Single‐Molecule Photoactivation FRET: A General and Easy‐To‐Implement Approach To Break the Concentration Barrier

Single‐molecule fluorescence resonance energy transfer (sm‐FRET) has become a widely used tool to reveal dynamic processes and molecule mechanisms hidden under ensemble measurements. However, the upper limit of fluorescent species used in sm‐FRET is still orders of magnitude lower than the association affinity of many biological processes under physiological conditions. Herein, we introduce single‐molecule photoactivation FRET (sm‐PAFRET), a general approach to break the concentration barrier by using photoactivatable fluorophores as donors. We demonstrate sm‐PAFRET by capturing transient FRET states and revealing new reaction pathways during translation using μm fluorophore labeled species, which is 2–3 orders of magnitude higher than commonly used in sm‐FRET measurements. sm‐PAFRET serves as an easy‐to‐implement tool to lift the concentration barrier and discover new molecular dynamic processes and mechanisms under physiological concentrations.     

Photobleaching pathways in single-molecule FRET experiments

To acquire accurate structural and dynamical information on complex biomolecular machines using single-molecule fluorescence resonance energy transfer (sm-FRET), a large flux of donor and acceptor photons is needed. To achieve such fluxes, one may use higher laser excitation intensity; however, this induces increased rates of photobleaching. Anti-oxidant additives have been extensively used for reducing acceptor's photobleaching. Here we focus on deciphering the initial step along the photobleaching pathway. Utilizing an array of recently developed single-molecule and ensemble spectroscopies and doubly labeled Acyl-CoA binding protein and double-stranded DNA as model systems, we study these photobleaching pathways, which place fundamental limitations on sm-FRET experiments. We find that: (i) acceptor photobleaching scales with FRET efficiency, (ii) acceptor photobleaching is enhanced under picosecond-pulsed (vs continuous-wave) excitation, and (iii) acceptor photobleaching scales with the intensity of only the short wavelength (donor) excitation laser. We infer from these findings that the main pathway for acceptor's photobleaching is through absorption of a short wavelength photon from the acceptor's first excited singlet state and that donor's photobleaching is usually not a concern. We conclude by suggesting the use of short pulses for donor excitation, among other possible remedies, for reducing acceptor's photobleaching in sm-FRET measurements.     

Self-assembly dynamics of a cylindrical capsule monitored by fluorescence resonance energy transfer

The constituent cavitands of a cylindrical capsule were labeled with donor and acceptor fluorophores, and fluorescence resonance energy transfer (FRET) was employed as a tool to study the dynamics of self-assembly. When donor and acceptor dyes are present in the same capsular assembly, they are brought within 25 A of each other, a distance suitable for efficient energy transfer to occur between them. This allowed for the study of interacting species at nanomolar concentrations providing information unattainable from NMR experiments. The kinetic stability of the capsule in the presence of various guest molecules was investigated which revealed a range of more than 4 orders of magnitude in the rates of cylindrical capsule exchange. While the thermodynamic stability of the capsule generally dictates the self-assembly dynamics, it was discovered that longer rigid guests can impart a significant kinetic barrier to monomer exchange.     

Determination of gas-phase ozonolysis rate coefficients of a number of sesquiterpenes at elevated temperatures using the relative rate method

The rates of ozonolysis of four sesquiterpenes, β-caryophyllene, α-humulene, isolongifolene and α-cedrene, are determined in the gas phase at an elevated temperature of 366 ± 3 K and a pressure of ~780 Torr using the EXTreme RAnge chamber (EXTRA). The experimentally obtained rate coefficients agree with extrapolated room temperature rate coefficients for isolongifolene and α-cedrene but not for β-caryophyllene and α-humulene, which were found to be three orders of magnitude slower than this in the literature. These new measurements support the hypothesis that operating under ambient conditions, kinetic measurements of condensable species can be influenced adversely by heterogeneous processes and should therefore be treated with caution.     

FRET- and PET-based sensing in a single material: expanding the dynamic range of an ultra-sensitive nitroaromatic explosives assay

A polyethylenimine polymer derivatized with pyrene moieties suitable for the fluorescence-based detection of nitroaromatic explosives (NAC) in aqueous systems is described. The system exhibits an exceptionally wide dynamic sensing range of 7 orders of magnitude (from 33 ppt to 225 ppm TNT or Tetryl). This broad range was achieved by the combination of FRET and PET sensing mechanisms in a single material. The sensing material is suitable for a paper strip assay. Simplicity, selectivity, and the wide dynamic range suggest this material for explosives detection in the field.     

