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

Amyloid-beta (Aß42) aggregates are characteristic signatures of Alzheimer's disease, 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 Aß42 fibrils. We quantify correlations between fibril architectures, measured by SMOLM, and their growth and decay visualized by single-molecule localization microscopy (SMLM). We discover that stable Aß42 fibrils tend to be well-ordered, signified by well-aligned NB orientations and small wobble. SMOLM also shows that increasing order and disorder are signatures of growing and decaying Aß42 fibrils, respectively. We also observe SMLM-invisible fibril remodeling, including steady growth and decay patterns that conserve ß-sheet organization. SMOLM reveals that increased heterogeneity in fibril architectures is correlated with more dynamic remodeling and that large-scale fibril remodeling tends to originate from local regions that exhibit strong heterogeneity.

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

Synthetic peptides that self-assemble into cross-ß 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 KFE8L and KFE8D and the pathological amyloid-beta peptide Aß42. Importantly, NR SMOLM reveals the helical (bilayer) ribbon structure of both KFE8 and Aß42 and quantifies the precise tilt of the fibrils' inner and outer backbones in relevant buffer conditions without the need for covalent labeling or sequence mutations. SMOLM also distinguishes polymorphic branched and curved morphologies of KFE8, whose backbones exhibit much more heterogeneity than those of typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross-ß-rich fibrils.

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. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.

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. We are therefore able to measure optically how redox mediators and the solid-state electrode modulate the redox state of fluorescent molecules, which follows an electrocatalytic (EC') mechanism, with SM sensitivity over a 900 µm2 field of view. Our observations indicate that redox mediator-assisted SM electrochemical imaging (SMEC) could be potentially used to sense any electroactive species. Combined with SM blinking and localization microscopy, SMEC imaging promises to resolve the nanoscale spatial distributions of redox species and their redox states, as well as the electron transfer kinetics of electroactive species in various bioelectrochemical processes.

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

Synthetic peptides that self-assemble into cross-ß 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, KFE8L and KFE8D, and the pathological amyloid-beta peptide Aß42. SMOLM reveals that the orientations of Nile red, as it transiently binds to both KFE8 and Aß42, are consistent with a helical (bilayer) ribbon structure and convey the precise tilt of the fibrils' inner and outer backbones. SMOLM also finds polymorphic branched and curved morphologies of KFE8 whose backbones exhibit much more heterogeneity than those of more typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross ß-rich fibrils.

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 10.9 nm and 2.0° over a 1.5 µm depth range. The raMVR microscope achieves 6D super-resolution imaging of Nile red (NR) molecules transiently bound to lipid-coated spheres, accurately resolving their spherical morphology despite refractive-index mismatch. By observing the rotational dynamics of NR, raMVR images also resolve the infiltration of lipid membranes by amyloid-beta oligomers without covalent labelling. Finally, we demonstrate 6D imaging of cell membranes, where the orientations of specific fluorophores reveal heterogeneity in membrane fluidity. With its nearly isotropic 3D spatial resolution and orientation measurement precision, we expect the raMVR microscope to enable 6D imaging of molecular dynamics within biological and chemical systems with exceptional detail.

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

Recent computations suggest that biomolecular condensates that form via macromolecular phase separation are network fluids featuring spatially inhomogeneous organization of the underlying molecules. Computations also point to unique conformations of molecules at condensate interfaces. Here, we test these predictions using high-resolution structural characterizations of condensates formed by intrinsically disordered prion-like low complexity domains (PLCDs). We leveraged the localization and orientational preferences of freely diffusing fluorogens and the solvatochromic effect whereby specific fluorogens are turned on in response to the physic-chemical properties of condensate microenvironments to facilitate single-molecule tracking and super-resolution imaging. We deployed three different fluorogens to probe internal microenvironments and molecular organization of PLCD condensates. The spatiotemporal resolution and environmental sensitivity afforded by single-fluorogen imaging shows that the internal environments of condensates are more hydrophobic than coexisting dilute phases. Molecules within condensates are organized in a spatially inhomogeneous manner featuring slow-moving nanoscale molecular clusters or hubs that coexist with fast-moving molecules. Finally, molecules at interfaces of condensates are found to have distinct orientational preferences when compared to the interiors. Our findings, which affirm computational predictions, help provide a structural basis for condensate viscoelasticity and dispel the notion of protein condensates being isotropic liquids defined by uniform internal densities.

