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The quenching of the fluorescence of carbon dots: A review on mechanisms and applications
Carbon dots (CDs) possess unique optical properties such as tunable photoluminescence (PL) and excitation dependent multicolor emission. The quenching and recovery of the fluorescence of CDs can be utilized for detecting analytes. The PL mechanisms of CDs have been discussed in previous articles, but the quenching mechanisms of CDs have not been summarized so far. Quenching mechanisms include static quenching, dynamic quenching, Förster resonance energy transfer (FRET), photoinduced electron transfer (PET), surface energy transfer (SET), Dexter energy transfer (DET) and inner filter effect (IFE). Following an introduction, the review (with 88 refs.) first summarizes the various kinds of quenching mechanisms of CDs (including static quenching, dynamic quenching, FRET, PET and IFE), the principles of these quenching mechanisms, and the methods of distinguishing these quenching mechanisms. This is followed by an overview on applications of the various quenching mechanisms in detection and imaging.
Graphical abstract
Schematic representation of the quenching mechanisms of carbon dots (CDs) which include static quenching, dynamic quenching, Förster resonance energy transfer (FRET), photoinduced electron transfer(PET), surface energy transfer (SET), Dexter energy transfer (DET) and inner filter effect (IFE). All these effects can be used to detect and image analytes.
Journal Article
Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer
Structural techniques such as x-ray crystallography and electron microscopy give insight into how macromolecules function by providing snapshots of different conformational states. Function also depends on the path between those states, but to see that path involves watching single molecules move. This became possible with the advent of single-molecule Förster resonance energy transfer (smFRET), which was first implemented in 1996. Lerner et al. review how smFRET has been used to study macromolecules in action, providing mechanistic insights into processes such as DNA repair, transcription, and translation. They also describe current limitations of the approach and suggest how future developments may expand the applications of smFRET. Science , this issue p. eaan1133 Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.
Journal Article
Single-molecule FRET imaging of GPCR dimers in living cells
Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.A suite of generally applicable methods and tools, developed to enable single-molecule FRET-based studies of transmembrane proteins diffusing in the cell membrane of living cells, was used to study the oligomerization and dynamics of GPCRs.
Journal Article
Fluorescence resonance energy transfer (FRET)-based biosensors: visualizing cellular dynamics and bioenergetics
Förster (or fluorescence) resonance energy transfer (FRET) is a process involving the radiation-less transfer of energy from a \"donor\" fluorophore to an \"acceptor\" fluorophore. FRET technology enables the quantitative analysis of molecular dynamics in biophysics and in molecular biology, such as the monitoring of protein-protein interactions, protein-DNA interactions, and protein conformational changes. FRET-based biosensors have been utilized to monitor cellular dynamics not only in heterogeneous cellular populations, but also at the single-cell level in real time. Lately, applications of FRET-based biosensors range from basic biological to biomedical disciplines. Despite the diverse applications of FRET, FRET-based sensors still face many challenges. There is an increasing need for higher fluorescence resolution and improved specificity of FRET biosensors. Additionally, as more FRET-based technologies extend to medical diagnostics, the affordability of FRET reagents becomes a significant concern. Here, we will review current advances and limitations of FRET-based biosensor technology and discuss future FRET applications.[PUBLICATION ABSTRACT]
Journal Article
FRET as a biomolecular research tool — understanding its potential while avoiding pitfalls
The applications of Förster resonance energy transfer (FRET) grow with each year. However, different FRET techniques are not applied consistently, nor are results uniformly presented, which makes implementing and reproducing FRET experiments challenging. We discuss important considerations for designing and evaluating ensemble FRET experiments. Alongside a primer on FRET basics, we provide guidelines for making experimental design choices such as the donor-acceptor pair, instrumentation and labeling chemistries; selecting control experiments to unambiguously demonstrate FRET and validate that the experiments provide meaningful data about the biomolecular process in question; analyzing raw data and assessing the results; and reporting data and experimental details in a manner that easily allows for reproducibility. Some considerations are also given for FRET assays and FRET imaging, especially with fluorescent proteins. Our goal is to motivate and empower all biologists to consider FRET for the powerful research tool it can be.
Journal Article
Dexter energy transfer pathways
Energy transfer with an associated spin change of the donor and acceptor, Dexter energy transfer, is critically important in solar energy harvesting assemblies, damage protection schemes of photobiology, and organometallic opto-electronic materials. Dexter transfer between chemically linked donors and acceptors is bridge mediated, presenting an enticing analogy with bridge-mediated electron and hole transfer. However, Dexter coupling pathways must convey both an electron and a hole from donor to acceptor, and this adds considerable richness to the mediation process. We dissect the bridge-mediated Dexter coupling mechanisms and formulate a theory for triplet energy transfer coupling pathways. Virtual donor–acceptor charge-transfer exciton intermediates dominate at shorter distances or higher tunneling energy gaps, whereas virtual intermediates with an electron and a hole both on the bridge (virtual bridge excitons) dominate for longer distances or lower energy gaps. The effects of virtual bridge excitons were neglected in earlier treatments. The two-particle pathway framework developed here shows how Dexter energy-transfer rates depend on donor, bridge, and acceptor energetics, as well as on orbital symmetry and quantum interference among pathways.
Journal Article
Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study
Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.
Journal Article
mScarlet: a bright monomeric red fluorescent protein for cellular imaging
An extremely bright, truly monomeric RFP, mScarlet, is described that outperforms existing RFPs in diverse labeling applications, especially in FRET with ratiometric imaging.
We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.
Journal Article
Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins
Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.
An international blind study confirms that smFRET measurements on dynamic proteins are highly reproducible across instruments, analysis procedures and timescales, further highlighting the promise of smFRET for dynamic structural biology.
Journal Article