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 can 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 by using molecular positions alone.

pixOL: pixel-wise dipole-spread function engineering for simultaneously measuring the 3D orientation and 3D localization of dipole-like emitters

“…Moreover, each NR exhibits relatively little rotational diffusion, i.e., small wobble angle Ω, (Fig. 3(f)), which is consistent with previous characterizations of NR within planar SLBs [19,35].…”

“…3(a), SI Section 6) [33,34] using two lipid compositions: DPPC (di(16:0) phosphatidylcholine) only vs. a mixture of DPPC and cholesterol (chol). Previous studies [19,35] have shown that NR orients itself perpendicular to the membrane when a high concentration (40% used here) of chol is present. Thus, a spherical SLB enables us to validate simultaneously orientation and position imaging performance, quantifying both precision and accuracy.…”

“…In addition, the vortex PSF measures SM 3D position and orientation by modulating fluorescence emission with a vortex phase plate, common in STED nanoscopy, and does not require a PBS [18]. However, its simple implementation requires comparatively bright emitters [13] or a PBS [19] to achieve high orientation measurement precision. Recent reports using estimation theory show that the aforementioned methods do not yet achieve optimal performance [13].…”

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

“…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.…”