Single-Molecule Localization Microscopy of 3D Orientation and Anisotropic Wobble Using a Polarized Vortex Point...

Area of Science:

• Condensed matter physics
• Single-molecule spectroscopy
• Soft matter science

Background:

• Single fluorophores are sensitive probes of their chemical environments.
• Simultaneous measurement of position, 3D orientation, and rotational diffusion is challenging due to limited photon budgets.

Purpose of the Study:

• To demonstrate a polarized vortex point spread function (PSF) for simultaneous measurement of single-molecule parameters.
• To characterize molecular wobble anisotropy from a single image.
• To enable nanoscale orientation imaging of soft matter.

Main Methods:

• Utilizing a polarized vortex PSF for single-molecule imaging.
• Imaging dim emitters with approximately 500 detected photons.
• Measuring localization precision, orientation precision, and wobble precision.

Main Results:

• Achieved 12 nm localization precision, 4°-8° orientation precision, and 26° wobble precision.
• Measured emission anisotropy of fluorescent beads.
• Characterized Nile red wobble dynamics in supported lipid bilayers.
• Distinguished orientation signatures of Nile red interacting with amyloid-beta species.

Conclusions:

• The polarized vortex PSF enables simultaneous measurement of single-molecule position, orientation, and wobble.
• This technique provides high precision even for dim emitters.
• It transforms single-molecule microscopy into nanoscale orientation imaging spectrometers, revealing soft matter structures through molecular orientation and wobble.