Single-molecule Fluorescence Intensity Research Articles

Symmetry-Breaking Charge Separation Mediated Triplet Population in a Perylenediimide Trimer at the Single-Molecule Level.

Herein, we demonstrate triplet excited-state population in a conformationally rigid perylenediimide trimer (PDI-T) via intramolecular symmetry-breaking charge separation (SB-CS) at the single-molecule level. The single-molecule fluorescence intensity trajectories of PDI-T in nonpolar polystyrene matrix (ε = 2.60) exhibit prolonged fluorescence with infrequent dark states, representing the triplet and/or the charge transfer states. In contrast, in a poly(vinyl alcohol) matrix (ε = 7.80), erratic blinking dynamics resulting in low photon counts were observed, corroborating the feasibility of charge separation in a polar environment. In agreement with the single-molecule measurements, transient absorption spectroscopy of PDI-T reveals ultrafast SB-CS (τCS < 5 ps) in polar tetrahydrofuran (ε = 7.58) and acetone (ε = 20.70), with the population of the triplet excited-state through charge recombination. The current investigation shows the utility of rigid and weakly coupled molecular constructs in controlling triplet generation and SB-CS for potential applications in optoelectronic devices.

Breakage of the oligomeric CaMKII hub by the regulatory segment of the kinase.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric enzyme with crucial roles in neuronal signaling and cardiac function. Previously, we showed that activation of CaMKII triggers the exchange of subunits between holoenzymes, potentially increasing the spread of the active state (Stratton et al., 2014; Bhattacharyya et al., 2016). Using mass spectrometry, we show now that unphosphorylated and phosphorylated peptides derived from the CaMKII-α regulatory segment bind to the CaMKII-α hub and break it into smaller oligomers. Molecular dynamics simulations show that the regulatory segments dock spontaneously at the interface between hub subunits, trapping large fluctuations in hub structure. Single-molecule fluorescence intensity analysis of CaMKII-α expressed in mammalian cells shows that activation of CaMKII-α results in the destabilization of the holoenzyme. Our results suggest that release of the regulatory segment by activation and phosphorylation allows it to destabilize the hub, producing smaller assemblies that might reassemble to form new holoenzymes.

Interplay of Nanoparticle Resonance Frequency and Array Surface Coverage in Live-Cell Plasmon-Enhanced Single-Molecule Imaging

Super-resolution imaging has provided new insights into nanoscale optics. Plasmonic gold nanotriangle arrays created by nanosphere lithography can enhance single-molecule fluorescence intensity to further improve imaging. Here, gold nanotriangle arrays on glass coverslips were used as inexpensive, facile, and broadly applicable imaging substrates for living Vibrio cholerae cells expressing photoactivatable fluorescent proteins—the red PAmCherry or the green PAGFP—and resulted in fluorescence enhancements upon coupling living cells to nanotriangle arrays. Within the requirements for this wide-field coupling geometry, we analyze and optimize the coupling as a function of local surface plasmon resonance frequency and particle coverage.

Probing single-molecule electron-hole transfer dynamics at a molecule-NiO semiconductor nanocrystalline interface.

Interfacial charge transfer dynamics in dye-sensitized NiO nanoparticles are being investigated for photocathodes in p-type dye-sensitized solar cells. In the photoreaction, after fast electron transfer from NiO to a molecule, the recombination of the hole in the nanoparticles with the electron in a reduced molecule plays an important role in the charge separation process and solar energy harvesting. Nevertheless, knowledge of the interfacial charge recombination (CR) rate and its mechanism is still limited due to the complex photoinduced electron and hole dynamics and lack of characterization of the inhomogeneity of the dynamics. Here, we report our work on probing interfacial charge recombination dynamics in Zn(ii)-5,10,15,20-tetra(3-carboxyphenyl)porphyrin (m-ZnTCPP) dye-sensitized NiO nanoparticles by correlating single-molecule fluorescence blinking dynamics with charge transfer dynamics using single-molecule photon-stamping spectroscopy. The correlated analyses of single-molecule fluorescence intensity, lifetime, and blinking reveal the intrinsic distribution and temporal fluctuation of interfacial charge transfer reactivity, which are closely related to site-specific molecular interactions and dynamics.

