Single-molecule Spectroscopy Research Articles

Deuterated oxazines are bright near-infrared fluorophores for mitochondrial imaging and single molecule spectroscopy.

Bright near-infrared fluorophores are in demand for microscopy. We showcase a deuterated oxazine being 23% brighter vs. ATTO700. With a longer lifetime of 1.85 nanoseconds, we find the best-in-class SulfoOxazine700-d10 to stain mitochondria for confocal microscopy, and demonstrate unaffected diffusion properties in single molecule fluorescence correlation spectroscopy.

Merging Vibrational Spectroscopy with Fluorescence Microscopy: Combining the Best of Two Worlds.

Vibrational spectroscopy and fluorescence spectroscopy have historically been two established but separate fields of molecular spectroscopy. While vibrational spectroscopy provides exquisite chemical information, fluorescence spectroscopy often offers orders of magnitude higher detection sensitivity. However, they each lack the advantages of each other. In recent years, a series of novel nonlinear optical spectroscopy studies have been developed that merge both spectroscopies into a single double-resonance process. These techniques combine the chemical specificity of Raman or infrared (IR) spectroscopy with the superb detection sensitivity and spatial resolution of fluorescence microscopy. Many facets have been explored, including Raman transition versus IR transition, time domain versus frequency domain, and spectroscopy versus microscopy. Notably, single-molecule vibrational spectroscopy has been achieved at room temperature without the need for plasmonics. Even super-resolution vibrational imaging beyond the diffraction limit was demonstrated. This review summarizes the growing field of vibrational-encoded fluorescence microscopy, including key technical developments, emerging applications, and future prospects.

Perspective: fluorescence lifetime imaging and single-molecule spectroscopy for studying biological condensates

Abstract Biological condensates, often formed via liquid-liquid phase separation (LLPS), are membraneless compartments organizing biochemical reactions. Recent advances have shifted the focus from identifying condensates to elucidating their dynamic biological functions, such as buffering concentrations, mediating reactions, and regulating signaling. These are critical for cellular processes and implicated in diseases like cancer and neurodegeneration. Advanced microscopy techniques, including fluorescence lifetime imaging microscopy (FLIM), FLIM-FRET, and fluorescence correlation spectroscopy (FCS), enable quantitative, real-time investigations of condensate composition, dynamics, material properties, and their responses to environmental stimuli in live cells. This perspective highlights the utility of time-resolved fluorescence and single-molecule spectroscopy techniques for shedding light on condensate functions, properties, and interactions with membranes, offering insights into cellular physiology and pathology.

Suppressing non-radiative relaxation in a NIR single photon emitter: the impact of deuteration and temperature.

The fluorescence quantum yield of organic NIR-emitters is typically limited by internal conversion (IC), restricting their applications in imaging and quantum technology. Here, we study the impact of deuteration and temperature on the emission properties of dibenzoterrylene (DBT) by bulk and single molecule spectroscopy. Based on simple photophysical modelling, we first clarify how IC affects the single molecule emission rate. Next, we show that deuteration of DBT leads to a concomitant increase in the fluorescence lifetime and quantum yield by up to 60%. This clear deuterium isotope effect indicates a significant contribution of C-H-vibrations in the IC process. The solvent-dependent changes in the IC rate of hydrogenated and deuterated compounds were found to follow the predictions of the energy gap law in the weak coupling limit. This view is supported by the very weak temperature dependence of the IC rate between 5 and 300 K. Our results not only shed light on the non-radiative relaxation of a topical polycyclic aromatic hydrocarbon, but also pave the way for single molecule quantum emitters with high emission yields in the NIR.

Interplay between conformational dynamics and substrate binding regulates enzymatic activity: a single-molecule FRET study.

Proteins often harness extensive motions of domains and subunits to promote their function. Deciphering how these movements impact activity is key for understanding life's molecular machinery. The enzyme adenylate kinase is an intriguing example for this relationship; it ensures efficient catalysis by large-scale domain motions that lead to the enclosure of the bound substrates ATP and AMP. Surprisingly, the enzyme is activated by urea, a compound commonly acting as a denaturant. We utilize this phenomenon to decipher the involvement of conformational dynamics in the mechanism of action of the enzyme. Combining single-molecule FRET spectroscopy and enzymatic activity studies, we find that urea promotes the open conformation of the enzyme, aiding the proper positioning of the substrates. Further, urea decreases AMP affinity, paradoxically facilitating a more efficient progression towards the catalytically active complex. These results allow us to define a complete kinetic scheme that includes the open/close transitions of the enzyme and to unravel the important interplay between conformational dynamics and chemical steps, a general property of enzymes. State-of-the-art tools, such as single-molecule fluorescence spectroscopy, offer new insights into how enzymes balance different conformations to regulate activity.

