatism.

Conclusions:

The developed algorithm overcomes limitations in current iPALM data analysis.
This approach enables 4Pi-SMS to reach its theoretical nanometer-scale 3D resolution limit.
The method simplifies microscope requirements while enhancing imaging performance.