Dipole-spread-function engineering for simultaneously measuring the 3D orientations and 3D positions of fluorescent...

Area of Science:

• Biophysics
• Microscopy
• Molecular Imaging

Background:

• Biomolecular interactions depend on spatial and angular organization.
• Microscope dipole-spread functions (DSF) blur spatial and angular information.
• Precisely measuring 3D orientation and location is crucial for understanding molecular behavior.

Purpose of the Study:

• To develop a novel algorithm (pixOL) for simultaneous optimization of phase mask pixels.
• To engineer a Green's tensor for enhanced point-spread function (PSF) engineering.
• To achieve high-precision simultaneous 3D localization and orientation measurements of single molecules.

Main Methods:

• Developed the pixOL algorithm for phase mask optimization.
• Engineered a Green's tensor, extending PSF engineering principles.
• Simulated performance using 2500 detected photons over a 700-nm depth range.

Main Results:

• Achieved optimal precision in simultaneous 3D orientation (4.1°), wobble angle (0.44 sr), lateral localization (23.2 nm), and axial localization (19.5 nm).
• Demonstrated accurate and precise 6D imaging of Nile red in a supported lipid bilayer.
• Resolved membrane defects and cholesterol concentration differences.

Conclusions:

• The pixOL microscope provides unprecedented precision for 6D single-molecule imaging.
• This technology enables detailed investigation of biomolecular organization and membrane dynamics.
• pixOL advances the understanding of lipid bilayer structure and function.