Dual-Wavelength On-Chip Integrated Metalens for Epi-Fluorescence Single-Molecule Sensing.

Single-molecule fluorescence spectroscopy offers unique capabilities for the low-concentration sensing and probing of molecular dynmics. However, employing such a methodology for versatile sensing and diagnostics under point-of-care demands device miniaturization to lab-on-a-chip size. In this study, we numerically design metalenses with high numerical aperture (NA = 1.1), which are composed of silicon nitride nanostructures deposited on a waveguide and can selectively focus guided light into an aqueous solution at two wavelengths of interest in the spectral range of 500-780 nm. Despite the severe chromatic focal shift in the lateral directions owing to the wavelength-dependent propagation constant in a waveguide, segmented on-chip metalenses provide perfectly overlapping focal volumes that meet the requirements for epi-fluorescence light collection. We demonstrate that the molecule detection efficiencies of metalenses designed for the excitation and emission wavelengths of ATTO 490LS, Alexa 555, and APC-Cy7 tandem fluorophores are sufficient to collect several thousand photons per second per molecule at modest excitation rate constants. Such sensitivity provides reliable diffusion fluorescence correlation spectroscopy analysis of single molecules on a chip to extract their concentration and diffusion properties in the nanomolar range. Achromatic on-chip metalenses open new avenues for developing ultra-compact and sensitive devices for precision medicine and environmental monitoring.

Nanocavity-based single-molecule plasmon-enhanced Raman spectroscopy: Features and advancements.

Since 1997, driven by advancements in nanoscience, single-molecule plasmon-enhanced Raman spectroscopy (SM-PERS) has developed into a powerful technique for ultrasensitive trace analysis through fingerprint vibrational chemical information. The nanocavity between the coupled plasmonic nanostructures, offering an exceptionally high Raman signal enhancement factor (i.e., plasmonic field hotspot), is crucial for the achievement of SM-PERS. Herein, we first briefly review the development of SM-PERS, followed by an introduction of the features and methodologies of SM-PERS, as well as the applications of SM-PERS in biological analysis, high-resolution chemical imaging, and the investigations of single-molecule reactions. Finally, a perspective highlighting the advancement of new methods and applications of nano-driven SM-PERS is presented.

Fine-tuning of Fgf8 morphogen gradient by heparan sulfate proteoglycans in the extracellular matrix.

Embryonic development is orchestrated by the action of morphogens, which spread out from a local source and activate, in a field of target cells, different cellular programs based on their concentration gradient. Fibroblast growth factor 8 (Fgf8) is a morphogen with important functions in embryonic organizing centers. It forms a gradient in the extracellular space by free diffusion, interaction with the extracellular matrix (ECM) and receptor-mediated endocytosis. However, morphogen gradient regulation by ECM is still poorly understood. Here we show that specific Heparan Sulfate Proteoglycans (HSPGs) bind Fgf8 with different affinities directly in the ECM of living zebrafish embryos, thus affecting its diffusion and signaling. Using single-molecule Fluorescence Correlation Spectroscopy, we quantify this binding in vivo, and find two different modes of interaction. First, reducing or increasing the concentration of specific HSPGs in the extracellular space alters Fgf8 diffusion, and thus, its gradient shape. Second, ternary complex formation of Fgf8 ligand with Fgf-receptors and HSPGs at the cell surface requires HSPG attachment to the cell membrane. Together, our results show that graded Fgf8 morphogen distribution is achieved by constraining free Fgf8 diffusion through successive interactions with HSPGs at the cell surface and in ECM space.

Real-Space Spectral Determination of Short Single-Stranded DNA Sequence Structures.

Resolving the sequence and structure of flexible biomolecules such as DNA is crucial to understanding their biological mechanisms and functions. Traditional structural biology methods remain challenging for the analysis of small and disordered biomolecules, especially those that are difficult to label or crystallize. Recent development of single-molecule tip-enhanced Raman spectroscopy (TERS) offers a label-free approach to identifying nucleobases in a single DNA chain. However, a clear demonstration of sequencing both spatially and spectrally at single-base resolution is still elusive due to the challenges caused by weak Raman signals and the flexibility of DNA molecules. Here, we report a proof-of-principle demonstration to this end, spectrally resolving in real space individual nucleobases and their sequence structures within a short, single-stranded DNA molecule artificially designed. This breakthrough is achieved through the development of subnanometer-resolved low-temperature TERS methodology for such thermally unstable flexible biomolecules. Further TERS mapping over individual nucleobases provides additional structural information about the molecular configurations and even the locations of functional groups, offering a way to track modification types and binding sites in biomolecules.