Quantifying the Distribution of the Stoichiometric Composition of Anticancer Peptide Lycosin-I on the Lipid Membrane with Single Molecule Spectroscopy.

Lycosin-I, a peptide toxin derived from spider venom, has been demonstrated to be a promising candidate for the inhibition of tumor cell growth in vitro and in vivo by interacting with and penetrating the cell membrane. Owing to the shortage of an efficient characterization strategy, however, there is still a lack of detailed knowledge about the distribution of the stoichiometric composition information on this peptide in solution and on lipid membrane prior to the cellular uptake process, which is fundamentally important for the understanding of the anticancer mechanism. In this work, with objective-type total internal reflection fluorescence microscopy (TIRF), the distribution of the stoichiometric composition of lycosin-I in different solutions as well as on the lipid membrane was explored extensively on the basis of a statistical single molecule fluorescence intensity analysis for the first time. It was found that lycosin-I is mainly present in a monomer state in diverse physiological solutions regardless of the concentration of the peptide and the incubation time. However, on the lipid membrane, the fraction of small size oligomers increased as a function of time. Fusion of movable peptide molecules to those peptide oligomers with restricted motion on the lipid membrane was also observed.

A novel drug delivery system of gold nanorods with doxorubicin and study of drug release by single molecule spectroscopy

The work presented here describes the fabrication of a novel drug delivery system, which consists of gold nanorods and doxorubicin, with the attachment of thioctic acid and folic acid, for the targeted release of drug to cancer cells. Doxorubicin, the potent anticancer drug, is widely used to treat various cancers. Gold nanorods were functionalized chemically to generate active groups for the attachment of drug molecules and subsequently attached to folic acid. The resulting nanostructure was characterized by UV-visible-NIR spectrophotometry, TEM techniques, zeta potential measurement and subsequently used to target folate receptor-expressing cancers cells for the delivery of doxorubicin. We generated a release profile for the release of doxorubicin from the nanostructures in KB cells using single-molecule fluorescence intensity images and fluorescence lifetime images. The results indicated that the nanorods were able to enter the target cells because of the attachment of folic acid and used as a carriers for the targeted delivery of doxorubicin.

Single-Molecule Interfacial Electron Transfer Dynamics of Porphyrin on TiO2 Nanoparticles: Dissecting the Complex Electronic Coupling Dependent Dynamics

The photosensitized interfacial electron transfer (ET) dynamics of the zinc(II)–5,10,15,20-tetra(3-carboxyphenyl)porphyrin (m-ZnTCPP)–TiO2 nanoparticle (NP) system has been studied using single-molecule photon-stamping spectroscopy. The single-molecule fluorescence intensity trajectories of m-ZnTCPP on TiO2 NP surface show fluctuations and blinking between bright and dark states, which are attributed to the variations in the reactivity of interfacial ET, i.e., intermittent interfacial electron transfer dynamics. Comparing the results with that from our earlier studied p-ZnTCPP–TiO2 nanoparticle system, we show the effect of anchoring group binding geometry (meta or para), hence electronic coupling of sensitizer (m-/p-ZnTCPP) and TiO2 substrate, on interfacial ET dynamics. Compared to p-ZnTCPP on TiO2 NP surface, with m-ZnTCPP, dark states are observed to dominate in single-molecule fluorescence intensity trajectories. This observation coupled with the large difference in lifetime derived from bright and dark states of m-ZnTCPP demonstrates higher charge injection efficiency of m-ZnTCPP than p-ZnTCPP. The nonexponential autocorrelation function decay and the power-law distribution of the dark-time probability density provide a detailed characterization of the inhomogeneous interfacial ET dynamics. The distribution of autocorrelation function decay times (τ) and power-law exponents (mdark) for m-ZnTCPP are found to be different from those for p-ZnTCPP, which indicates the sensitivity of τ and mdark on the molecular structure, molecular environment, and molecule–substrate electronic coupling of the interfacial electron transfer dynamics. Overall, our results strongly suggest that the fluctuation and even intermittency of excited-state chemical reactivity are intrinsic and general properties of molecular systems that involve strong molecule–substrate interactions.

Single-molecule enzymatic conformational dynamics: spilling out the product molecules.

