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

Physical Review Letters, Journal Year: 2025, Volume and Issue: 134(9)

Published: March 7, 2025

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 illumination or detection dipole-spread function (DSF). If target known be dipole pair, existing imaging methods perform poorly for measuring their angular separation. propose simultaneously modulating excitation and DSF, which demonstrates robust discrimination between pairs versus molecules. Our method improves precision centroid orientation by 50% separation two- four-fold over techniques.

Language: Английский

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Fundamental Limits in Measuring the Anisotropic Rotational Diffusion of Single Molecules

The Journal of Physical Chemistry A, Journal Year: 2024, Volume and Issue: 128(28), P. 5808 - 5815

Published: July 9, 2024

Many biophysical techniques, such as single-molecule fluorescence correlation spectroscopy, Förster resonance energy transfer, and anisotropy, measure the translation rotation of biomolecules to quantify molecular processes at nanoscale. These methods often simplify data analysis by assuming isotropic rotational diffusion, e.g., that molecules wobble within a circular cone. This simplification ignores anisotropy present in many biological contexts may cause exhibit different degrees diffusion directions. Here, we loosen this assumption establish theoretical framework for describing measuring anisotropic using imaging. We show is directly quantified eigenvalues 3-by-3 positive-semidefinite Hermitian matrix M consisting second-order moments molecule's transition dipole μ. formalism enables us model influence unavoidable shot noise perturbation E; E bound errors measurements via Weyl's inequality. Quantifying various perturbations reveals are generally more sensitive compared quantifying wobble. Moreover, severe can induce negative estimates M, thereby causing measurement fail. Our analysis, Fisher information, shows techniques with worse orientation sensitivity experience stronger require larger signal background ratios accurately. work provides deep insights improving state art imaging orientations single molecules.

Language: Английский

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Single-molecule orientation-localization microscopy: Applications and approaches

Quarterly Reviews of Biophysics, Journal Year: 2024, Volume and Issue: 57

Published: Jan. 1, 2024

Abstract Single-molecule orientation-localization microscopy (SMOLM) builds upon super-resolved localization by imaging orientations and rotational dynamics of individual molecules in addition to their positions. This added dimensionality provides unparalleled insights into nanoscale biophysical biochemical processes, including the organization actin networks, movement molecular motors, conformations DNA strands, growth remodeling amyloid aggregates, composition changes within lipid membranes. In this review, we discuss recent innovations SMOLM cover three key aspects: (1) enabled labeling strategies that endow fluorescent probes bind targets with orientation specificity; (2) advanced techniques leverage physics light-matter interactions estimation theory encode information high fidelity microscope images; (3) computational methods ensure accurate precise data analysis interpretation, even presence severe shot noise. Additionally, compare approaches, hardware, publicly available software aid community choosing best implementation for specific application. Finally, highlight future directions SMOLM, such as development improved photostability specificity, design “smart” adaptive use approaches handle large, complex datasets. review underscores significant current potential impact deepening our understanding dynamics, paving way breakthroughs fields biophysics, biochemistry, materials science.

Language: Английский

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Determining the Recruiting Rate of Spontaneously Blinking Rhodamines by Density Functional Calculations

The Journal of Physical Chemistry A, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 10, 2025

A recruiting rate (krc) of 0.1–5 s–1 has been proposed as the criterion for super-resolution spontaneously blinking rhodamines. Accurate prediction rhodamines is very important developing However, far we know, there no reliable theoretical method to predict krc. Herein, meticulously investigated effect intermolecular hydrogen bonds on spirocyclization reactions Moreover, a descriptor (ΔEC–T) was reliably assess ΔEC–T quantified ring-opening energy barrier reactions. robust linear correlation established between values and experimentally krc values. Based this correlation, designed screened five sulfonamide rhodamine dyes with optimized We expected that these findings could enable targeted design

Language: Английский

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Vibrational spectroscopy of single molecules/single entities without plasmonic enhancement

Applied Spectroscopy Reviews, Journal Year: 2025, Volume and Issue: unknown, P. 1 - 15

Published: March 24, 2025

Language: Английский

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Advancing Single-Molecule Biophysics: Next-Generation Organic Fluorophores with Tailored Labeling Strategies

Chemical & Biomedical Imaging, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Language: Английский

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Super-Resolution Mapping and Quantification of Molecular Diffusion via Single-Molecule Displacement/Diffusivity Mapping (SMdM)

