Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane

Area of Science:

• Optical microscopy
• Super-resolution imaging
• Biophysics

Background:

• Three-dimensional (3D) localization microscopy is crucial for understanding biological processes.
• Existing methods like astigmatic and biplane imaging have limitations in axial localization precision and depth of field.
• The double-helix point spread function (DH-PSF) offers a potential improvement for 3D super-resolution imaging.

Purpose of the Study:

• To theoretically compare the localization precision of the DH-PSF with astigmatic and biplane imaging.
• To evaluate the impact of pixelation and background noise on localization accuracy.
• To determine the optimal imaging strategy for high-precision 3D single-molecule localization.

Main Methods:

• Fisher information analysis was employed to calculate theoretical localization precision.
• Simulations incorporated pixelation effects and varying signal-to-background ratios.
• Performance was assessed for DH-PSF, astigmatic, and biplane imaging modalities.

Main Results:

• DH-PSF achieved nearly constant localization precision across all three dimensions within a 2 micrometer depth of field.
• Astigmatic and biplane imaging demonstrated improved axial precision but over smaller axial ranges.
• DH-PSF exhibited superior average localization precision, especially at high signal-to-background ratios.

Conclusions:

• The DH-PSF is a highly effective method for achieving robust 3D single-molecule localization.
• It offers advantages in terms of depth of field and consistent precision over other common 3D localization techniques.
• DH-PSF microscopy represents a significant advancement for high-resolution 3D biological imaging.