Bleaching-resistant,Near-continuous Single-molecule Fluorescence and FRET Based on Fluorogenic and Transient DNA Binding

Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach enables capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.     

The dynamic nature of amyloid beta (1-40) aggregation

In this paper, we characterize the dynamic nature of the full amyloid beta (1-40) (Aβ (1-40)) aggregates. We labeled the peptide with either 5-carboxytetramethylrhodamine (TAMRA) or with fluorescein-isothiocyanate (FITC). The labeled peptides were mixed after separate fibrillization, and the dynamic changes in the structure of the fibrils were imaged using confocal microscopy. Fluorescence resonance energy transfer (FRET) measurements showed that the Aβ (1-40) peptides detach from and reattach to the fibrils in a biologically relevant timescale (days). With time, the two peptides mix at the molecular level. This process is concentration dependent and occurs primarily in the external parts of the aggregates with a half time between 4 and 7 days. This study shows that the combination of confocal microscopy and FRET analysis is a facile method for studying dynamic processes in supra-molecular aggregates.     

A new approach to the measurement of the momentum densities in solids using an electron microscope

A new approach to the direct determination of electron momentum distributions in solids, using a combination of electron microscopy and electron spectroscopy, is described. The technique is several orders of magnitude more sensitive than currently available ones. The results of measurements on amorphous carbon are presented and used to demonstrate the potential of this new technique.     

Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins.

Detection of F?rster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater F?rster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET.     

Determination of cluster composition in heteroaggregation of binary particle systems by flow cytometry

Cluster composition in aggregation processes of multiple particle species can be dynamically determined by flow cytometry if particle populations are fluorescently labeled. By flow cytometric single particle analysis, aggregates can be characterized according to the exact amount of constituent particles, allowing the detailed and separate quantification of homo- and heteroaggregation. This contribution demonstrates the application of flow cytometry for the experimental detection of heteroaggregation in a binary particle mixture of oppositely charged polystyrene (PS) particles and Rhodamine-B labeled melamine-formaldehyde (MF-RhB) particles. Experiments with different particle concentration, temperature, mixing mode, ionic strength and particle mixing ratio are presented. Aggregation kinetics are enhanced with increasing particle concentration and temperature as well as by increased shear of mixing. These results represent well-known behavior published in previous investigations and validate the performance of flow cytometry for probing heteroaggregation processes. Physical insight with a novel level of detail is gained by the quantification of de- and restabilization phenomena. At low ionic strength, "raspberry"-type aggregates with PS cores are formed by primary heteroaggregation. At moderate particle number ratios, these aggregates are electrostatically destabilized and form more complex aggregates in a secondary heteroaggregation process. At high particle number ratios (> or =50:1), the raspberry-type aggregates are electrostatically restabilized and secondary heteroaggregation is prevented. The dynamic change of aggregate charge was verified by zeta-potential measurements. The elevation of salt concentration over several orders of magnitude retards aggregation dynamics, since attractive interparticle forces are diminished by an electrostatic double layer. This indicates that heteroaggregation induced by attractive interparticle forces is faster than aggregation due to random Brownian motion. Destabilization at high ionic strength is facilitated by charged ions and no longer by MF-RhB coverage. This results in a species independent one step aggregation process.     

On-line reaction monitoring by extractive electrospray ionisation

The design and development of a novel extractive electrospray ionisation (EESI) device for on-line reaction monitoring is described. The EESI apparatus uses a secondary, grounded nebuliser to produce an analyte aerosol and a Venturi pump is then used to transfer a sample of the aerosol to an electrospray source where it is ionised. The EESI apparatus was then tested with a variety of small, organic molecules to assess sensitivity, linearity and dynamic range. The performance of the technique will depend on the mass spectrometer used for the experiments; in the configurations used here it has a usable dynamic range of around 3.5 orders of magnitude with a linear range of around 2.5 orders of magnitude and is capable of analysing species present down to low μg/mL with signal-to-noise ratio greater than 2.5. The use of EESI for reaction monitoring was validated using a series of mock reaction mixtures and then used to monitor the base hydrolysis of ethyl salicylate to salicylic acid.     