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 (3.8° orientation, 0.32 sr wobble angle, and 8.5 nm lateral position using 1000 detected photons). Deep-SMOLM also demonstrates state-of-art estimation performance on overlapping images of emitters, e.g., a 0.95 Jaccard index for emitters separated by 139 nm, corresponding to a 43% image overlap. Deep-SMOLM accurately and precisely reconstructs 5D information of both simulated biological fibers and experimental amyloid fibrils from images containing highly overlapped DSFs at a speed ~10 times faster than iterative estimators.

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. One PSF is very similar to the existing Tetrapod PSFs; the other is a rotating single-spot PSF, which we call the crescent PSF. The latter exhibits excellent performance for localizing single emitters throughout a 1-µm focal volume (localization precisions of 7.3 nm in x, 7.7 nm in y, and 18.3 nm in z using 1000 detected photons), and it distinguishes between one and two closely spaced emitters with superior accuracy (25-53% lower error rates than the best-performing Tetrapod PSF, averaged throughout a 1-µm focal volume). Our study provides additional insights into optimal strategies for encoding 3D spatial information into optical PSFs.

In Situ 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 in situ 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. Rather than blocking active sites, we observed a significant enhancement in activity for photocatalytic water oxidation after treatment with ascorbic acid. While the initial nanowires contain inactive regions dispersed along their length, the functionalized nanowires show high uniformity in their photocatalytic activity. Spatial colocalization of the active regions with their surface chemical properties shows that oxidation of ascorbic acid during photocatalysis generates new oxygen vacancies along the nanowire surface. We demonstrate that controlling surface-ligand redox chemistry during photocatalysis can enhance the active site concentration on nanocrystal catalysts.

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. However, spatial and angular information are mixed within the image of a fluorescent molecule-the microscope's dipole-spread function (DSF). We demonstrate the pixOL algorithm for simultaneously optimizing all pixels within a phase mask to produce an engineered Green's tensor-the dipole extension of point-spread function engineering. The pixOL DSF achieves optimal precision for measuring simultaneously the 3D orientation and 3D location of a single molecule, i.e., 4.1° orientation, 0.44 sr wobble angle, 23.2 nm lateral localization, and 19.5 nm axial localization precisions in simulations over a 700-nm depth range using 2500 detected photons. The pixOL microscope accurately and precisely resolves the 3D positions and 3D orientations of Nile red within a spherical supported lipid bilayer, resolving both membrane defects and differences in cholesterol concentration in 6 dimensions.

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, 1.5° orientation precision, and 0.17 sr precision in estimating rotational wobble. Within DPPC SLBs, SMOLM imaging reveals the existence of randomly oriented binding pockets that prevent NR from freely exploring all orientations. Treating the SLBs with cholesterol-loaded methyl-ß-cyclodextrin (MßCD-chol) causes NR's orientational diffusion to be dramatically reduced, but curiously NR's median lateral displacements drastically increase from 20.8 to 75.5 nm (200 ms time lag). These jump diffusion events overwhelmingly originate from cholesterol-rich nanodomains within the SLB. These detailed measurements of single-molecule rotational and translational dynamics are made possible by raPol's high measurement precision and are not detectable in standard SMLM.

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. 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.

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. Through a coordinate-based colocalization analysis, we quantify the spatial correlation between the locations where fluorogenic probe molecules are oxidized and reduced on the surface of individual nanoplates. Surprisingly, we observed two distinct photochemical behaviors for BiOBr particles prepared within the same batch, which exhibit either predominantly uncorrelated activity where electrons and holes are extracted from different sites or colocalized activity in which oxidation and reduction take place within the same nanoscale regions. By analyzing the emissive properties of the fluorogenic probes, we propose that electrons and holes colocalize at defect-deficient regions, while defects promote the selective extraction of one carrier type by trapping either electrons or holes. Although previous work has used defect engineering to enhance the activity of bismuth oxyhalides and other semiconductor photocatalysts for useful reductive half-reactions (e.g., CO2 or N2 reduction), our results show that defect-free regions are needed to promote both oxidation and reduction in fuel-generating photocatalysts that do not rely on sacrificial reagents.

Single-molecule orientation localization microscopy I: fundamental limits.