Product releasing is an essential step of an enzymatic reaction, and a mechanistic understanding primarily depends on the active-site conformational changes and molecular interactions that are involved in this step of the enzymatic reaction. Here we report our work on the enzymatic product releasing dynamics and mechanism of an enzyme, horseradish peroxidase (HRP), using combined single-molecule time-resolved fluorescence intensity, anisotropy, and lifetime measurements. Our results have shown a wide distribution of the multiple conformational states involved in active-site interacting with the product molecules during the product releasing. We have identified that there is a significant pathway in which the product molecules are spilled out from the enzymatic active site, driven by a squeezing effect from a tight active-site conformational state, although the conventional pathway of releasing a product molecule from an open active-site conformational state is still a primary pathway. Our study provides new insight into the enzymatic reaction dynamics and mechanism, and the information is uniquely obtainable from our combined time-resolved single-molecule spectroscopic measurements and analyses.

A multi-property fluorescent probe for the investigation of polymer dynamics near the glass transition

AbstractIn addition to the commonly observed single molecule fluorescence intensity fluctuations due to molecular reorientation dynamics, a perylene bisimide-calixarene compound (1) shows additional on-off fluctuations due to its ability to undergo intramolecular excited state electron transfer (PET). This quenching process is turned on rather sharply when a film of poly(vinylacetate) containing 1 is heated above its glass transition temperature (T g), which indicates that the electron transfer process depends on the availability of sufficient free volume. Spatial heterogeneities cause different individual molecules to reach the electron transfer regime at different temperatures, but these heterogeneities also fluctuate in time: in the matrix above T g molecules that are mostly nonfluorescent due to PET can become fluorescent again on timescales of seconds to minutes.The two different mechanisms for intensity fluctuation, rotation and PET, thus far only observed in compound 1, make it a unique probe for the dynamics of supercooled liquids.

Combined Single Molecule Force and Fluorescence Spectroscopy of the Unfolding and Refolding of Green Fluorescent Protein

We have used optical tweezers to study the free energy surface for unfolding-refolding of the green fluorescent protein (EGFP) and simultaneously monitored the loss and recovery of its fluorescence. This conformational landscape shows unfolding intermediates, molten globe refolding intermediates, as well as misfolded states. As force is used to drive transitions between conformational substates, single molecule fluorescence is probed. In its native state, the emission of EGFP is punctuated by transient dark states (blinking); this emission is lost and regained as the protein is unfolded and subsequently refolded. These results provide a full understanding of the unfolding-refolding energy landscape of EGFP and how the conformational state affects the environment of the fluorophore, which reconciles three classes of previous experiments: (1) bulk unfolding/refolding with fluorescence (2) single molecule force-unfolding and (3) single molecule fluorescence intensity fluctuations. This investigation is relevant to efforts at developing EGFP as a genetically encoded force sensor, as well as its use in single molecule imaging and fluorescence recovery after photobleaching experiments.

Probing redox proteins on a gold surface by single molecule fluorescence spectroscopy

The interaction between the fluorescently labeled redox protein, azurin, and a thin gold film is characterized using single-molecule fluorescence intensity and lifetime measurements. Fluorescence quenching starts at distances below 2.3 nm from the gold surface. At shorter distances the quantum yield may decrease down to fourfold for direct attachment of the protein to bare gold. Outside of the quenching range, up to fivefold enhancement of the fluorescence is observed on average with increasing roughness of the gold layer. Fluorescence-detected redox activity of individual azurin molecules, with a lifetime switching ratio of 0.4, is demonstrated for the first time close to a gold surface.

In Vitro Motility Assay Studies at Low [MgATP] - Evidence For Inter-Head Cooperativity in Fast Skeletal Myosin II