Bohmian mechanics with complex action: a new trajectory-based formulation of quantum mechanics

In recent years there has been a resurgence of interest in Bohmian mechanics as a numerical tool because of its local dynamics, which suggest the possibility of significant computational advantages for the simulation of large quantum systems. However, closer inspection of the Bohmian formulation reveals that the nonlocality of quantum mechanics has not disappeared-it has simply been swept under the rug into the quantum force. In this paper we present a new formulation of Bohmian mechanics in which the quantum action, S, is taken to be complex. This leads to a single equation for complex S, and ultimately complex x and p but there is a reward for this complexification-a significantly higher degree of localization. The quantum force in the new approach vanishes for Gaussian wave packet dynamics, and its effect on barrier tunneling processes is orders of magnitude lower than that of the classical force. In fact, the current method is shown to be a rigorous extension of generalized Gaussian wave packet dynamics to give exact quantum mechanics. We demonstrate tunneling probabilities that are in virtually perfect agreement with the exact quantum mechanics down to 10(-7) calculated from strictly localized quantum trajectories that do not communicate with their neighbors. The new formulation may have significant implications for fundamental quantum mechanics, ranging from the interpretation of non-locality to measures of quantum complexity.     

Characterizing Single‐Molecule FRET Dynamics with Probability Distribution Analysis

Probability distribution analysis (PDA) is a recently developed statistical tool for predicting the shapes of single‐molecule fluorescence resonance energy transfer (smFRET) histograms, which allows the identification of single or multiple static molecular species within a single histogram. We used a generalized PDA method to predict the shapes of FRET histograms for molecules interconverting dynamically between multiple states. This method is tested on a series of model systems, including both static DNA fragments and dynamic DNA hairpins. By fitting the shape of this expected distribution to experimental data, the timescale of hairpin conformational fluctuations can be recovered, in good agreement with earlier published results obtained using different techniques. This method is also applied to studying the conformational fluctuations in the unliganded Klenow fragment (KF) of Escherichia coli DNA polymerase I, which allows both confirmation of the consistency of a simple, two‐state kinetic model with the observed smFRET distribution of unliganded KF and extraction of a millisecond fluctuation timescale, in good agreement with rates reported elsewhere. We expect this method to be useful in extracting rates from processes exhibiting dynamic FRET, and in hypothesis‐testing models of conformational dynamics against experimental data.     

Dynamic range optimization in SIMS analyses of arsenic and antimony dopants in silicon

Summary The application of two sample preparation techniques leads to a better understanding of the profile tailing in SIMS analyses of As and Sb distributions measured with Cs primary ions. The dynamic range in the SIMS depth profiles in terms of the detected concentration range is improved nearly by three orders of magnitude on samples with extremely high surface concentrations compared to measurements without preparation. More than five orders of magnitude are obtained. The application of the preparation techniques to As ion implants in silicon reveals a channeling tail below a concentration level of 5·10¹⁶ at/cm³ which is nearly independent of the implanted ion dose if it exceeds 5·10¹² at/cm².     

Autophagy Caught in the Act: A Supramolecular FRET Pair Based on an Ultrastable Synthetic Host–Guest Complex Visualizes Autophagosome–Lysosome Fusion

A supramolecular FRET pair based on the ultrahigh binding affinity between cyanine 3 conjugated cucurbit[7]uril (CB[7]‐Cy3) and cyanine 5 conjugated adamantylamine (AdA‐Cy5) was exploited as a new synthetic tool for imaging cellular processes in live cells. Confocal laser scanning microscopy revealed that CB[7]‐Cy3 and AdA‐Cy5 were intracellularly translocated and accumulated in lysosomes and mitochondria, respectively. CB[7]‐Cy3 and AdA‐Cy5 then formed a host–guest complex, reported by a FRET signal, as a result of the fusion of lysosomes and mitochondria. This observation not only indicated that CB[7] forms a stable complex with AdA in a live cell, but also suggested that this FRET pair can visualize dynamic organelle fusion processes, such as those involved in the degradation of mitochondria through autophagy (mitophagy), by virtue of its small size, chemical stability, and ease of use.     

Simple antilog converter for conventional and flow-injection measurements with ion-selective electrodes

The antilog converter for potentiometric measurements produces an output voltage which is linearly dependent on the concentration of the sensed species over two orders of magnitude. Signals obtained in measurements with chloride, fluoride, potassium and copper ion-selective electrodes range from 1.0 to 10.0 V. The converter is successfully applied for flow-injection potentiometry within one concentration decade but its sensitivity depends on the dispersion in the flow system.     