Precisely measuring the three-dimensional position and orientation of individual fluorophores is challenging due to the substantial photon shot noise in single-molecule experiments. Facing this limited photon budget, numerous techniques have been developed to encode 2D and 3D position and 2D and 3D orientation information into fluorescence images. In this work, we adapt classical and quantum estimation theory and propose a mathematical framework to derive the best possible precision for measuring the position and orientation of dipole-like emitters for any fixed imaging system. We find that it is impossible to design an instrument that achieves the maximum sensitivity limit for measuring all possible rotational motions. Further, our vectorial dipole imaging model shows that the best quantum-limited localization precision is 4%-8% worse than that suggested by a scalar monopole model. Overall, we conclude that no single instrument can be optimized for maximum precision across all possible 2D and 3D localization and orientation measurement tasks.

Single-molecule orientation localization microscopy II: a performance comparison.

Various techniques have been developed to measure the 2D and 3D positions and 2D and 3D orientations of fluorescent molecules with improved precision over standard epifluorescence microscopes. Due to the challenging signal-to-background ratio in typical single-molecule experiments, it is essential to choose an imaging system optimized for the specific target sample. In this work, we compare the performance of multiple state-of-the-art and commonly used methods for orientation localization microscopy against the fundamental limits of measurement precision. Our analysis reveals optimal imaging methods for various experiment conditions and sample geometries. Interestingly, simple modifications to the standard fluorescence microscope exhibit superior performance in many imaging scenarios.

Quantifying accuracy and heterogeneity in single-molecule super-resolution microscopy.

The resolution and accuracy of single-molecule localization microscopes (SMLMs) are routinely benchmarked using simulated data, calibration rulers, or comparisons to secondary imaging modalities. However, these methods cannot quantify the nanoscale accuracy of an arbitrary SMLM dataset. Here, we show that by computing localization stability under a well-chosen perturbation with accurate knowledge of the imaging system, we can robustly measure the confidence of individual localizations without ground-truth knowledge of the sample. We demonstrate that our method, termed Wasserstein-induced flux (WIF), measures the accuracy of various reconstruction algorithms directly on experimental 2D and 3D data of microtubules and amyloid fibrils. We further show that WIF confidences can be used to evaluate the mismatch between computational models and imaging data, enhance the accuracy and resolution of reconstructed structures, and discover hidden molecular heterogeneities. As a computational methodology, WIF is broadly applicable to any SMLM dataset, imaging system, and localization algorithm.

Single-molecule orientation localization microscopy for resolving structural heterogeneities between amyloid fibrils.

Simultaneous measurements of single-molecule positions and orientations provide critical insight into a variety of biological and chemical processes. Various engineered point spread functions (PSFs) have been introduced for measuring the orientation and rotational diffusion of dipole-like emitters, but the widely used Cramér-Rao bound (CRB) only evaluates performance for one specific orientation at a time. Here, we report a performance metric, termed variance upper bound (VUB), that yields a global maximum CRB for all possible molecular orientations, thereby enabling the measurement performance of any PSF to be computed efficiently (~1000× faster than calculating average CRB). Our VUB reveals that the simple polarized standard PSF provides robust and precise orientation measurements if emitters are near a refractive index interface. Using this PSF, we measure the orientations and positions of Nile red (NR) molecules transiently bound to amyloid aggregates. Our super-resolved images reveal the main binding mode of NR on amyloid fiber surfaces, as well as structural heterogeneities along amyloid fibrillar networks, that cannot be resolved by single-molecule localization alone.

Quantum limits for precisely estimating the orientation and wobble of dipole emitters.

Precisely measuring molecular orientation is key to understanding how molecules organize and interact in soft matter, but the maximum theoretical limit of measurement precision has yet to be quantified. We use quantum estimation theory and Fisher information (QFI) to derive a fundamental bound on the precision of estimating the orientations of rotationally fixed molecules. While direct imaging of the microscope pupil achieves the quantum bound, it is not compatible with wide-field imaging, so we propose an interferometric imaging system that also achieves QFI-limited measurement precision. Extending our analysis to rotationally diffusing molecules, we derive conditions that enable a subset of second-order dipole orientation moments to be measured with quantum-limited precision. Interestingly, we find that no existing techniques can measure all second moments simultaneously with QFI-limited precision; there exists a fundamental trade-off between precisely measuring the mean orientation of a molecule versus its wobble. This theoretical analysis provides crucial insight for optimizing the design of orientation-sensitive imaging systems.