Single-molecule Spectroscopy Techniques Research Articles

Assessing the impact of increase in the number of hydroxyl groups on the microscopic behaviors of ammonium-based room temperature ionic liquids: A combined fluorescence up-conversion, fluorescence correlation and NMR spectroscopic study

The current study aims to understand the impact of variation of number of hydroxyl groups on the cationic head of ammonium-based room temperature ionic liquids (RTILs) towards inter/intra molecular hydrogen bonding interaction, local structural organization and dynamics of the solvent systems. For this purpose, three hydroxyl functionalized ammonium based RTILs (HFILs) bearing different numbers of hydroxyl groups on the cationic head as well as a non-hydroxyl ammonium based RTIL have been examined using both ensembled average and single-molecule spectroscopy techniques. Investigation of solvent relaxation dynamics of all the concerned RTILs by the combination of femto-second fluorescence up-conversion techniques and time corelated single photon counting techniques reveals a bimodal solvent relaxation behaviour having a very fast sub-picosecond and a relatively slower picosecond to nanosecond solvation time component for all the RTILs. Interestingly, analysis of the rotational and translational diffusion dynamics of a few particular solutes shows that the solvent–solvent interaction in the poly-hydroxyl-based HFILs is substantially stronger than the solute–solvent interaction. Additionally, analysis of the rotational diffusion data reveals that all RTILs exhibit significant dynamic heterogeneity, which increases with increase the number of hydroxyl groups on the cationic head of HFILs. Moreover, PFG-NMR study have shown that the HFILs had considerably greater hydrodynamic radii than the non-hydroxyl RTILs, which is likely due to the result of stronger hydrogen bonding interaction between the hydroxyl groups and the constituent of the HFILs. The outcomes of all these investigations have essentially demonstrated that subsequent addition of hydroxyl functionalities to the cationic head of the RTILs significantly alter the intra/inter molecular interaction, local structural organisation and heterogeneity of the medium.

Protein Folding Dynamics as Diffusion on a Free Energy Surface: Rate Equation Terms, Transition Paths, and Analysis of Single-Molecule Photon Trajectories.

The rates of protein (un)folding are often described as diffusion on the projection of a hyperdimensional energy landscape onto a few (ideally one) order parameters. Testing such an approximation by experiment requires resolving the reactive transition paths of individual molecules, which is now becoming feasible with advanced single-molecule spectroscopic techniques. This has also sparked the interest of theorists in better understanding reactive transition paths. Here we focus on these issues aiming to establish (i) practical guidelines for the mechanistic interpretation of transition path times (TPT) and (ii) methods to extract the free energy surface and protein dynamics from the maximum likelihood analysis of photon trajectories (MLA-PT). We represent the (un)folding rates as diffusion on a 1D free energy surface with the FRET efficiency as a reaction coordinate proxy. We then perform diffusive kinetic simulations on surfaces with two minima and a barrier, but with different shapes (curvatures, barrier height, and symmetry), coupled to stochastic simulations of photon emissions that reproduce current SM-FRET experiments. From the analysis of transition paths, we find that the TPT is inversely proportional to the barrier height (difference in free energy between minimum and barrier top) for any given surface shape, and that dividing the TPT into climb and descent segments provides key information about the barrier's symmetry. We also find that the original MLA-PT procedure used to determine the TPT from experiments underestimates its value, particularly for the cases with smaller barriers (e.g., fast folders), and we suggest a simple strategy to correct for this bias. Importantly, we also demonstrate that photon trajectories contain enough information to extract the 1D free energy surface's shape and dynamics (if TPT is >4-5-fold longer than the interphoton time) using the MLA-PT directly implemented with a diffusive free energy surface model. When dealing with real (unknown) experimental data, the comparison between the likelihoods of the free energy surface and discrete kinetic three-state models can be used to evaluate the statistical significance of the estimated free energy surface.