Accounts of Chemical Research, Journal Year: 2025, Volume and Issue: unknown

Published: April 4, 2025

ConspectusDiffusion underlies vital physicochemical and biological processes provides a valuable window into molecular states interactions. However, it remains challenge to map diffusion at subcellular submicrometer scales. Whereas single-particle tracking of fluorescent molecules path quantify motion the nanoscale, its typical pursuit long trajectories limits wide-field mapping slow bound molecules.Single-molecule displacement/diffusivity (SMdM) rises challenge. Rather than following each molecule longitudinally as randomly visits potentially heterogeneous environments, SMdM flips question ask, for every location (e.g., 100 × nm2 spatial bin) in wide field, how different single identical nature move locally. This location-centered strategy is naturally effective diffusivity. Moreover, by focusing on local motion, only needs be detected transient displacement within fixed short time achieve statistics. task fulfilled fast-diffusing using tandem excitation scheme which pair closely timed stroboscopic pulses are applied across two frames, so that single-molecule images recorded pulse-defined ≲1 ms separation unlimited camera frame rate. With fitting models robust against mismatched anisotropy, thus successfully achieves super-resolution D fluorescently labeled contrasting sizes properties diverse cellular vitro systems.For intracellular protein diffusion, uncovers nanoscale heterogeneities mammalian cytoplasm nucleus further elucidates their origins from macromolecular crowding effects cytoskeletal chromatin ultrastructures, respectively, through correlated localization microscopy (SMLM). Across compartments cell, including cytoplasm, nucleus, endoplasmic reticulum (ER) lumen, mitochondrial matrix, unveils striking charge effect, positively charged proteins biasedly impeded. For membranes, integration with fluorogenic probes enables diffusivity fine-mapping, which, combination spectrally resolved SMLM (SR-SMLM), diffusional origins. biomolecular condensates, another synergy SR-SMLM gradual formation diffusion-suppressed, hydrophobic amyloid nanoaggregates surface FUS (fused sarcoma) condensates during aging. Beyond mapping, mass accumulation displacements affords means exceptional precision. advantage harnessed show no enhanced enzymes reactions, uncover ubiquitous net charge-driven protein-protein interactions solution, strategically manipulated cytoplasmic extracts interaction crowded cell defined an overwhelmingly negatively environment dense meshworks, echoing our parallel results cell.Together, uniquely enabling high-precision quantification systems, opens new door reveal fascinating spatiotemporal living cells beyond.

Language: Английский

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

Nano Letters, Journal Year: 2024, Volume and Issue: 24(24), P. 7276 - 7283

Published: June 3, 2024

Amyloid-beta (Aβ42) aggregates are characteristic Alzheimer's disease signatures, but probing how their nanoscale architectures influence growth and decay remains challenging using current technologies. Here, we apply time-lapse single-molecule orientation-localization microscopy (SMOLM) to measure the orientations rotational "wobble" of Nile blue (NB) molecules transiently binding Aβ42 fibrils. We correlate fibril measured by SMOLM with over course 5 20 min visualized localization (SMLM). discover that stable fibrils tend be well-ordered signified well-aligned NB small wobble. also shows increasing order disorder signatures growing decaying fibrils, respectively. observe SMLM-invisible remodeling, including steady patterns conserve β-sheet organization. reveals increased architectural heterogeneity is correlated dynamic remodeling large-scale tends originate from strongly heterogeneous local regions.

Language: Английский

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Single-molecule diffusivity quantification in Xenopus egg extracts elucidates physicochemical properties of the cytoplasm

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(50)

Published: Dec. 5, 2024

The living cell creates a unique internal molecular environment that is challenging to characterize. By combining single-molecule displacement/diffusivity mapping (SM d M) with physiologically active extracts prepared from Xenopus laevis eggs, we sought elucidate properties of the cytoplasm. Quantification diffusion coefficients 15 diverse proteins in extract showed that, compared water, negatively charged diffused ~50% slower, while positively was reduced by ~80 90%. Adding increasing concentrations salt progressively alleviated suppressed observed for proteins, signifying electrostatic interactions within predominately macromolecular environment. To investigate contribution RNA, an abundant, component cytoplasm, were treated ribonuclease, which resulted low diffusivity domains indicative aggregation, likely due liberation RNA-binding such as ribosomal since this effect could be mimicked adding polypeptides. Interestingly, under typical conditions inhibit actin polymerization, different sizes similar suppression consistent our separately measured 2.22-fold higher viscosity over water. Restoring or enhancing polymerization larger recapitulating behaviors cells. Together, these results indicate crowded are defined overwhelmingly containing cytoskeletal networks.

Language: Английский

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

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 27, 2024

ABSTRACT Amyloid-beta (Aβ42) aggregates are characteristic signatures of Alzheimer’s disease, but probing how their nanoscale architectures influence growth and decay remains challenging using current technologies. Here, we apply time-lapse single-molecule orientation-localization microscopy (SMOLM) to measure the orientations rotational “wobble” Nile blue (NB) molecules transiently binding Aβ42 fibrils. We quantify correlations between fibril architectures, measured by SMOLM, visualized localization (SMLM). discover that stable fibrils tend be well-ordered, signified well-aligned NB small wobble. SMOLM also shows increasing order disorder growing decaying fibrils, respectively. observe SMLM-invisible remodeling, including steady patterns conserve β-sheet organization. reveals increased heterogeneity in is correlated with more dynamic remodeling large-scale tends originate from local regions exhibit strong heterogeneity.

Language: Английский

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