Imaging the three-dimensional orientation and rotational mobility of fluorescent emitters using the Tri-spot point spread function

Abstract

Fluorescence photons emitted by single molecules contain rich information regarding their rotational motions, but adapting single-molecule localization microscopy (SMLM) to measure their orientations and rotational mobilities with high precision remains a challenge. Inspired by dipole radiation patterns, we design and implement a Tri-spot point spread function (PSF) that simultaneously measures the three-dimensional orientation and the rotational mobility of dipole-like emitters across a large field of view. We show that the orientation measurements done using the Tri-spot PSF are sufficiently accurate to correct the anisotropy-based localization bias, from 30 nm to 7 nm, in SMLM. We further characterize the emission anisotropy of fluorescent beads, revealing that both 20-nm and 100-nm diameter beads emit light significantly differently from isotropic point sources. Exciting 100-nm beads with linearly polarized light, we observe significant depolarization of the emitted fluorescence using the Tri-spot PSF that is difficult to detect using other methods. Finally, we demonstrate that the Tri-spot PSF detects rotational dynamics of single molecules within a polymer thin film that are not observable by conventional SMLM.

Dense Super-Resolution Imaging of Molecular Orientation Via Joint Sparse Basis Deconvolution and Spatial Pooling

“…Fluorescent beads were utilized to validate our method's detection and localization capabilities. A sparse layer of beads was imaged using the standard and Tri-spot PSFs with the system described in [5] (Fig. 2).…”

“…Henceforth we drop the corresponding constraint sets. This approximation is physically justified since the energy radiated by a dipole is mostly contained in the first three basis images [5,10]. Consequently, it renders Eq.…”

“…Besides their position, fluorescent molecules interacting with their nano-environment also report information such as orientation and emission spectra that are hidden from standard, diffraction-limited optical imaging systems. Augmenting the PSF, or the impulse response of the microscope, to uncover such fine details remains a vibrant research topic [4,5]. In addition, biophysical measurements utilizing SMLM are more accurate when a physically-realistic image-formation model and recovery problem are utilized that include parameters such as molecular orientation and rotational mobility.…”

“…In addition, the problem of PSF overlap in the presence of multi-channel measurement errors has not been considered before. In this work, we tackle these challenges by 1) constructing a forward model that decomposes molecular parameters (i.e., brightness, position, and orientation) into six basis images corresponding to the six second moments of orientation dynamics [5]; 2) building a novel sparse basis deconvolution algorithm that exploits a group-sparsity norm to jointly recover all molecular parameters; and 3) applying spatial pooling across the six basis images to avoid false localizations due to experimental mismatches. We applied the proposed method to resolve DNA conformation via super-resolution imaging of DNA intercalating probes.…”

Computational Correction of Spatially-Variant Optical Aberrations in 3D Single Molecule Localization Microscopy

“…A major advancement of single-molecule localization microscopy has been the ability to use PSF engineering to encode the 3D position of an emitter and/or its emission wavelength [16][17][18] or its molecular orientation [19][20][21]. To achieve emitter localization below the diffraction limit in both the lateral (x,y) and axial (z) dimensions, PSFs have been engineered to change shape rapidly along the z-dimension, either through stretching or through rotation [22][23][24][25][26][27][28][29][30].…”