Nitric oxide release from a cucurbituril encapsulated NO-donor.

Controlling S-nitrosothiol decomposition, with the consequent release of nitric oxide, is a topic of great research interest. The incorporation of nitrosomercaptopyridine (SNO+) into the cucurbit[7]uril cavity results in a large increase of its nitrosation equilibrium constant. This effect being a consequence of the preferential stabilization of organic cations by the formation of host : guest complexes with CB7 results in a drastic reduction of the SNO+ denitrosation rate constant. Moreover, SNO+ encapsulation also prevents its decomposition yielding disulfide and nitric oxide. The expulsion of SNO+ from the cucurbituril cavity through the application of a chemical stimulus (competitive binding) results in controlled nitric oxide release as was confirmed by using a NO selective electrode.

Nonergodic subdiffusion from transient interactions with heterogeneous partners.

Spatiotemporal disorder has been recently associated to the occurrence of anomalous nonergodic diffusion of molecular components in biological systems, but the underlying microscopic mechanism is still unclear. We introduce a model in which a particle performs continuous Brownian motion with changes of diffusion coefficients induced by transient molecular interactions with diffusive binding partners. In spite of the exponential distribution of waiting times, the model shows subdiffusion and nonergodicity similar to the heavy-tailed continuous time random walk. The dependence of these properties on the density of binding partners is analyzed and discussed. Our work provides an experimentally testable microscopic model to investigate the nature of nonergodicity in disordered media.

Transient subdiffusion from an Ising environment.

We introduce a model in which a particle performs a continuous-time random walk (CTRW) coupled to an environment with Ising dynamics. The particle shows locally varying diffusivity determined by the geometrical properties of the underlying Ising environment, that is, the diffusivity depends on the size of the connected area of spins pointing in the same direction. The model shows anomalous diffusion when the Ising environment is at critical temperature. We show that any finite scale introduced by a temperature different from the critical one, or a finite size of the environment, cause subdiffusion only during a transient time. The characteristic time, at which the system returns to normal diffusion after the subdiffusive plateau depends on the limiting scale and on how close the temperature is to criticality. The system also displays apparent ergodicity breaking at intermediate time, while ergodicity breaking at longer time occurs only under the idealized infinite environment at the critical temperature.

γ-Cyclodextrin modulates the chemical reactivity by multiple complexation.

Multiple complexation by γ-CD has been studied by self-diffusion coefficients (DOSY) and chemical kinetics experiments in which 4-methoxybenzenesulfonyl chloride (MBSC) solvolysis was used as a chemical probe. The addition of a surfactant as a third component to the reaction mixture induced a very complex reactivity pattern that was explained on the basis of multiple complexation phenomena and surfactant self-assembly to form micelles. A cooperative effect that yielded a ternary complex formed by cyclodextrin-surfactant-MBSC was observed. The larger cavity of γ-CD in comparison with ß-CD is responsible for the change from the competitive complexation mechanism predominant with ß-CD to a cooperative/competitive mixed mechanism operating for the larger derivative. The cavity size in γ-CD is large enough to bind two surfactant alkyl chains with a cooperative effect. Water molecules released by the formation of 1:1 host-guest complexes made the cavity more hydrophobic and promoted further inclusion. A reduction in the available volume of the cavity should be considered on binding a second guest.

Nonergodic subdiffusion from Brownian motion in an inhomogeneous medium.

Nonergodicity observed in single-particle tracking experiments is usually modeled by transient trapping rather than spatial disorder. We introduce models of a particle diffusing in a medium consisting of regions with random sizes and random diffusivities. The particle is never trapped but rather performs continuous Brownian motion with the local diffusion constant. Under simple assumptions on the distribution of the sizes and diffusivities, we find that the mean squared displacement displays subdiffusion due to nonergodicity for both annealed and quenched disorder. The model is formulated as a walk continuous in both time and space, similar to the Lévy walk.

Competition between surfactant micellization and complexation by cyclodextrin.