Reaction kinetics of dye decomposition processes monitored inside a photocatalytic microreactor

The photocatalytic decomposition processes of several kinds of dyes were monitored in real-time, in a TiO(2)-immobilized microcapillary. Their fluorescence spectra were measured directly from the UV-irradiated area. The photocatalytic reactions proceeded two orders of magnitude faster in the microcapillary than in a bulk reaction, and intermediate species were easily observed, due to their high concentrations compared with those of the reactants. Even for molecules that were not originally fluorescent, fluorescence was detected for the reactants or intermediate species of all the molecules studied. Photocatalytic reactions are typically analyzed in terms of pseudo-first-order or Langmuir-Hinshelwood reaction mechanisms, but it was ascertained that all of the dyes investigated in this study decomposed via a multi-step reaction such as a simple multi-step reaction, a self-catalytic reaction, and further, a more complicated reaction, depending on the molecular structure. These reactions were simulated using models based on the reaction kinetics, and reaction mechanisms were assigned to each type of dye. The fact that intermediate species (which are difficult to observe using conventional analytical methods) were successfully detected meant that mechanisms for different dyes could be further clarified.     

Multiscale spectroscopy using a monolithic liquid core waveguide with laterally attached fiber ports

In conventional absorption spectrometers, the range of accessible concentrations of analytes in aqueous solution is significantly limited by the dynamic range of the measurement system. Here we introduce the concept of multiscale spectroscopy allowing extending that range by orders of magnitude within one single device. The concept relies on using multiple light-sample interaction lengths, boosting the accessible concentration range by a particular extension factor. We experimentally implement our concept by a liquid core waveguide having multiple fiber ports side-wise attached to the waveguide, thus probing the light propagating inside the core at predefined distances from the input. This configuration provides three orders of magnitude of interaction length in one device. To verify the concept we exemplarily determine the concentrations of nitrate and of Rhodamine 6G in water, showing one hundred times improved measurement capabilities. The multiscale spectrometer uses the entire sample volume and allows the simultaneous measurement of fluorescence and attenuance. Due to its integrated design and the extended measurements capabilities, we anticipate application of our device in many application-relevant areas such as water quality analysis or environmental science.     

Fluorescence resonance energy transfer in gaseous, mass-selected polyproline peptides

Despite the many successes of mass spectrometry in the analysis of biological samples, the need to better understand the correlation between condensed-phase properties and those of electrospray species remains. In particular, the link between structures in the condensed phase and in the gaseous environment of the mass spectrometer is still elusive. Here, we show that fluorescence resonance energy transfer (FRET) can be used to probe the conformations of gaseous biopolymers which are formed by electrospray ionization (ESI) and manipulated in a quadrupole ion trap mass spectrometer. A rhodamine dye pair suitable for gas-phase FRET is characterized. Both steady state spectra and lifetime measurements are used to monitor energy transfer in a series of dye-labeled polyproline-based peptides. FRET efficiency is explored as a function of peptide chain length and charge state. For the peptide with eight proline repeats, virtually complete energy transfer is observed. For the peptide with 14 proline repeats, energy transfer decreases as the charge state increases, consistent with Coulomb repulsion induced elongation of the peptide backbone. FRET measurements of the longest peptide examined, which has 20 proline repeats, indicates that the peptide adopts a bent configuration. Evidence for multiple conformations present within the ensemble of trapped ions is provided by fluorescence lifetime measurements. Gas-phase FRET measurements promise to be a new route to probe the conformations of large gaseous ions.     

A sensitivity enhanced high-performance liquid chromatography fluorescence method for the detection of nephrotoxic and carcinogenic aristolochic acid in herbal medicines

A new, sensitive and selective HPLC method with fluorescence detector (HPLC-FLD) for the determination of nephrotoxic and carcinogenic aristolochic acid (AA) in herbal medicines by using pre-column derivatization with zinc powder in acetic acid is presented. Variables governing the derivatization reaction, such as the amount of zinc powder and acetic acid, as well as the derivatization time were studied and optimized. An extended linear dynamic range over three orders of magnitude was observed for AA-I and AA-II (R(2)>0.9998). Method accuracy at low, medium and high spiked AA levels determined by the percentage mean deviation was below 4.4% and 7.2% for AA-I and AA-II, respectively. The detection limits of 0.39 ng/mL (AA-I) and 0.52 ng/mL (AA-II) were 2 orders of magnitude lower than those obtained from HPLC-MS or CE-ECD analyses, 3-4 orders of magnitude lower than those from HPLC-UV or CE-UV methods. The developed method has been applied for the determination of AA in herbal medicines. Among the tested samples, Guanmutong had the highest AA concentration (2607.0 microg/g AA-I, 711.2 microg/g AA-II). Comparison studies between HPLC-FLD and HPLC-MS/MS demonstrated that the two methods gave similar quantitative results for the selected herb samples.