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

Abstract

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 (~500 photons detected), the polarized vortex PSF is able to obtain 12 nm localization precision, 4-8° orientation precision, and 26° 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 using molecular positions alone.

Six-Dimensional Single-Molecule Imaging with Isotropic Resolution using a Multi-View Reflector Microscope

“…This strategy yields isotropic 3D spatial resolution, with raMVR achieving ~26-59% smaller standard deviations on average for measuring the 3D positions and orientations compared to recent, state-of-the-art methods. [11][12][13]15,17 We believe the raMVR pupil-splitting approach has many exciting possibilities for further development. It can be adapted for real-time orientation-resolved epifluorescence microscopy (Figure S14) of cellular structures.…”

“…While the mirror configuration is similar to that of a Cassegrain reflector (Figure 1c), here we mount our piecewise-planar pyramid mirror from the diagonal directions to avoid blocking any fluorescence (see Figure S1), achieving a photon efficiency close to 100% and superior to that of metasurfaces 30 and spatial light modulators. 15,[25][26][27] Figure 1e shows raMVR images captured from SMs with various orientations. Each SM orientation and degree of rotational diffusion is clearly distinguishable by the varying brightnesses of the focused DSF in each channel.…”

“…We incubated Aβ42 monomers with lipid-coated 350-nm spheres at 37 • C with constant shaking (200 rpm). While NR binds to both lipid membranes and Aβ42 aggregates, the sensitivity of NR's rotational dynamics to its chemical environment 15,16 enables us to resolve pure SLBs [Figure 4c(i)] from those that have been infiltrated by Aβ42. When in contact with amyloid aggregates on the spheres' surfaces, NR shows larger wobble angles [Figure 4c(ii-iv), Ω = 2.98 ± 1.14 sr in panel (ii), 2.33 ± 1.20 sr in panel (iii), and 2.09 ± 1.15 sr in panel (iv)] compared to those within DPPC SLBs (Figure 3d, Ω = 1.13 ± 0.95 sr).…”

“…A hallmark of biological systems is their careful control and regulation of biomolecular interactions at the nanoscale, via mechanical forces, 1 lipid membranes, 2 and biomolecular condensates, 3,4 to name a few examples. Owing to their sensitivity and versatility, 5,6 single fluorescent molecules and their translational and rotational dynamics have been used to study a variety of biochemical processes, including the motions of molecular motors, 7,8 conformations of supercoiled DNA, [9][10][11] and the structure and organization of actin filaments, 12,13 amyloid aggregates, 14,15 and lipid membranes. 16,17 Simultaneously resolving the 3D position, 3D orientation, and rotational diffusion of single fluorophores, termed single molecule orientation localization microscopy (SMOLM), requires careful manipulation of the phase and polarization of only hundreds or thousands of photons.…”

“…Limited by challenging signal-to-background ratios (SBRs) in typical single molecule (SM) experiments, microscopists have developed various SMOLM techniques to improve measurement performance, 18 approaching the theoretical limits under ideal conditions. [19][20][21] To use precious fluorescence photons more efficiently and maximize signal-to-noise ratios (SNRs), most recently developed techniques create dipolespread functions (DSFs), or images of dipole emitters, with compact footprints 11,12,15,17,22 and thusly achieve higher precision and accuracy. One key challenge with these designs is that orientation estimation requires fitting subtle features within noisy images.…”