Supramolecular property systems composed of alkyltrimethylammonium surfactants and ß-cyclodextrin were studied by means of a chemical probe. Solvolysis of 4-methoxybenzenesulfonyl chloride (MBSC) was used in the mixed systems with the aim of being able to determine the concentration of uncomplexed cyclodextrin in equilibrium with the micellar system. The surfactants used enabled us to vary the length of the hydrocarbon chain between 6 and 18 carbon atoms. In all cases the existence of a significant concentration of uncomplexed CD was observable in equilibrium with the micellar system. The percentage of uncomplexed cyclodextrin increases both on increasing and decreasing the surfactant alkyl chain length, being minimal for alkyl chains between 10-12 carbon atoms. This behavior is a consequence of two simultaneous processes: complexation of surfactant monomers by the cyclodextrin and surfactant self-assembly to form micellar aggregates. By using Gibbs free energies for micellization and surfactant complexation by ß-CD, we can quantitatively explain the observed behavior.

Influence of changes in water properties on reactivity in strongly acidic microemulsions.

Replacing the counterion in sodium bis(2-ethylhexyl)sulfosuccinate (NaOT, usually known as AOT or Aerosol OT) with H+ (HOT) allows strongly acidic microemulsions to be obtained through the effect of a change in the solvation mechanism of the surfactant, where the Na+...OH2 interaction is displaced by a stronger H+...OH2 interaction. This raises the proportion of water bound to the counterion, which is reflected in the FT-IR spectrum for water trapped in the microemulsion and the 1H NMR spectrum for the hydrogen atoms in the water molecules. In NaOT microemulsions, the resonance signal for hydrogen atoms in the water molecules increases from delta approximately 3.9 ppm at W = 2 (with W = [H2O]/[NaOT]) to delta approximately 4.8 ppm at W = 50. In HOT microemulsions, the disparate strength of Na+...OH2 and H+...OH2 interactions results in a decrease in the resonance signal for the hydrogen atoms in the water molecules from delta approximately 8.6 ppm at W = 2 to delta approximately 4.9 ppm at W = 50. These changes in the physical properties of water alter chemical reactivity in a way that is clearly apparent in solvolytic processes in NaOT and HOT microemulsions. Thus, the rate constants of reactions involving an associative mechanism increase with decreasing W in NaOT microemulsions, but decrease with decreasing W in HOT microemulsions. The disparate behavior is a result of a decreased nucleophilicity of interfacial water in HOT microemulsions relative to NaOT microemulsions. For a dissociative process the rate constants are greater in HOT microemulsions than in NaOT ones, and increase with increasing W in both types of microemulsions, which can be ascribed to an increased electrophilicity of interfacial water in HOT microemulsions.

Determination of pyridine-2-azo-p-dimethylaniline acidity constants by spectra resolution methodology.

The influence of acidity upon the pyridine-2-azo-p-dimethylaniline (PADA) absorption spectrum has been studied. The obtained results allowed us to calculate the acidity constants of PADA. The spectra resolution method has been used to determinate the constants. The absorption spectrum was decomposed in two sub-bands for the neutral form of the indicator, one for the monoprotonated molecule and another more for the diprotonated structure. The quantitative analysis of relative areas variation with the medium acidity allows us to obtain the equilibrium constants of PADA prolongation. The obtained values are in good agreement with the values reported in the literature.

Single-molecule pump-probe experiments reveal variations in ultrafast energy redistribution.

Single-molecule pump probe (SM2P) is a novel, fluorescence-based technique that allows the study of ultrafast processes on the single-molecule level. Exploiting SM2P we have observed large variations (from 1 ps to below 100 fs) in the energy redistribution times of chemically identical molecules in the same sample. Embedding the molecules in a different matrix or changing the excitation wavelength does not lead to significant changes in the average redistribution time. However, chemically different molecules exhibit different characteristic redistribution times. We therefore conclude that the process measured with the SM2P technique is dominated by intramolecular energy redistribution and not intermolecular transfer to the surrounding matrix. The matrix though is responsible for inducing conformational changes in the molecule, which affect the coupling between electronic and vibrational modes. These conformational changes are the main origin of the observed broad distribution of redistribution times.

Monolayer-functionalized microfluidics devices for optical sensing of acidity.