Chemically Interrogating N-Heterocyclic Carbenes at the Single-Molecule Level Using Tip-Enhanced Raman Spectroscopy.

N-heterocyclic carbenes (NHCs) have been established as powerful modifiers to functionalize metal surfaces for a wide variety of energy and nanoelectronic applications. To fundamentally understand and harness NHC modification, it is essential to identify suitable methods to interrogate NHC surface chemistry at the spatial limit. Here, we demonstrate tip-enhanced Raman spectroscopy (TERS) as a promising tool for chemically probing the surface properties of NHCs at the single-molecule scale. We show that with subnanometer resolution, TERS measurements are capable of not only unambiguously identifying the chemical structure of individual NHCs by their vibrational fingerprints but also definitively determining the binding mode of NHCs on metal surfaces. In particular, by investigating low-temperature NHC adsorption on Ag(111), our TERS studies provide insights into the temperature dependence of the adsorption properties of NHCs. This work suggests the potential of single-molecule vibrational spectroscopy for investigations of NHC surface modification at the most fundamental level.

Concept of a Convex on-Chip Metalens as a Miniature Sensor of Fluorescence of Single Molecules

Fluorescence spectroscopy of single molecules is of fundamental significance to determine a small amount of matter and to study molecular dynamic processes. However, the applications of this method in medicine require new solutions for the miniaturization of a sensor planform. The most promising direction in this area seems to be the development of photonic integrated circuits with a high molecule detection efficiency in a volume of about cubic micron. In this work, a concept of a dielectric metalens on a waveguide, which has a high efficiency of the focusing/collection of radiation from an aqueous solution, has been presented. The structure of the metalens with a numerical aperture of above 1.1 operating in the optical range near the fluorescence maximum of the Alexa Fluor 647 dye has been simulated. After the calculation of the molecule detection efficiency, diffusion autocorrelation functions of Alexa Fluor 647 molecules have been calculated to characterize the possibility of measuring the brightness, as well as the number and dynamics of single molecules in the focal volume of the metalens. This concept provides the foundation for the development of future sensors of single molecules as biomedical and environment screening tools.

A sequential binding mechanism for 5′ splice site recognition and modulation for the human U1 snRNP

Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how human U1 snRNP recognizes particular 5′ splice sites (5′SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5′SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5′SS modulation. We have determined a sequential binding mechanism for modulation of −1A bulged 5′SS by branaplam using a combination of ensemble kinetic measurements and colocalization single molecule spectroscopy (CoSMoS). Our mechanism establishes that U1-C protein binds reversibly to U1 snRNP, and branaplam binds to the U1 snRNP/U1-C complex only after it has engaged with a −1A bulged 5′SS. Obligate orders of binding and unbinding explain how reversible branaplam interactions cause formation of long-lived U1 snRNP/5′SS complexes. Branaplam targets a ribonucleoprotein, not only an RNA duplex, and its action depends on fundamental properties of 5′SS recognition.

Disordered regions of human eIF4B orchestrate a dynamic self-association landscape

Eukaryotic translation initiation factor eIF4B is required for efficient cap-dependent translation, it is overexpressed in cancer cells, and may influence stress granule formation. Due to the high degree of intrinsic disorder, eIF4B is rarely observed in cryo-EM structures of translation complexes and only ever by its single structured RNA recognition motif domain, leaving the molecular details of its large intrinsically disordered region (IDR) unknown. By integrating experiments and simulations we demonstrate that eIF4B IDR orchestrates and fine-tunes an intricate transition from monomers to a condensed phase, in which large-size dynamic oligomers form before mesoscopic phase separation. Single-molecule spectroscopy combined with molecular simulations enabled us to characterize the conformational ensembles and underlying intra- and intermolecular dynamics across the oligomerization transition. The observed sensitivity to ionic strength and molecular crowding in the self-association landscape suggests potential regulation of eIF4B nanoscopic and mesoscopic behaviors such as driven by protein modifications, binding partners or changes to the cellular environment.

