Simultaneous orientation and 3D localization microscopy with a Vortex point spread function.

Area of Science:

• Optical microscopy
• Biophysics
• Nanotechnology

Background:

• Estimating 3D position and orientation of single molecules is crucial for understanding biological processes.
• Conventional methods often require complex setups like polarization splitting or large engineered Point Spread Functions (PSFs).

Purpose of the Study:

• To develop a simplified and efficient method for simultaneously determining 3D position, dipole orientation, and rotational constraint of single emitters.
• To integrate this method with existing localization microscopy techniques.

Main Methods:

• Utilized a compact, modified PSF (Vortex PSF) created using an affordable phase plate, altering the emission light path.
• Implemented a vectorial PSF fitting routine and calibrated for field-dependent aberrations.
• Applied the technique to single molecules, λ-DNA with DNA intercalators, and supercoiled DNA structures.

Main Results:

• Achieved simultaneous estimation of 3D position, dipole orientation, and rotational constraint from single 2D images.
• The Vortex PSF requires no polarization splitting and has a compact size, facilitating integration with localization microscopy.
• Demonstrated high accuracy, within 30% of the Cramér-Rao bound, over a 66 μm field of view.
• Successfully visualized reorienting single molecules, DNA structures, and periodicity in supercoiled DNA.

Conclusions:

• The Vortex PSF offers a robust and accessible approach for advanced single-molecule analysis in localization microscopy.
• This technique simplifies experimental setups while maintaining high precision in determining molecular properties.
• The method has broad applicability for studying molecular dynamics and structural organization in biological systems.