Single-molecule excitation–emission spectroscopy

Single-molecule spectroscopy (SMS) provides a detailed view of individual emitter properties and local environments without having to resort to ensemble averaging. While the last several decades have seen substantial refinement of SMS techniques, recording excitation spectra of single emitters still poses a significant challenge. Here we address this problem by demonstrating simultaneous collection of fluorescence emission and excitation spectra using a compact common-path interferometer and broadband excitation, which is implemented as an extension of a standard SMS microscope. We demonstrate the technique by simultaneously collecting room-temperature excitation and emission spectra of individual terrylene diimide molecules and donor-acceptor dyads embedded in polystyrene. We analyze the resulting spectral parameters in terms of optical lineshape theory to obtain detailed information on the interactions of the emitters with their nanoscopic environment. This analysis finally reveals that environmental fluctuations between the donor and acceptor in the dyads are not correlated.

Understanding the Organization and Dynamics of KRAS4b on a Complex 8-Lipid Reconstituted Model Membrane using Microscopy and Spectroscopy Methods

The enrichment of negatively charged phospholipids, particular phosphatidylserine (PS) and phosphoinositides, in the inner leaflet of plasma membrane (PM) is one of the underlying principle behind recruitment of numerous intracellualr proteins to PM, thus regulating key cellular events. One such protein of interest is KRAS4b, a member of Ras superfamily of small GTPases, which is an oncoprotein implicated in 95% of pancreatic cancer, 45% of colorectal cancer, 35% of lung cancer and high extent in other forms of cancer. When Ras is active and bound to the membrane, it binds to its downstream effectors and triggers multiple signal cascading pathways that are essential for cell growth, proliferation and survival. KRAS4b localizes to PM predominantly by virtue of (i) the electrostatic interaction between six positively charged lysine residues located in the hypervariable region (C-terminal) of KRAS4b and negatively charged phospholipids in PM and (ii) the hydrophobicity of the farnesyl group, post-translational modification in C185 amino acid of KRAS4b, which anchors the protein into the disordered lipid domains. In this study, we aim to explore the molecular mechanism of KRAS4b interaction with the membrane using recombinant protein on an artificial supported lipid bilayer. The biomimetic surface is composed of complex 8-lipids composition that closely resemble the lipid composition of the inner leaflet of biological PM. We use a combination of fluorescence-based microscopy and single molecule spectroscopy techniques as well as high resolution scanning probe microscopy to study the lateral organization and dynamics of KRAS4b as well as the lipid bilayer. Using a very complex reconstituted model membrane to study KRAS4b, our research provides mechanistic insight into RAS biology and possibly a nobel platform for targeting RAS.

Free and Chaperone Bound Unfolded States of Outer Membrane Proteins

Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with chaperones. In the case of bacterial outer-membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance, because they remain unfolded for extended periods of time during their biogenesis and rely on interactions with periplasmic chaperones to prevent aggregation and support correct folding. Here, we study the unfolded-state properties of outer-membrane proteins. Using a combination of ensemble and single-molecule spectroscopy techniques including single-molecule FRET, we find that under strongly denaturing conditions and in the absence of chaperones, OMPs adopt conformationally heterogeneous unfolded states that lack the fast chain reconfiguration motions expected for an unstructured, fully unfolded chain. Interestingly, when complexed with periplasmic chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA), we observe for the protein OmpLA adopts an expanded, yet squished conformational ensemble, in which the overall broad distribution of unfolded states is preserved. Differently sized OMPs like OmpX (8 β-strands) and OmpF (16 β-strands) allow a broader perspective on chaperone enabled conformations of OMPs. These findings indicate that periplasmic chaperones Skp and SurA prestructure OMPs for their next binding partner (e.g., the β-barrel assembly machinery (BAM)) to facilitate insertion and proper folding into the outer membrane.