Mechanistic basis of activation and inhibition of protein disulfide isomerase by allosteric antithrombotic compounds

BackgroundProtein disulfide isomerase (PDI) is a promising target for combating thrombosis. Extensive research over the past decade has identified numerous PDI-targeting compounds. However, limited information exists regarding how these compounds control PDI activity, which complicates further development. ObjectivesTo define the mechanism of action of 2 allosteric antithrombotic compounds of therapeutic interest, quercetin-3-O-rutinoside and bepristat-2a. MethodsA multipronged approach that integrates single-molecule spectroscopy, steady-state kinetics, single-turnover kinetics, and site-specific mutagenesis. ResultsPDI is a thiol isomerase consisting of 2 catalytic a domains and 2 inactive b domains arranged in the order a-b-b'-a'. The active sites CGHC are located in the a and a' domains. The binding site of quercetin-3-O-rutinoside and bepristat-2a is in the b' domain. Using a library of 9 Förster resonance energy transfer sensors, we showed that quercetin-3-O-rutinoside and bepristat-2a globally alter PDI structure and dynamics, leading to ligand-specific modifications of its shape and reorientation of the active sites. Combined with enzyme kinetics and mutagenesis of the active sites, Förster resonance energy transfer data reveal that binding of quercetin-3-O-rutinoside results in a twisted enzyme with reduced affinity for the substrate. In contrast, bepristat-2a promotes a more compact conformation of PDI, in which a greater enzymatic activity is achieved by accelerating the nucleophilic step of the a domain, leading to faster formation of the covalent enzyme–substrate complex. ConclusionThis work reveals the mechanistic basis underlying PDI regulation by antithrombotic compounds quercetin-3-O-rutinoside and bepristat-2a and points to novel strategies for furthering the development of PDI-targeting compounds into drugs.

Fluorescence-Detected Two-Dimensional Electronic Spectroscopy of a Single Molecule.

Single-molecule fluorescence spectroscopy is a powerful method that avoids ensemble averaging, but its temporal resolution is limited by the fluorescence lifetime to nanoseconds at most. At the ensemble level, two-dimensional spectroscopy provides insight into ultrafast femtosecond processes, such as energy transfer and line broadening, even beyond the Fourier limit, by correlating pump and probe spectra. Here, we combine these two techniques and demonstrate coherent 2D spectroscopy of individual dibenzoterrylene (DBT) molecules at room temperature. We excite the molecule in a confocal microscope with a phase-modulated train of femtosecond pulses and detect the emitted fluorescence with single-photon counting detectors. Using a phase-sensitive detection scheme, we were able to measure the nonlinear 2D spectra of most of the DBT molecules that we studied. Our method is applicable to a wide range of single emitters and opens new avenues for understanding energy transfer in single quantum objects on ultrafast time scales.

Controlled single-electron transfer enables time-resolved excited-state spectroscopy of individual molecules

An increasing number of scanning-probe-based spectroscopic techniques provides access to diverse electronic properties of single molecules. Typically, these experiments can only study a subset of all electronic transitions, which obscures the unambiguous assignment of measured quantities to specific quantum transitions. Here we develop a single-molecule spectroscopy that enables the access to many quantum transitions of different types, including radiative, non-radiative and redox, that is, charge-related, transitions. Our method relies on controlled alternating single-charge attachment and detachment. For read-out, the spin states are mapped to charge states, which we can detect by atomic force microscopy. We can determine the relative energies of ground and excited states of an individual molecule and can prepare the molecule in defined excited states. After a proof-of-principle demonstration of the technique on pentacene, we apply it to PTCDA, the scanning-probe luminescence of which has been interpreted controversially. The method may be used to guide, understand and engineer tip-induced chemical reactions as well as phosphorescence and fluorescence of individual molecules.

Mei5-Sae3 stabilizes Dmc1 nucleating clusters for efficient Dmc1 assembly on RPA-coated single-stranded DNA.

