Super-resolution optical fluctuation imaging

Nature Photonics, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 10, 2025

Language: Английский

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Doubling the resolution of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy

Nature Photonics, Journal Year: 2024, Volume and Issue: 18(10), P. 1059 - 1066

Published: Aug. 2, 2024

Language: Английский

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Bayesian Inference of Binding Kinetics from Fluorescence Time Series

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 3, 2025

Abstract The study of binding kinetics via the analysis fluorescence time traces is often con-founded by measurement noise and photophysics. Although photoblinking can be mitigated using labels less likely to photoswitch, photobleaching generally cannot eliminated. Current methods for measuring unbinding rates are therefore limited concurrent events. Here, we propose a method infer alongside intensity traces. Our approach two-stage process involving analyzing individual regions interest (ROIs) with Hidden Markov Model levels each trace. We then use inferred level state trajectory from all ROIs kinetic rates. has several advantages, including ability analyze noisy traces, account presence events, provide uncertainties associated kinetics. demonstrate effectiveness reliability our through simulations data DNA origami experiments.

Language: Английский

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Enabling Hours-Long Drift Correction with Nanometer Resolution in Optical Microscopy through Reflection Interferometry of Fiducial Beads

Nano Letters, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 3, 2025

Optical microscopy excels at revealing the dynamics of biological systems in real time. However, focal drift limits most approaches to only several minutes absence drift-mitigation strategies. Here we introduce focus readjustment for enhanced vertical resolution (FREVR), a method which uses micrometer-sized fiducial beads alongside specimen. By tracking interference fringes generated by these beads, FREVR can detect changes position with nanometer precision. We showcase this approach on microscope equipped high-speed CMOS camera used bead and highly sensitive EMCCD tasked quantify fluorescence spectral resolution. Using approach, measured samples hours single-molecule systems, as well mammalian cells having their actin filaments or membrane proteins fluorescently labeled. has potential dramatically increase temporal spatial long-term stability techniques.

Language: Английский

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Oblique line scan illumination enables expansive, accurate and sensitive single-protein measurements in solution and in living cells

Nature Methods, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 18, 2025

Language: Английский

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Maximum Likelihood Analysis of Diffusing Molecules with Conformational Dynamics in Single-Molecule FRET

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

Published: Feb. 18, 2025

In single-molecule Förster resonance energy transfer (FRET) experiments, characterizing conformational dynamics from photon bursts emitted by diffusing molecules can be challenging due to the interplay of molecular transitions, translational diffusion, and background noise. This paper extends maximum likelihood analysis (burstML) incorporate both diffusion through laser spot, offering a comprehensive single molecules. The new approach integrates two previously developed methods: one accounting for without other addressing diffusion. By combining these approaches, extended burstML method allows determination brightness, time, FRET efficiency in each state, transition rates, even under conditions, such as fast (comparable count rates) slow (one per several bursts) high noise, unequal brightness or diffusivity states. performance was demonstrated on simulated data two-state molecule compared with colorML method, which simplifies excluding While is computationally efficient performs well ideal conditions (low noise equal states), its accuracy diminishes when are not met. contrast, remains accurate across broader range experimental scenarios. Both were applied analyze folding proteins (Pin1 WW domain, FiP35 FBP28 villin, synthetic protein α3D) various highlighting where differs providing insights into applicability methods diverse settings.

Language: Английский

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Statistical analysis of fluorescence intensity transients with Bayesian methods

Science Advances, Journal Year: 2025, Volume and Issue: 11(16)

Published: April 18, 2025

Molecular movement and interactions at the single-molecule level, particularly in live cells, are often studied using fluorescence correlation spectroscopy (FCS). While powerful, FCS has notable drawbacks: It requires high laser intensities long acquisition times, increasing phototoxicity, relies on problematic statistical assumptions data fitting. We introduce intensity trace analysis (FITSA), a Bayesian method that directly analyzes traces. FITSA offers faster, more stable convergence than previous approaches provides robust parameter estimation from far shorter measurements conventional FCS. Our results demonstrate achieves comparable precision to while requiring substantially fewer photons. This advantage becomes even pronounced when accounting for dependencies analysis, which overlooked but necessary accurate error estimation. By reducing exposure, minimizes phototoxicity effects, representing major advancement quantitative of molecular processes across fields.

Language: Английский

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Denoising for single-cell resolution highly multiplexed imaging: Deep learning safeguards cell spatial heterogeneity analysis

TrAC Trends in Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown, P. 118282 - 118282

Published: April 1, 2025

Language: Английский

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BNP-Track: a framework for superresolved tracking

Nature Methods, Journal Year: 2024, Volume and Issue: 21(9), P. 1716 - 1724

Published: July 22, 2024

Superresolution tools, such as PALM and STORM, provide nanoscale localization accuracy by relying on rare photophysical events, limiting these methods to static samples. By contrast, here, we extend superresolution dynamics without photodynamics simultaneously determining emitter numbers their tracks (localization linking) with the same per frame widefield immobilized emitters under similar imaging conditions (≈50 nm). We demonstrate our Bayesian nonparametric track (BNP-Track) framework both in cellulo synthetic data. BNP-Track develops a joint (posterior) distribution that learns quantifies uncertainty over associated propagated from shot noise, camera artifacts, pixelation, background out-of-focus motion. In doing so, integrate spatiotemporal information into distribution, which is otherwise compromised modularly localizing linking positions across frames. For this reason, remains accurate crowding regimens beyond those accessible other single-particle tracking tools.

Language: Английский

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Bayesian Inference of Binding Kinetics from Fluorescence Time Series

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

Published: May 7, 2025

The study of binding kinetics via the analysis fluorescence time traces is often confounded by measurement noise and photophysics. Although photoblinking can be mitigated using labels less likely to photoswitch, photobleaching generally cannot eliminated. Current methods for measuring unbinding rates are, therefore, limited concurrent events. Here, we propose a method infer alongside intensity traces. Our approach two-stage process involving analyzing individual regions interest (ROIs) with hidden Markov model levels each trace. We then use inferred level state trajectory from all ROIs kinetic rates. has several advantages, including ability analyze noisy traces, account presence events, provide uncertainties associated kinetics. demonstrate effectiveness reliability our through simulations data DNA origami experiments.

Language: Английский

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