Ultrafast energy relaxation in single light-harvesting complexes

Energy relaxation in light-harvesting complexes has been extensively studied by various ultrafast spectroscopic techniques, the fastest processes being in the sub-100-fs range. At the same time, much slower dynamics have been observed in individual complexes by single-molecule fluorescence spectroscopy (SMS). In this work, we use a pump-probe-type SMS technique to observe the ultrafast energy relaxation in single light-harvesting complexes LH2 of purple bacteria. After excitation at 800 nm, the measured relaxation time distribution of multiple complexes has a peak at 95 fs and is asymmetric, with a tail at slower relaxation times. When tuning the excitation wavelength, the distribution changes in both its shape and position. The observed behavior agrees with what is to be expected from the LH2 excited states structure. As we show by a Redfield theory calculation of the relaxation times, the distribution shape corresponds to the expected effect of Gaussian disorder of the pigment transition energies. By repeatedly measuring few individual complexes for minutes, we find that complexes sample the relaxation time distribution on a timescale of seconds. Furthermore, by comparing the distribution from a single long-lived complex with the whole ensemble, we demonstrate that, regarding the relaxation times, the ensemble can be considered ergodic. Our findings thus agree with the commonly used notion of an ensemble of identical LH2 complexes experiencing slow random fluctuations.

Chain-Length-Dependent Exciton Dynamics in Linear Oligothiophenes Probed Using Ensemble and Single-Molecule Spectroscopy

Exciton dynamics in π-conjugated molecular systems is highly susceptible to conformational disorder. Using time-resolved and single-molecule spectroscopic techniques, the effect of chain length on the exciton dynamics in a series of linear oligothiophenes, for which the conformational disorder increased with increasing chain length, was investigated. As a result, extraordinary features of the exciton dynamics in longer-chain oligothiophene were revealed. Ultrafast fluorescence depolarization processes were observed due to exciton self-trapping in longer and bent chains. Increase in exciton delocalization during dynamic planarization processes was also observed in the linear oligothiophenes via time-resolved fluorescence spectra but was restricted in L-10T because of its considerable conformational disorder. Exciton delocalization was also unexpectedly observed in a bent chain using single-molecule fluorescence spectroscopy. Such delocalization modulates the fluorescence spectral shape by attenuating the 0-0 peak intensity. Collectively, these results provide significant insights into the exciton dynamics in conjugated polymers.

Molecular approaches to chromatography using single molecule spectroscopy.

Molecular approaches to chromatography using single molecule spectroscopy.

Singlet-triplet annihilation limits exciton yield in poly(3-hexylthiophene).

Control of chain length and morphology in combination with single-molecule spectroscopy techniques provides a comprehensive photophysical picture of excited-state losses in the prototypical conjugated polymer poly(3-hexylthiophene) (P3HT). Our examination reveals a universal self-quenching mechanism, based on singlet-triplet exciton annihilation, which accounts for the dramatic loss in fluorescence quantum yield of a single P3HT chain between its solution (unfolded) and bulklike (folded) state. Triplet excitons fundamentally limit the fluorescence of organic photovoltaic materials, which impacts the conversion of singlet excitons to separated charge carriers, decreasing the efficiency of energy harvested at high excitation densities. Interexcitonic interactions are so effective that a single P3HT chain of order 100 kDa weight behaves like a 2-level system, exhibiting perfect photon antibunching.

Plasmonic antennas and zero‐mode waveguides to enhance single molecule fluorescence detection and fluorescence correlation spectroscopy toward physiological concentrations

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as Förster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero-mode waveguides (ZMW) and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometer scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET, and FCS. Single molecule spectroscopy techniques greatly benefit from ZMW and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics.

