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

Within condensed matter, single fluorophores are sensitive probes of their chemical environments, but it is difficult to use their limited photon budget to image precisely their positions, 3D orientations, and rotational diffusion simultaneously. We demonstrate the polarized vortex point spread function (PSF) for measuring these parameters, including characterizing the anisotropy of a molecule's wobble, simultaneously from a single image. Even when imaging dim emitters (similar to 500 photons detected), the polarized vortex PSF can obtain 12 nm localization precision, 4 degrees-8 degrees orientation precision, and 26 degrees wobble precision. We use the vortex PSF to measure the emission anisotropy of fluorescent beads, the wobble dynamics of Nile red (NR) within supported lipid bilayers, and the distinct orientation signatures of NR in contact with amyloid-beta fibrils, oligomers, and tangles. The unparalleled sensitivity of the vortex PSF transforms single-molecule microscopes into nanoscale orientation imaging spectrometers, where the orientations and wobbles of individual probes reveal structures and organization of soft matter that are nearly impossible to perceive by using molecular positions alone.

Automated summary

The polarized vortex point spread function (PSF) allows for precise measurement of position, orientation, and wobble of molecules simultaneously from a single image, even for dim emitters. This method can be used to measure emission anisotropy, wobble dynamics, and orientation signatures of different substances, transforming single-molecule microscopes into nanoscale orientation imaging spectrometers.