Biology subjects

Lew, M. D.

Publications and source records attributed to Lew, M. D..

15 recordsLinked to original sources

bioRxiv2025

Nuclear speckle proteins form intrinsic and MALAT1-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. This gives rise to size-limited, ordered assemblies, that are 30 - 45 nm in diameter. Depending on the protein, each microphase comprises several tens to hundreds of molecules. The sub-micron scale territories observed in cells are shown to be clusters of microphases. The regulatory lncRNA MALAT1 binds preferentially to SRSF1 microphases to enhance microphase separation and alter microphase structures. Microphase separation enables the concentration of finite numbers of splicing factors into assemblies with distinct nanoscale structures that can be modulated by MALAT1. Our findings provide a structural framework for the functional organization of splicing factors.

biophysics↗

bioRxiv2024

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

Biomolecular condensates form via macromolecular phase separation, giving rise to coexisting phases delineated by interfaces. Here, we characterize the structures of interfaces formed by phase separation driven by heterotypic interactions in ternary mixtures of two types of RNA molecules and polyethylene glycol. We find that purine-rich RNAs are scaffolds that drive phase separation via strong heterotypic interactions. Conversely, pyrimidine-rich RNA molecules are defined by weaker heterotypic interactions. They function as adsorbents that accumulate at and wet the interfaces of coexisting phases formed by phase separation of scaffolds. Our computations predict that scaffolds and adsorbents have different non-random orientational preferences at interfaces. We tested these predictions using single-molecule super resolution imaging that tracks the motions of fluorogenic probes that are bound to RNA molecules. Motions parallel to the interface were found to be faster than motions perpendicular to the interface. These findings support previous predictions regarding anisotropies of motions at interfaces.

biophysics↗

bioRxiv2024

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

Amyloid-beta (A{beta}42) aggregates are characteristic signatures of Alzheimers 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 A{beta}42 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 A{beta}42 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 A{beta}42 fibrils, respectively. We also observe SMLM-invisible fibril remodeling, including steady growth and decay patterns that conserve {beta}-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.

biophysics↗

bioRxiv2023

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

Synthetic peptides that self-assemble into cross-{beta} 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 A{beta}42. SMOLM reveals that the orientations of Nile red, as it transiently binds to both KFE8 and A{beta}42, 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 {beta}-rich fibrils.

biophysics↗

bioRxiv2023

POLCAM: Instant molecular orientation microscopy for the life sciences

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting the widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, that can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field of view errors, used simulations to optimize experimental design, and developed a fast algorithm based on Stokes parameter estimation which can operate over 1000 fold faster than the state of the art, enabling near instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software, and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study alpha-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.

biophysics↗

bioRxiv2023

Single fluorogen imaging reveals spatial inhomogeneities within 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. Specifically, we leverage the localization and orientational preferences of freely diffusing fluorogens and the solvatochromic effect whereby specific fluorogens are turned on in response to condensate microenvironments. We deployed three different fluorogens to probe the microenvironments and molecular organization of different protein-based 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, and this gives rise to slow-moving nanoscale molecular clusters that coexist with fast-moving molecules. Fluorogens that localize preferentially to the interface help us map their distinct features. Our findings provide a structural and dynamical basis for the viscoelasticity of condensates.

biophysics↗

bioRxiv2022

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 archives superior 3D orientation and 2D position measurement precision within 3% of the theoretical limit (3.8{whitebullet} orientation, 0.32 sr wobble angle, and 8.5 nm lateral position using 1000 detected photons). Deep-SMOLM also achieves 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.

molecular biology↗

bioRxiv2022

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

We report a radially and azimuthally polarized multi-view reflector (raMVR) microscope for precise imaging of the 3D positions and 3D orientations of single molecules (SMs, 10.9 nm and 2.0{degrees} precisions using 5000 photons). These precisions are [~]1.5 times better than those of existing methods for SM orientation-localization microscopy. The raMVR microscope achieves 6D super-resolution imaging of Nile red (NR) molecules transiently bound to 150 nm, 350 nm, and 1 {micro}m-diameter lipid-coated spheres, accurately resolving their spherical morphology despite refractive-index mismatch. Simply by observing the rotational dynamics o raMVR images also resolve the infiltration of lipid membranes by amyloid-beta oligomers without covalent labeling. Finally, we demonstrate 6D imaging of HEK-293T cell membranes, where the orientations of merocyanine 540 molecules reveal heterogeneities in membrane fluidity. With its [~]2 {micro}m depth range, nearly isotropic 3D spatial resolution, and superior orientation measurement precision, we expect the raMVR microscope to enable 6D imaging of molecular dynamics within biological and chemical systems with unprecedented detail.