Structure‐Dependent Electronic Nature of Star‐Shaped Oligothiophenes, Probed by Ensemble and Single‐Molecule Spectroscopy

We have investigated the photophysical properties of star-shaped oligothiophenes with three terthiophene arms (meta to each other, S3) or six terthiophene arms (ortho-, meta-, and para-arranged, S6) connected to an ethynylbenzene core to elucidate the relationship between their molecular structure and electronic properties by using a combination of ensemble and single-molecule spectroscopic techniques. We postulate two different conformations for molecules S3 and S6 on the basis of the X-ray structure of hexakis(5-hexyl-2-thienlyethynyl)benzene and suggest the coexistence of these conformers by using spectroscopic methods. From the steady-state spectroscopic data of compound S6, we show that the exciton is delocalized over the core structure, but that the meta-linkage in compound S3 prevents the electronic communication between the arms. However, in single-molecule spectroscopic measurements, we observed that some molecules of compound S3 showed long fluorescence lifetimes (about 1.4 ns) in the fluorescence-intensity trajectories, which indicated that π electrons were delocalized along the meta linker. Based on these observations, we suggest that the delocalized exciton is intensely sensitive towards the dihedral angle between the core and the adjacent thiophene ring, as well as to the substituted position of the terthiophene arms. Our results highlight that the fluorescence lifetimes of compounds S3 and S6 are strongly correlated with the spatial location of their excitons, which is mainly affected by their conformation, that is, whether the innermost thiophene rings are facing each other or not. More interestingly, we observed that the difference between the degrees of ring-torsional flexibility of compounds S3 and S6 results in their sharply contrasting fluorescence properties, such as a change in fluorescence intensity as a function of temperature.

Single molecule detection of nanomechanical motion.

We investigate theoretically how single molecule spectroscopy techniques can be used to perform fast and high resolution displacement detection and manipulation of nanomechanical oscillators, such as singly clamped carbon nanotubes. We analyze the possibility of real time displacement detection by the luminescence signal and of displacement fluctuations by the degree of second order coherence. Estimates of the electromechanical coupling constant indicate that intriguing regimes of strong backaction between the two-level system of a molecule and the oscillator can be realized.

Modeling of Emission Spectra for Molecular Rings - LH2 And LH4 Complexes

Computer simulation of steady state fluorescence spectra of the ring molecular systems (resembling, e.g. the light harvesting rings from LH2 and LH4 photosynthetic complexes of purple bacteria) is presented in this paper. The general organization of the LH2 and LH4 complexes is the same: identical subunits are repeated cyclically in such a way that a ring-shaped structure is formed. However, the symmetries of these rings are different: LH2 is usually nonameric but LH4 is octameric. The other difference is the presence of four bacteriochlorophyll molecules per repeating unit in LH4 rather than three ones found in LH2. Transi- tion dipole moments of bacteriochlorophylls in B850 ring of LH2 have nearly tangential orientation whereas in LH4 they are organized in a more radial fashion. The dynamical aspects in ensemble of rings are reflected in optical line shapes of electronic transitions. The observed linewidths reflect the combined influence of different types of static and dynamic disorder. To avoid the broadening of lines due to ensemble averaging one uses the single-molecule spectroscopy technique to obtain a fluorescence-excitation spectrum. For our simulations we have used the ring of tightly bound two-level systems. Static disorder is taken into account simultaneously with dynamic disorder in Markovian approximation. The cumulant-expansion method of Mukamel et al. is used for the calculation of spectral responses of the system with exciton-phonon coupling. Comparison of steady state fluorescence spectra for B850 ring from LH2 and LH4 ring is done.

Solvent Vapor Annealing of Single Conjugated Polymer Chains: Building Organic Optoelectronic Materials from the Bottom Up.

