Simultaneous orientation and 3D localization microscopy with a Vortex point spread function

Abstract

We have developed an engineered Point Spread Function (PSF) to enable the simultaneous estimation of dipole orientation, 3D position and degree of rotational constraint of single-molecule emitters from a single 2D focal plane. Besides giving access to orientation information, the Vortex PSF along with the vectorial PSF fitter avoids localization bias common in localization microscopy for fixed dipole emitters. We demonstrate this technique on reorienting single-molecules and using binding-activated localization microscopy on DNA intercalators, corroborating perpendicular azimuthal angles to the DNA axis for in-plane emitters but find a non-uniform distribution as a function of the polar angle. The Vortex PSF is realized by an affordable glass phase mask and has a compact footprint that can easily be combined with localization microscopy techniques on rotationally constrained emitters.

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

“…We used a liquidcrystal spatial light modulator to create the vortex phase mask within a bespoke 4f system with two polarized imaging channels, as described previously. 31,44 An unpolarized vortex PSF was recently reported by Hulleman and coworkers 23 for simultaneously imaging the 3D positions and orientations of SMs. While this method is easy to implement because it does not require two polarized imaging channels, it shows reduced sensitivity for measuring the orientational second moments in terms of the Cramér-Rao bound (Figure S3).…”

“…4,10,11 The field continues to evolve with theo-retical studies to determine fundamental performance limits, [15][16][17] the assistance of machine learning to design optimal PSFs for 3D SMLM, [18][19][20] and new PSFs for measuring the 3D positions and 3D orientations of SMs. [21][22][23] The central challenge facing SM spectroscopists is the finite photon budget afforded to each individual fluorophore before irreversible photobleaching; due to photon shot noise, it is difficult to measure experimentally the many properties of SM emission, e.g., the position, arrival time, wavelength, and polarization of each incoming photon, simultaneously with high precision. Tradeoffs are unavoidable, and one's sensitivity to a particular parameter can be improved to incredible degrees by reducing sensitivity to others, e.g., achieving~1 nm localization precision in MINFLUX by making the measurement largely independent of emission wavelength and polarization.…”

“…Second, existing techniques are largely limited to estimating rotational diffusion, or the "wobble" of an SM, as a scalar parameter averaged over all directions. 16,17,21,23,[25][26][27] However, we know molecules wobble in full 3D space and, in certain environments, perhaps anisotropically, i.e., more in one direction versus another. 28,29 Is it possible to robustly quantify the degree of wobble anisotropy of an SM experimentally?…”

“…Here, we adopt the vortex phase mask, typically used for generating a donut-shaped illumination ring in STED 30 and MINFLUX, 24 and place it in the detection path of an epifluorescence microscope to create the PSF. We improve the orientation sensitivity of an existing implementation 23 by splitting the detected fluorescence into two orthogonally polarized channels. The polarized vortex PSF dramatically changes shape with molecular orientation, thus enabling precise estimation of SM rotational diffusion in full 3D space using a single camera snapshot.…”

Elucidating the Role of Topological Constraint on the Structure of Overstretched DNA Using Fluorescence Polarization Microscopy

“… 11 , 14 , 47 It is our hope that the new data presented here motivate further inquiry and discussion. More broadly, our current work, alongside emerging techniques in polarization microscopy, 48 , 49 single-molecule orientation microscopy, 50 − 54 and spatio-angular image analysis, 55 , 56 provides a methodological blueprint to nondestructively investigate a wide variety of biological and material systems.…”