Single-fluorogen imaging reveals distinct environmental and structural features of biomolecular condensates.

Biomolecular condensates are viscoelastic materials. Simulations predict that condensates formed by intrinsically disordered proteins are network fluids defined by spatially inhomogeneous organization of the underlying molecules. Here, we test these predictions and find that molecules within condensates are organized into slow-moving nanoscale clusters and fast-moving dispersed molecules.

Differential interactions determine anisotropies at interfaces of RNA-based biomolecular condensates.

Biomolecular condensates form via macromolecular phase separation. Here, we report results from our characterization of synthetic condensates formed by phase separation of mixtures comprising two types of RNA molecules and the biocompatible polymer polyethylene glycol. Purine-rich RNAs are scaffolds that drive phase separation via heterotypic interactions.

Resolving the Orientations of and Angular Separation Between a Pair of Dipole Emitters.

We prove that it is impossible to distinguish two spatially coinciding fluorescent molecules from a single rotating molecule using polarization-sensitive imaging, even if one modulates the polarization of the illumination or the detection dipole-spread function (DSF). If the target is known to be a dipole pair, existing imaging methods perform poorly for measuring their angular separation. We propose simultaneously modulating the excitation polarization and DSF, which demonstrates robust discrimination between dipole pairs versus single molecules.

Nuclear speckle proteins form intrinsic and -dependent microphases.

Nuclear speckles are enriched in serine / arginine rich splicing factors (SRSFs), such as SRSF1. Splicing factors and proteins such as TDP-43 concentrate into distinct speckle territories to enable pre-mRNA processing. We have discovered that SRSFs and TDP-43 are block copolymers and the protein-specific interplay of inter-block repulsions and attractions drives spontaneous microphase separation.

Single-molecule orientation-localization microscopy: Applications and approaches.

Single-molecule orientation-localization microscopy (SMOLM) builds upon super-resolved localization microscopy by imaging orientations and rotational dynamics of individual molecules in addition to their positions. This added dimensionality provides unparalleled insights into nanoscale biophysical and biochemical processes, including the organization of actin networks, movement of molecular motors, conformations of DNA strands, growth and remodeling of amyloid aggregates, and composition changes within lipid membranes. In this review, we discuss recent innovations in SMOLM and cover three key aspects: (1) biophysical insights enabled by labeling strategies that endow fluorescent probes to bind to targets with orientation specificity; (2) advanced imaging techniques that leverage the physics of light-matter interactions and estimation theory to encode orientation information with high fidelity into microscope images; and (3) computational methods that ensure accurate and precise data analysis and interpretation, even in the presence of severe shot noise.

Fundamental Limits in Measuring the Anisotropic Rotational Diffusion of Single Molecules.

Many biophysical techniques, such as single-molecule fluorescence correlation spectroscopy, Förster resonance energy transfer, and fluorescence anisotropy, measure the translation and rotation of biomolecules to quantify molecular processes at the nanoscale. These methods often simplify data analysis by assuming isotropic rotational diffusion, e.g.

Single-Molecule Orientation Imaging Reveals the Nano-Architecture of Amyloid Fibrils Undergoing Growth and Decay.

Amyloid-beta (Aβ42) aggregates are characteristic Alzheimer's disease signatures, but probing how their nanoscale architectures influence their growth and decay remains challenging using current technologies. Here, we apply time-lapse single-molecule orientation-localization microscopy (SMOLM) to measure the orientations and rotational "wobble" of Nile blue (NB) molecules transiently binding to Aβ42 fibrils. We correlate fibril architectures measured by SMOLM with their growth and decay over the course of 5 to 20 min visualized by single-molecule localization microscopy (SMLM).

Macromolecular condensation organizes nucleolar sub-phases to set up a pH gradient.

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass.

Resolving the Nanoscale Structure of β-Sheet Peptide Self-Assemblies Using Single-Molecule Orientation-Localization Microscopy.

Synthetic peptides that self-assemble into cross-β fibrils are versatile building blocks for engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarities to amyloid species have been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize by using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize Nile red (NR), an amyloidophilic fluorogenic probe, and single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers KFE8 and KFE8 and the pathological amyloid-beta peptide Aβ42.

Single-molecule electrochemical imaging resolves the midpoint potentials of individual fluorophores on nanoporous antimony-doped tin oxide.

