Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from structural dynamics

Proceedings of the National Academy of Sciences, Год журнала: 2023, Номер 120(4)

Опубликована: Янв. 18, 2023

Fluorescence correlation spectroscopy is a versatile tool for studying fast conformational changes of biomolecules especially when combined with Förster resonance energy transfer (FRET). Despite the many methods available identifying structural dynamics in FRET experiments, determination forward and backward transition rate constants thereby also equilibrium constant difficult two intensity levels are involved. Here, we combine analysis fluorescence lifetime information by including only subset photons autocorrelation based on their arrival time respect to excitation pulse (microtime). By fitting amplitude as function microtime gate, from fluorescence-intensity level systems corresponding obtained. This shrinking-gate (sg-FCS) approach demonstrated using simulations DNA origami-based model system experiments immobilized freely diffusing molecules. We further show that sg-FCS can distinguish photophysics dynamic even if dark quencher, this case graphene, Finally, unravel mechanism FRET-based membrane charge sensor indicating broad potential method. With sg-FCS, present an algorithm does not require prior knowledge therefore easily implemented carried out time-correlated single-photon data.

Язык: Английский

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Recent Advances in Single-Molecule Tracking and Imaging Techniques

Annual Review of Analytical Chemistry, Год журнала: 2023, Номер 16(1), С. 253 - 284

Опубликована: Июнь 14, 2023

Since the early 1990s, single-molecule detection in solution at room temperature has enabled direct observation of single biomolecules work real time and under physiological conditions, providing insights into complex biological systems that traditional ensemble methods cannot offer. In particular, recent advances tracking techniques allow researchers to follow individual their native environments for a timescale seconds minutes, revealing not only distinct pathways these take downstream signaling but also roles supporting life. this review, we discuss various imaging developed date, with an emphasis on advanced three-dimensional (3D) achieve ultrahigh spatiotemporal resolution provide sufficient working depths suitable molecules 3D tissue models. We then summarize observables can be extracted from trajectory data. Methods perform clustering analysis future directions are discussed.

Язык: Английский

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Combining pMINFLUX, graphene energy transfer and DNA-PAINT for nanometer precise 3D super-resolution microscopy

Light Science & Applications, Год журнала: 2023, Номер 12(1)

Опубликована: Март 10, 2023

3D super-resolution microscopy with nanometric resolution is a key to fully complement ultrastructural techniques fluorescence imaging. Here, we achieve by combining the 2D localization of pMINFLUX axial information graphene energy transfer (GET) and single-molecule switching DNA-PAINT. We demonstrate <2 nm precision in all 3 dimension reaching below 0.3 nm. In DNA-PAINT measurements, structural features, i.e., individual docking strands at distances nm, are directly resolved on DNA origami structures. GET represent particular synergetic combination for imaging near surface such as cell adhesion membrane complexes each photon used both information. Furthermore, introduce local PAINT (L-PAINT), which imager equipped an additional binding sequence upconcentration improving signal-to-background ratio speed clusters. L-PAINT demonstrated triangular structure 6 side lengths within seconds.

Язык: Английский

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Monitoring Dynamic Conformations of a Single Fluorescent Molecule Inside a Protein Cavity

Small Methods, Год журнала: 2025, Номер unknown

Опубликована: Фев. 2, 2025

Abstract Fluorescence nanoscopy and single‐molecule methods are entering the realm of structural biology, breaking new ground for dynamic measurements at room temperature liquid environments. Here, localization microscopy, polarization‐dependent excitation, protein engineering combined to determine orientation a fluorophore forming hydrogen bonds inside cavity. The observed conformations in good agreement with molecular dynamics simulations, enabling new, more realistic interplay between experiments simulations identify stable key interactions involved. Furthermore, jumps can be monitored precision 3° time resolution few seconds, confirming potential this methodology retrieving information nanoscopic biological systems under physiologically compatible conditions.

Язык: Английский

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Super-resolution Imaging of Energy Transfer by Intensity-Based STED-FRET

Nano Letters, Год журнала: 2021, Номер 21(5), С. 2296 - 2303

Опубликована: Фев. 23, 2021

Förster resonance energy transfer (FRET) imaging methods provide unique insight into the spatial distribution of and (bio)molecular interaction events, though they deliver average information for an ensemble events included in a diffraction-limited volume. Coupling super-resolution fluorescence microscopy FRET has been challenging elusive task. Here, we present STED-FRET, method general applicability to obtain super-resolved images. In addition higher resolution, STED-FRET provides more accurate quantification capacity suppressing contributions noninteracting partners, which are otherwise masked by averaging conventional imaging. The capabilities were first demonstrated on DNA-origami model systems, verified uniformly double-labeled microtubules, then utilized image biomolecular interactions membrane-associated periodic skeleton (MPS) neurons.