The idea that fast skeletal myosin II exhibits processivity with sequential actions of the two myosin heads during muscle contraction has long been a topic of discussion but with appreciable difficulties to obtain conclusive experimental results. The difficulties may be related to a limited processivity (few sequential head actions) that is of greatest importance at low velocity (e.g. high resistive load), difficult to study accurately in vitro. In order to aid the investigations we here make use of recent evidence that the bipyridine drug amrinone inhibits the strain-dependent ADP-release step of myosin II with expected enhancement of processivity (Albet-Torres et al., J Biol Chem., 2009); Månsson, Biophys J., 2010). For accurate velocity measurements, the recombinant expressed capping protein CapZ (Soeno et. al., J Muscle Res Cell Motil., 1998) was fluorescence labeled. Nanometer tracking was achieved by two-dimensional Gaussian fits to single molecule fluorescence intensity profiles representing CapZ attached to the trailing actin filament end. Importantly, the heavy meromyosin (HMM) propelled actin filament sliding velocity (1mM MgATP) for CapZ-capped filaments was comparable to that of uncapped filaments at different ionic strengths and HMM surface densities. Studies at low [MgATP] (5-30μM) suggests a non-linearity in the [MgATP]-velocity plot that was enhanced by 1mM amrinone. The result is in contrast to the linearity expected for independent myosin heads and could be interpreted as an apparent velocity dependence of the myosin step length. This is in accordance with the idea of limited processivity of myosin II if it is assumed that a doubled apparent step length corresponds to sequential action of the two heads. Further insight into the mechanism will be obtained using proteolytically prepared one-headed HMM and e.g. studies with external loads on actin.

Single-molecule charge transfer dynamics in dye-sensitized p-type NiO solar cells: influences of insulating Al2O3layers

In this study, we investigated interfacial charge transfer dynamics in water-soluble perylenediimide (WS-PDI) dye sensitized p-type semiconductor NiO nanoparticle films to better understand how molecular interactions influence photoconversion processes involved in solar cells by means of ensemble-averaged and single-molecule spectroscopies. Transient absorption data showed that strong and weak electronic couplings coexist between WS-PDI molecules and NiO nanoparticles, resulting in fast (within several picoseconds) and slow (requiring tens of picoseconds to nanoseconds) hole transfer from the excited dye to NiO, followed by charge recombination occurring at pico- to microsecond time scales. The correlated analyses of single-molecule fluorescence intensity, lifetime, blinking, and anisotropy revealed the intrinsic distribution and temporal fluctuation of interfacial charge transfer reactivity, which are closely related to site-specific molecular interactions and dynamics. It was also found that a suitable insulating Al2O3 layer can weaken the electronic interaction between WS-PDI and NiO, thereby retarding charge recombination and significantly enhancing photoelectric conversion efficiency. The results presented here will provide a reliable basis for design of highly efficient p-type solar cells and other molecule/semiconductor systems for their use in optoelectronic and solar energy applications.

Single-molecule fluorescence dynamics of a butadiyne-linked porphyrin dimer: the effect of conformational flexibility in host polymers

We have investigated the single-molecule fluorescence dynamics of a butadiyne-linked porphyrin dimer (Z2B) depending on the density of the poly(methyl methacrylate) (PMMA) matrix (5, 10, 25, and 50 mg ml(-1)). By recording single-molecule fluorescence intensity trajectories and fluorescence lifetimes, we observed more frequent one-step photobleaching behavior, less frequent on-off behavior, and narrower fluorescence lifetime distributions in lower densities of PMMA polymer. In contrast, more enhanced photostability was observed in higher densities of PMMA polymer. These results are explained by a difference in the molecular surroundings depending on the change in PMMA polymer density, suggesting that the individual photophysical properties of Z2B are strongly associated with their conformations and molecular surroundings in the solid state. Our studies will provide further information on the structure/surroundings relationship of single molecules in the solid state.

Two-Photon Fluorescence Imaging of Single Fluorescent Proteins Inside Mammalian Cells

Exciting progress has been made recently in biophysical techniques that allow optical imaging of single molecules in live cells. However, imaging of single fluorescent proteins in live cells is usually limited to bacteria that are small in size or proteins that are on the cell surface, where excitation through total internal reflection can be used to reduce fluorescence background and achieve single-molecule sensitivity. Eukaryotic cells are typically larger in size with higher autofluorescence background. To achieve single-molecule sensitivity deep inside a eukaryotic cell is thus challenging. We have used two-photon fluorescence excited by infrared lasers to reduce autofluorescence background. We show that single green fluorescent proteins can be imaged deep inside a mammalian cell using two-photon fluorescence. Discrete stepwise photobleaching of enhanced green fluorescent proteins was observed. The single-molecule fluorescence intensity analysis and on-time distribution indicate that two-photon fluorescence is detectable at the single-molecule level deep inside a eukaryotic cell. Moreover, it is not necessary to use a pulsed laser for two-photon fluorescence excitation. A continuous-wave laser that is routinely used for optical tweezers can be used to excite two-photon fluorescence and achieve single-molecule sensitivity. These advantages could significantly benefit future application of this single-molecule technique in biological studies. We present applications of our technique, and demonstrate two-photon fluorescence imaging of eukaryotic cells at single-molecule level.