We report reversible switching of oxazine, cyanine, and rhodamine dyes by a nanoporous antimony-doped tin oxide electrode that enables single-molecule (SM) imaging of electrochemical activity. Since the emissive state of each fluorophore is modulated by electrochemical potential, the number of emitting single molecules follows a sigmoid function during a potential scan, and we thus optically determine the formal redox potential of each dye. We find that the presence of redox mediators (phenazine methosulfate and riboflavin) functions as an electrochemical switch on each dye's emissive state and observe significantly altered electrochemical potential and kinetics.

Resolving the nanoscale structure of β-sheet assemblies using single-molecule orientation-localization microscopy.

Synthetic peptides that self-assemble into cross-β fibrils have remarkable utility as engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarity to amyloid species has been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers, KFE8 and KFE8, and the pathological amyloid-beta peptide Aβ42.

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

Imaging both the positions and orientations of single fluorophores, termed single-molecule orientation-localisation microscopy, is a powerful tool to study biochemical processes. However, the limited photon budget associated with single-molecule fluorescence makes high-dimensional imaging with isotropic, nanoscale spatial resolution a formidable challenge. Here, we realise a radially and azimuthally polarized multi-view reflector (raMVR) microscope for the imaging of the 3D positions and 3D orientations of single molecules, with precision of 10.

Single fluorogen imaging reveals distinct environmental and structural features of biomolecular condensates.

Biomolecular condensates are viscoelastic materials. Simulations predict that fluid-like condensations are defined by spatially inhomogeneous organization of the underlying molecules. Here, we test these predictions using single-fluorogen tracking and super-resolution imaging.

Towards optimal point spread function design for resolving closely spaced emitters in three dimensions.

The past decade has brought many innovations in optical design for 3D super-resolution imaging of point-like emitters, but these methods often focus on single-emitter localization precision as a performance metric. Here, we propose a simple heuristic for designing a point spread function (PSF) that allows for precise measurement of the distance between two emitters. We discover that there are two types of PSFs that achieve high performance for resolving emitters in 3D, as quantified by the Cramér-Rao bounds for estimating the separation between two closely spaced emitters.

Deep-SMOLM: deep learning resolves the 3D orientations and 2D positions of overlapping single molecules with optimal nanoscale resolution.

Dipole-spread function (DSF) engineering reshapes the images of a microscope to maximize the sensitivity of measuring the 3D orientations of dipole-like emitters. However, severe Poisson shot noise, overlapping images, and simultaneously fitting high-dimensional information-both orientation and position-greatly complicates image analysis in single-molecule orientation-localization microscopy (SMOLM). Here, we report a deep-learning based estimator, termed Deep-SMOLM, that achieves superior 3D orientation and 2D position measurement precision within 3% of the theoretical limit (3.

Imaging of Catalytic Reactions on Tungsten Oxide Nanowires Connects Surface-Ligand Redox Chemistry with Photocatalytic Activity.

Semiconductor nanocrystals are promising candidates for generating chemical feedstocks through photocatalysis. Understanding the role of ligands used to prepare colloidal nanocrystals in catalysis is challenging due to the complexity and heterogeneity of nanocrystal surfaces. We use single-molecule fluorescence imaging to map the spatial distribution of active regions along individual tungsten oxide nanowires before and after functionalizing them with ascorbic acid.

Dipole-spread-function engineering for simultaneously measuring the 3D orientations and 3D positions of fluorescent molecules.

Interactions between biomolecules are characterized by both where they occur and how they are organized, e.g., the alignment of lipid molecules to form a membrane.

Resolving the Three-Dimensional Rotational and Translational Dynamics of Single Molecules Using Radially and Azimuthally Polarized Fluorescence.

We report a radially and azimuthally polarized (raPol) microscope for high detection and estimation performance in single-molecule orientation-localization microscopy (SMOLM). With 5000 photons detected from Nile red (NR) transiently bound within supported lipid bilayers (SLBs), raPol SMOLM achieves 2.9 nm localization precision, 1.

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 (∼500 photons detected), the polarized vortex PSF can obtain 12 nm localization precision, 4°-8° orientation precision, and 26° wobble precision.

Single-Molecule Colocalization of Redox Reactions on Semiconductor Photocatalysts Connects Surface Heterogeneity and Charge-Carrier Separation in Bismuth Oxybromide.

The surface structure of semiconductor photocatalysts controls the efficiency of charge-carrier extraction during photocatalytic reactions. However, understanding the connection between surface heterogeneity and the locations where photogenerated charge carriers are preferentially extracted is challenging. Herein we use single-molecule fluorescence imaging to map the spatial distribution of active regions and quantify the activity for both photocatalytic oxidation and reduction reactions on individual bismuth oxybromide (BiOBr) nanoplates.