Язык: Английский

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Isotropic three-dimensional dual-color super-resolution microscopy with metal-induced energy transfer

Science Advances, Год журнала: 2022, Номер 8(23)

Опубликована: Июнь 8, 2022

Over the past two decades, super-resolution microscopy has seen a tremendous development in speed and resolution, but for most of its methods, there exists remarkable gap between lateral axial which is by factor 2 to 3 worse. One recently developed method close this metal-induced energy transfer (MIET) imaging, achieves an resolution down nanometers. It exploits distance-dependent quenching fluorescence when fluorescent molecule brought metal surface. In present manuscript, we combine extreme MIET imaging with extraordinary single-molecule localization microscopy, particular direct stochastic optical reconstruction ( d STORM). This combination allows us achieve isotropic three-dimensional subcellular structures. Moreover, used spectral demixing implementing dual-color MIET- STORM that image colocalize, three dimensions, different cellular structures simultaneously.

Язык: Английский

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Exploring the Synergies of Single‐Molecule Fluorescence and 2D Materials Coupled by DNA

Advanced Materials, Год журнала: 2023, Номер 35(41)

Опубликована: Сен. 5, 2023

The world of 2D materials is steadily growing, with numerous researchers attempting to discover, elucidate, and exploit their properties. Approaches relying on the detection single fluorescent molecules offer a set advantages, for instance, high sensitivity specificity, that allow drawing conclusions unprecedented precision. Herein, it argued how study benefits from fluorescence-based single-molecule modalities, vice versa. A special focus placed DNA, serving as versatile adaptor when anchoring dye materials. existing literature fruitful combination two fields reviewed, an outlook additional synergies can be created between them provided.

Язык: Английский

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Advancements and Practical Considerations for Biophysical Research: Navigating the Challenges and Future of Super-resolution Microscopy

Chemical & Biomedical Imaging, Год журнала: 2024, Номер 2(5), С. 331 - 344

Опубликована: Апрель 19, 2024

The introduction of super-resolution microscopy (SRM) has significantly advanced our understanding cellular and molecular dynamics, offering a detailed view previously beyond reach. Implementing SRM in biophysical research, however, presents numerous challenges. This review addresses the crucial aspects utilizing effectively, from selecting appropriate fluorophores preparing samples to analyzing complex data sets. We explore recent technological advancements methodological improvements that enhance capabilities SRM. Emphasizing integration with other analytical methods, we aim overcome inherent limitations expand scope biological insights achievable. By providing comprehensive guide for choosing most suitable methods based on specific research objectives, empower researchers processes enhanced precision clarity, thereby advancing frontiers research.

Язык: Английский

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A common framework for single-molecule localization using sequential structured illumination

Biophysical Reports, Год журнала: 2021, Номер 2(1), С. 100036 - 100036

Опубликована: Ноя. 24, 2021

Localization of single fluorescent molecules is key for physicochemical and biophysical measurements, such as single-molecule tracking super-resolution imaging by localization microscopy. Over the last two decades, several methods have been developed in which position a emitter interrogated with sequence spatially modulated patterns light. Among them, recent MINFLUX technique outstands achieving ∼10-fold improvement compared wide-field camera-based localization, reaching ∼1-2 nm precision at moderate photon counts. Here, we present common framework this type measurement. Using Cramér-Rao bound limit achievable precision, benchmark reported methods, including developments, MINSTED, long-established orbital tracking. In addition, characterize new proposed schemes, raster scanning, minimum intensity. Overall, found that approaches using an intensity similar performance central region excitation pattern, independent geometry they outperform featuring maximum.

Язык: Английский

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Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live‐Cell MINFLUX Nanoscopy

Small Methods, Год журнала: 2024, Номер 8(9)

Опубликована: Март 18, 2024

Abstract The single‐molecule localization concept MINFLUX has triggered a reevaluation of the features fluorophores for attaining nanometer‐scale resolution. nanoscopy benefits from temporally controlled fluorescence (“on”/“off”) photoswitching. Combined with an irreversible switching behavior, process is expected to turn highly efficient and quantitative data analysis simple. potential in recently reported photoactivable xanthone (PaX) dyes recognized extend list molecular switches used 561 nm excitation beyond fluorescent protein mMaple. success rates PaX 560 , PaX+ 560, mMaple are quantitatively compared by analyzing effective labeling efficiency endogenously tagged nuclear pore complexes. prove be superior on par best reversible routinely microscopy. Moreover, rationally designed 595 introduced complementing dual color imaging based spectral classification deterministic, irreversible, additive‐independent nature photoactivation showcased fast live‐cell imaging. meet demands robust readout each label position fill void reliable dedicated

Язык: Английский

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