Optoelectronic devices based on organic materials show a strong relationship between the morphological structure of the material and the function of the device. One of the grand challenges in improving the efficiencies of these devices is hence achieving morphological control throughout the entire course of processing. One of the most important postprocessing methods is solvent vapor annealing, which has repeatedly demonstrated its utility in improving the efficiency of organic-material-based devices by changing bulk-film morphology. This Perspective discusses the recent impact of single-molecule spectroscopy techniques in unraveling morphological changes and molecular dynamics and presents solvent vapor annealing as a tool to build organic optoelectronic materials from the bottom up. In particular, we discuss examples of how solvent vapor annealing at the single-chain level can be split into two different regimes, (i) the solvation regime, in which intrachain interactions and molecular dynamics during solvent vapor annealing can be probed, and (ii) the aggregation regime, in which the influence of interchain interactions can be probed. Finally, it will be shown that solvent vapor annealing in the aggregation regime can be used to build highly ordered mesoscopic objects with distinct properties such as long-range energy transfer.

Enhancing the Sensitivity of Single-Particle Photothermal Imaging with Thermotropic Liquid Crystals.

Individual molecules and nanoparticles can be imaged based on their absorption using photothermal microscopy. This technique relies on the heating-induced changes in the refractive index of the surrounding medium. Here, we demonstrate an order of magnitude larger enhancement of the signal-to-noise ratio in photothermal imaging of 20 nm gold nanoparticles when using a thermotropic liquid crystal (5CB). We show quantitatively that this increase is due to the large change in the thermo-optical properties of 5CB mainly along the nematic director. Enhancing the sensitivity is important for the further development of absorption-based single-molecule spectroscopy techniques.

Single Molecule Studies of Streptavidin-Biotin Dissociation at Elevated Temperatures

Over the past 10 years single molecule spectroscopic techniques have matured and are widely used in the biomolecular sciences. More recently, temperature control has been incorporated, increasing the dimensionality of the possible experimental parameters. However, conventional immobilization techniques rely on biotin-streptavidin linkages to localize single molecules on surfaces. While this ligand binding interaction is one of the strongest non-covalent linkages in nature (KD≈10-14 M) and is extremely stable at room temperature, the kinetics of dissociation at elevated temperatures are poorly understood. Pulsed laser based IR absorbance heating and fluorescence techniques are used to measure the dissociation rate at temperatures well above room temperature. The rate constants of dissociation are found to increase significantly from ∼2x10-5 s-1 at 20°C to ∼1x10-3 s-1 at 55°C. Eyring analysis reveals that the dissociation reaction has a large enthalpic contribution (ΔH‡ = 20 (1) kcal/mol), the magnitude of which is on the order of the previously measured enthalpies of formation. This result indicates that the biotin binding occurs over a nearly barrier-less transition state.

Hole transfer dynamics from dye molecules to p-type NiO nanoparticles: effects of processing conditions

Hole transfer dynamics of Atto647N sensitized p-type NiO nanoparticle (NP) thin films is investigated using both ensemble-averaged and single-molecule spectroscopy techniques. The rate of hole transfer is dependent on the processing conditions and is enhanced when the NiO is pre-annealed in air as compared to vacuum. This is possibly due to an upward shift of the valence band of the semiconductor and an increase in the free energy for hole transfer as more Ni(2)O(3) are formed in the presence of air. The stretched exponential fluorescence decay profile of Atto647N on NiO NP suggests the presence of a distribution of hole transfer rates. This is in agreement with the observed emission lifetime and intensity fluctuations and non-monoexponential fluorescence decays for individual Atto647N molecules on NiO NP films. A plausible explanation for the heterogeneous hole transfer rates is an inhomogeneous distribution of (defect) sites on the metal oxide due to the processing conditions and a fluctuation in the intermolecular interaction.