Single-molecule detection using continuous wave excitation of two-photon fluorescence

Two-photon fluorescence (TPF) is one of the most important discoveries for biological imaging. Although a cw laser is known to excite TPF, its application in TPF imaging has been very limited due to the perceived low efficiency of excitation. Here we directly excited fluorophores with an IR cw laser used for optical trapping and achieved single-molecule fluorescence sensitivity: discrete stepwise photobleaching of enhanced green fluorescent proteins was observed. The single-molecule fluorescence intensity analysis and on-time distribution strongly indicate that a cw laser can generate TPF detectable at the single-molecule level, and thus opens the door to single-molecule TPF imaging using cw lasers.

Excimer Formation Dynamics of Intramolecular π-Stacked Perylenediimides Probed by Single-Molecule Fluorescence Spectroscopy

Pi-stacked perylenediimides (PDIs) have strong electronic communication between the individual molecules and show great promise as organic electronic materials for applications in field effect transistors, photovoltaics, and liquid crystal displays. To gain further insight into the relationship between conformational behaviors and electronic structures of pi-stacked PDIs, we have investigated changes in the excimer-like state of cofacial PDI oligomers that result from pi-stacking in real time by monitoring the single-molecule fluorescence intensity and lifetime trajectories in a PMMA polymer matrix. The fluorescence intensity and lifetime of pi-stacked perylenediimides are sensitive to the degree of pi-orbital interactions among PDI units, which is strongly associated with molecular conformations in the polymer matrix. Furthermore, our results can be applied to probe the conformational motions of biomolecules such as proteins.

Nanometer resolution imaging by single molecule switching.

The fluorescence intensity of single molecules can change dramatically even under constant laser excitation. The phenomenon is frequently called as ‘blinking,’ and involves molecules switching between high- and low-intensity states (1). In addition to spontaneous blinking, the fluorescence of some special fluorophores, such as cyanine dyes and photoactivatable fluorescent proteins, can be switched on and off, by choice, using a second laser. Recent single-molecule spectroscopy investigations have shed light on mechanisms of single-molecule blinking and photoswitching. This ability to controllably switch single molecules led to the invention of a novel fluorescence microscopy with nanometer spatial resolution well beyond the diffraction limit. (Published: 2 April 2010) Citation: Nano Reviews 2010, 1: 5122 - DOI: 10.3402/nano.v1i0.5122

Fluorescence Dynamics of Chlorophyll Trefoils in the Solid State Studied by Single-Molecule Fluorescence Spectroscopy

We have comparatively investigated the single-molecule photophysical properties of two chlorophyll trefoils that feature distinctive electronic couplings induced by differences in linkage: one is an ethynyl-linked chlorophyll trefoil (1) in which relatively short and rigid linkage between the chromophores promotes effective electronic coupling, and the other is a phenyl−ethynyl-linked chlorophyll trefoil (2) in which the phenyl addition induces an orthogonal geometry impeding π-conjugation and provides a longer interchlorophyll distance reducing through-space interaction. By recording single-molecule fluorescence intensity trajectories and their corresponding lifetimes, we observed one-step photobleaching behaviors, less frequent on−off behaviors, a narrower fluorescence lifetime distribution, and higher photostability in 1 as compared with 2. These results indicate that the performance of molecular photosynthetic systems in the solid state is strongly associated with electronic couplings and, thus, give ...

Protein dynamics from single-molecule fluorescence intensity correlation functions

Fluorescence intensity correlation functions contain information about photophysical and conformational dynamics. We propose and implement a simple procedure to analyze such functions measured in the presence of resonance energy transfer. When there is a separation of time scales and the conformational dynamics is modeled as diffusion in the potential of mean force along the interdye distance, we obtain an analytic expression for the conformational correlation time. This can be used to find the diffusion coefficient describing conformational fluctuations given the photon count rate and equilibrium distribution.