This paper describes the integration of opto-chemosensors in microfluidics networks. Our technique exploits the internal surface of the network as a platform to build a sensing system by coating the surface with a self-assembled monolayer and subsequently binding a fluorescent sensing molecule to the monolayer. Fluorescent molecules were used that can switch between a fluorescent and a non-fluorescent state, depending on the acidity of the surrounding solution. Two systems were investigated. The first employs surface confinement of a Rhodamine B dye in a glass micro channel that serves as a molecular switch in organic solutions. Upon rinsing the micro channels with acidic or basic solutions it was possible to switch between the fluorescent and non-fluorescent forms reversibly. Moreover, this system could be used to monitor the mixing of two solutions of different acidity along the micro channel. To widen the scope of optical sensing in micro channels an Oregon Green dye derivative was immobilized, which functions as a sensing molecule for pH differences in aqueous solutions. In this case, a hybrid system was used consisting of a glass slide and PDMS channels. The fluorescence intensity was found to be directly correlated to the pH of the solution in contact, indicating the possibility of using such a system as a pH sensor. These systems allow real-time measurements and can be easily implemented in micro- and nanofluidics systems thus enabling analysis of extremely small sample volumes in a fast and reproducible manner.

Single molecule photobleaching probes the exciton wave function in a multichromophoric system.

The exciton wave function of a trichromophoric system is investigated by means of single molecule spectroscopy at room temperature. Individual trimers exhibit superradiance and loss of vibronic structure in emission spectrum, features proving exciton delocalization. We identify two distinct photodegradation pathways for single trimers upon sequential photobleaching of the chromophores. The rate of each pathway is a measure for the contribution of the separate dyes to the collective excited state of the system, in this way probing the wave function of the delocalized exciton.

Near-field scanning optical microscopy in liquid for high resolution single molecule detection on dendritic cells.

Clustering of cell surface receptors into micro-domains in the plasma membrane is an important mechanism for regulating cellular functions. Unfortunately, these domains are often too small to be resolved with conventional optical microscopy. Near-field scanning optical microscopy (NSOM) is a relatively new technique that combines ultra high optical resolution, down to 70 nm, with single molecule detection sensitivity. As such, the technique holds great potential for direct visualisation of domains at the cell surface. Yet, NSOM operation under liquid conditions is far from trivial. In this contribution, we show that the performance of NSOM can be extended to measurements in liquid environments using a diving bell concept. For the first time, individual fluorescent molecules on the membrane of cells in solution are imaged with a spatial resolution of 90 nm. Furthermore, using this technique we have been able to directly visualise nanometric sized domains of the C-type lectin DC-SIGN on the membrane of dendritic cells, both in air and in liquid.

Cell biology beyond the diffraction limit: near-field scanning optical microscopy.

Throughout the years, fluorescence microscopy has proven to be an extremely versatile tool for cell biologists to study live cells. Its high sensitivity and non-invasiveness, together with the ever-growing spectrum of sophisticated fluorescent indicators, ensure that it will continue to have a prominent role in the future. A drawback of light microscopy is the fundamental limit of the attainable spatial resolution--approximately 250 nm--dictated by the laws of diffraction. The challenge to break this diffraction limit has led to the development of several novel imaging techniques. One of them, near-field scanning optical microscopy (NSOM), allows fluorescence imaging at a resolution of only a few tens of nanometers and, because of the extremely small near-field excitation volume, reduces background fluorescence from the cytoplasm to the extent that single-molecule detection sensitivity becomes within reach. NSOM allows detection of individual fluorescent proteins as part of multimolecular complexes on the surface of fixed cells, and similar results should be achievable under physiological conditions in the near future.

The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection.

Recent studies on the newly cloned red fluorescence protein DsRed from the Discosoma genus have shown its tremendous advantages: bright red fluorescence and high resistance against photobleaching. However, it has also become clear that the protein forms closely packed tetramers, and there is indication for incomplete protein maturation with unknown proportion of immature green species. We have applied single-molecule methodology to elucidate the nature of the fluorescence emission in the DsRed. Real-time fluorescence trajectories have been acquired with polarization sensitive detection. Our results indicate that energy transfer between identical monomers occurs efficiently with red emission arising equally likely from any of the chromophoric units. Photodissociation of one of the chromophores weakly quenches the emission of adjacent ones. Dual color excitation (at 488 and 568 nm) single-molecule microscopy has been performed to reveal the number and distribution of red vs. green species within each tetramer. We find that 86% of the DsRed contain at least one green species with a red-to-green ratio of 1.2-1.5. On the basis of our findings, oligomer suppression would not only be advantageous for protein fusion but will also increase the fluorescence emission of individual monomers.