Interhomolog recombination in meiosis requires a meiosis-specific recombinase, Dmc1. In Saccharomyces cerevisiae, the Mei5-Sae3 complex facilitates the loading of Dmc1 onto the replication protein A (RPA)-coated single-stranded DNA (ssDNA) to form nucleoprotein filaments. In vivo, Dmc1 and Mei5-Sae3 are interdependent in their colocalization on the chromosomes. However, the mechanistic role of Mei5-Sae3 in mediating Dmc1 activity remains unclear. We used single-molecule fluorescence resonance energy transfer and colocalization single-molecule spectroscopy experiments to elucidate how Mei5-Sae3 stimulates Dmc1 assembly on ssDNA and RPA-coated ssDNA. We showed that Mei5-Sae3 stabilized Dmc1 nucleating clusters with two to threemolecules on naked DNA by preferentially reducing Dmc1 dissociation rates. Mei5-Sae3 also stimulated Dmc1 assembly on RPA-coated DNA. Using green fluorescent protein-labeled RPA, we showed the coexistence of an intermediate with Dmc1 and RPA on ssDNA before RPA dissociation. Moreover, the displacement efficiency of RPA depended on Dmc1 concentration, and its dependence was positively correlated with the stability of Dmc1 clusters on short ssDNA. These findings suggest a molecular model that Mei5-Sae3 mediates Dmc1 binding on RPA-coated ssDNA by stabilizing Dmc1 nucleating clusters, thus altering RPA dynamics on DNA to promote RPA dissociation.

Enzyme activation by urea reveals the interplay between conformational dynamics and substrate binding: a single-molecule FRET study.

Proteins often harness extensive motions of domains and subunits to promote their function. Deciphering how these movements impact activity is key for understanding life's molecular machinery. The enzyme adenylate kinase is an intriguing example for this relationship; it ensures efficient catalysis by large-scale domain motions that lead to the enclosure of the bound substrates ATP and AMP. At high concentrations, AMP also operates as an allosteric inhibitor of the protein. Surprisingly, the enzyme is activated by urea, a compound commonly acting as a denaturant. Combining single-molecule FRET spectroscopy and enzymatic activity studies, we find that urea interferes with two key mechanisms that contribute to enzyme efficacy. First, urea promotes the open conformation of the enzyme, aiding the proper positioning of the substrates. Second, urea decreases AMP affinity, paradoxically facilitating a more efficient progression towards the catalytically active complex. These results signify the important interplay between conformational dynamics and chemical steps, including binding, in the activity of enzymes. State-of-the-art tools, such as single-molecule fluorescence spectroscopy, offer new insights into how enzymes balance different conformations to regulate activity.

Single-Molecule Surface-Enhanced Raman Spectroscopy: Challenges, Opportunities, and Future Directions.

Single-molecule surface-enhanced Raman spectroscopy (SM-SERS) is a powerful experimental technique for label-free sensing, imaging, and chemical analysis. Although Raman spectroscopy itself is an extremely "feeble" phenomenon, the intense interaction of optical fields with metallic nanostructures in the form of plasmonic hotspots can generate Raman signals from single molecules. While what constitutes a true single-molecule signal has taken some years for the scientific community to establish, many SERS experiments, even those not specifically attempting single-molecule sensitivity, have observed fluctuation in both the SERS intensity and spectral features. In this Perspective, we discuss the impact that fluctuating SERS signals have had on the continuing advancement of SM-SERS, along with challenges and current and potential future applications.

Single-Molecule Characterization of Heterogeneous Oligomer Formation during Co-Aggregation of 40- and 42-Residue Amyloid-β.

The two most abundant isoforms of amyloid-β (Aβ) are the 40- (Aβ40) and 42-residue (Aβ42) peptides. Since they coexist and there is a correlation between toxicity and the ratio of the two isoforms, quantitative characterization of their interactions is crucial for understanding the Aβ aggregation mechanism. In this work, we follow the aggregation of individual isoforms in a mixture using single-molecule FRET spectroscopy by labeling Aβ42 and Aβ40 with the donor and acceptor fluorophores, respectively. We found that there are two phases of aggregation. The first phase consists of coaggregation of Aβ42 with a small amount of Aβ40, while the second phase results mostly from aggregation of Aβ40. We also found that the aggregation of Aβ42 is slowed by Aβ40 while the aggregation of Aβ40 is accelerated by Aβ42 in a concentration-dependent manner. The formation of oligomers was monitored by incubating mixtures in a plate reader and performing a single-molecule free-diffusion experiment at several different stages of aggregation. The detailed properties of the oligomers were obtained by maximum likelihood analysis of fluorescence bursts. The FRET efficiency distribution is much broader than that of the Aβ42 oligomers, indicating the diversity in isoform composition of the oligomers. Pulsed interleaved excitation experiments estimate that the fraction of Aβ40 in the co-oligomers in a 1:1 mixture of Aβ42 and Aβ40 varies between 0 and 20%. The detected oligomers were mostly co-oligomers especially at the physiological ratio of Aβ42 and Aβ40 (1:10), suggesting the critical role of Aβ40 in oligomer formation and aggregation.