biophysics↗

bioRxiv2021

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

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 microscopes dipolespread function (DSF). We demonstrate the pixOL algorithm for simultaneously optimizing all pixels within a phase mask to produce an engineered Greens 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., 1.14{degrees} orientation, 0.24 sr wobble angle, 8.17 nm lateral localization, and 12.21 nm axial localization precisions over an 800-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.

molecular biology↗

bioRxiv2021

Resolving the 3D 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{degrees} 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-{beta}-cyclodextrin (M{beta}CD-chol) causes NRs orientational diffusion to be dramatically reduced, but curiously, NRs median lateral displacements drastically increase from 20.8 nm 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 raPols high measurement precision and are not detectable in standard SMLM.

biophysics↗

bioRxiv2021

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 molecules wobble, simultaneously from a single image. Even when imaging dim emitters ([~]500 photons detected), the polarized vortex PSF is able to obtain 12 nm localization precision, 4-8{degrees} orientation precision, and 26{degrees} 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 using molecular positions alone.

biophysics↗

bioRxiv2020

Single-molecule 3D orientation imaging reveals nanoscale compositional heterogeneity in lipid membranes

In soft matter, thermal energy causes molecules to continuously translate and rotate, even in crowded environments, impacting the spatial organization and function of most molecular assemblies, such as lipid membranes. Directly measuring the orientation and spatial organization of large collections (>3000 molecules/m2) of single molecules with nanoscale resolution remains elusive. We present SMOLM, single-molecule orientation localization microscopy, to directly measure the orientation spectra (3D orientation plus "wobble") of lipophilic probes transiently bound to lipid membranes, revealing that Nile reds (NR) orientation spectra are extremely sensitive to membrane chemical composition. SMOLM images resolve nanodomains and enzyme-induced compositional heterogeneity within membranes, where NR within liquid-ordered vs. liquid-disordered domains shows a ~4{degrees} difference in polar angle and a ~0.3{pi} sr difference in wobble angle. As a new type of imaging spectroscopy, SMOLM exposes the organizational and functional dynamics of lipid-lipid, lipid-protein, and lipid-dye interactions with single-molecule, nanoscale resolution.

biophysics↗

bioRxiv2020

Single-molecule orientation localization microscopy for resolving structural heterogeneities within 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 Cramer-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 (~1000x 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.

biophysics↗

bioRxiv2019

Measuring localization confidence for quantifying accuracy and heterogeneity in single-molecule super-resolution microscopy

The resolution and accuracy of single-molecule localization micro-scopes (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 recon-structed structures, and discover hidden molecular heterogeneities. As a computational methodology, WIF is broadly applicable to any SMLM dataset, imaging system, and localization algorithm.

biophysics↗

bioRxiv2018

Minimizing Structural Bias in Single-Molecule Super-Resolution Microscopy

Single-molecule localization microscopy (SMLM) depends on sequential detection and localization of individual molecular blinking events. Due to the stochasticity of single-molecule blinking and the desire to improve SMLMs temporal resolution, algorithms capable of analyzing frames with a high density (HD) of active molecules, or molecules whose images overlap, are a prerequisite for accurate location measurements. Thus far, HD algorithms are evaluated using scalar metrics, such as root-mean-square error, that fail to quantify the structure of errors caused by the structure of the sample. Here, we show that the spatial distribution of localization errors within super-resolved images of biological structures are vectorial in nature, leading to systematic structural biases that severely degrade image resolution. We further demonstrate that the shape of the microscopes point-spread function (PSF) fundamentally affects the characteristics of imaging artifacts. We built a Robust Statistical Estimation algorithm (RoSE) to minimize these biases for arbitrary structures and PSFs. RoSE accomplishes this minimization by estimating the likelihood of blinking events to localize molecules more accurately and eliminate false localizations. Using RoSE, we measure the distance between crossing microtubules, quantify the morphology of and separation between vesicles, and obtain robust recovery using diverse 3D PSFs with unmatched accuracy compared to state-of-the-art algorithms.

bioinformatics↗