Single-Photon Emission Behavior of Isolated CdSe/ZnS Quantum Dots Interacting with the Localized Surface Plasmon Resonance of Silver Nanoparticles

To reveal the exciton dynamics, particularly multiexciton dynamics in single colloidal CdSe/ZnS quantum dots (QDs) interacting with the localized surface plasmon resonance (LSPR) of silver nanoparticles (AgNPs), we investigated the single-photon emission behavior in the fluorescence from the single QDs using a single-molecule spectroscopy technique in combination with a femtosecond-pulsed laser excitation. By applying simultaneous measurements of the photon-correlation (photon antibunching), the fluorescence intensity, and the fluorescence lifetime to the single QD near AgNPs, we revealed that the probability of single-photon emission strongly depended on the fluorescence lifetime; that is, the probability of single-photon emission decreased when the lifetime was shorter than subnanoseconds. On the basis of the estimation of both radiative and nonradiative decay rates enhanced by the LSPR, the following mechanism was suggested. In the absence of AgNPs, multiple excitons generated by a high-power excitation lead to single-photon emission via nonradiative Auger recombination process between the excitons. In the presence of AgNPs, even when the excitation power is low, multiple excitons are initially generated in a single QD by the enhanced electromagnetic field of the AgNPs’ LSPR. Subsequently, a portion of these excitons radiatively decay via plasmon, that is, the radiative decay rate enhanced by LSPR competes with the Auger recombination process. When the enhanced radiative decay rate is faster than that of the Auger process, multiphoton emission can be observed. Therefore, a decrease in the probability of single-photon emission is observed when the fluorescence lifetime is shortened. This result will improve our understanding of fluorescence enhancement by the LSPR of metal nanostructures, assist in the creation of effective single-photon sources, and allow for the utilization of the multiexcitons in QDs for optoelectronic applications.

Probing the Role of Chain Length on the Diffusion Dynamics of π-Conjugated Polymers by Fluorescence Correlation Spectroscopy

We investigate the role of the chain length and molecular weight distribution on the diffusion dynamics of freshly synthesized MEH-PPV polymer chains. For the above purpose, a new technique based on combination of size exclusion chromatography (SEC) with fluorescence correlation spectroscopy (FCS) is developed to probe the diffusion dynamics of a narrow molecular weight distribution of fractionated samples of 20-500 kDa. The narrow dispersed samples were characterized by absorbance, emission, and time-resolved fluorescence decay techniques. The results revealed that the properties of fractionated samples were almost uniform for a wide range of molecular weights. A maximum entropy based method for FCS data analysis is employed to obtain the correct diffusion coefficients of the polymer chains with heterogeneous dynamics. The FCS experiment on the unfractionated broad molecular weight sample is not enough to establish the correlation between the molecular weight of the chains with diffusion dynamics and emphasized the need for relatively monodispersed π-conjugated polymers. FCS results show that higher molecular weight chains diffuse much faster than shorter ones. Atomic force microscopy revealed that 300 kDa polymers produced 130 nm particles, whereas 50 kDa polymer chains formed micrometer size aggregates. At higher molecular weights, the strong chain interactions promote the formation of globular (or tightly packed) particles which diffuse faster in solution. The low molecular weight chains experience strong interparticle interaction; as a consequence, the diffusion of chains becomes slower. In the present investigation, we demonstrate the need for the narrow polydisperse sample for establishing the correlation between diffusion dynamics and chain length (or molecular weights) of π-conjugated polymers using a single molecule spectroscopy technique such as FCS.

Single‐Molecule Spectroscopy for Plastic Electronics: Materials Analysis from the Bottom‐Up

pi-conjugated polymers find a range of applications in electronic devices. These materials are generally highly disordered in terms of chain length and chain conformation, besides being influenced by a variety of chemical and physical defects. Although this characteristic can be of benefit in certain device applications, disorder severely complicates materials analysis. Accurate analytical techniques are, however, crucial to optimising synthetic procedures and assessing overall material purity. Fortunately, single-molecule spectroscopic techniques have emerged as an unlikely but uniquely powerful approach to unraveling intrinsic material properties from the bottom up. Building on the success of such techniques in the life sciences, single-molecule spectroscopy is finding increasing applicability in materials science, effectively enabling the dissection of the bulk down to the level of the individual molecular constituent. This article reviews recent progress in single molecule spectroscopy of conjugated polymers as used in organic electronics.