Near-field effects in single molecule emission.

We present the first experimental proof of the influence of a nearby nano-sized metal object on the angular photon emission by a single molecule. A novel angular sensitive detection scheme is implemented in an existing near-field scanning optical microscope (NSOM). The positioning accuracy ( approximately 1 nm) of the NSOM allows a systematic investigation of the intensity ratio between two different half-spaces as a function of the position of the metal-glass interfaces of the probe with respect to the single emitter. The observed effects are shown to be particularly strong for molecules that are excited mainly below the rims of the aperture. An excellent agreement is found between experiments and numerical simulations for these molecules. The observed angular redistribution of the angular emission of a single molecule could explain the alteration of the emission polarization observed for certain molecules in earlier experiments (Veerman et al. (1999) J. Microsc. 194, 477-482).

Moulded photoplastic probes for near-field optical applications.

The inexpensive fabrication of high-quality probes for near-field optical applications is still unsolved although several methods for integrated fabrication have been proposed in the past. A further drawback is the intensity loss of the transmitted light in the 'cut-off' region near the aperture in tapered optical fibres typically used as near-field probes. As a remedy for these limitations we suggest here a new wafer-scale semibatch microfabrication process for transparent photoplastic probes. The process starts with the fabrication of a pyramidal mould in silicon by using the anisotropic etchant potassium hydroxide. This results in an inverted pyramid limited by < 111 > silicon crystal planes having an angle of approximately 54 degrees. The surface including the mould is covered by a approximately 1.5 nm thick organic monolayer of dodecyltrichlorosilane (DTS) and a 100-nm thick evaporated aluminium film. Two layers of photoplastic material are then spin-coated (thereby conformal filling the mould) and structured by lithography to form a cup for the optical fibre microassembly. The photoplastic probes are finally lifted off mechanically from the mould with the aluminium coating. Focused ion beam milling has been used to subsequently form apertures with diameters in the order of 80 nm. The advantage of our method is that the light to the aperture area can be directly coupled into the probe by using existing fibre-based NSOM set-ups, without the need for far-field alignment, which is typically necessary for cantilevered probes. We have evidence that the aluminium layer is considerably smoother compared to the 'grainy' layers typically evaporated on free-standing probes. The optical throughput efficiency was measured to be about 10-4. This new NSOM probe was directly bonded to a tuning fork sensor for the shear force control and the topography of a polymer sample was successfully obtained.

Optical probing of single fluorescent molecules and proteins.

Single-molecule detection and analysis of organic fluorescent molecules and proteins are presented, with emphasis on the underlying principles, methodology and the application of single-molecule analysis at room temperature. This Minireview is mainly focused on the application of confocal and near-field optical microscopy to investigate the photodynamics of individual molecules embedded in ultrathin polymer layers. We discuss rotational mobility of individual probe molecules in polystyrene and poly(methylmethacrylate) thin films, fluorescence lifetime trajectories and their spatial distribution, and real-time singlet-triplet dynamics. As a whole, the single-molecule photodynamics observed is due to the dynamic nature of both polymers at room temperature, where local polymer conformational dynamics modulates the oxygen concentration and diffusion on a molecular scale, influencing the fluorescence lifetime and intersystem crossing parameters. We also discuss the photodynamics of individual autofluorescent proteins, in particular the on/off blinking and the apparent stability of the protein against bleaching. These studies illustrate the unique information obtainable with the single-molecule approach, information that is otherwise hidden in ensemble-averaged measurements.

Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules.

Real-time single-molecule fluorescence detection using confocal and near-field scanning optical microscopy has been applied to elucidate the nature of the "on-off" blinking observed in the Ser-65 --> Thr (S65T) mutant of the green fluorescent protein (GFP). Fluorescence time traces as a function of the excitation intensity, with a time resolution of 100 micros and observation times up to 65 s, reveal the existence of a nonemissive state responsible for the long dark intervals in the GFP. We find that excitation intensity has a dramatic effect on the blinking. Whereas the time during which the fluorescence is on becomes shorter as the intensity is increased, the off-times are independent of excitation intensity. Statistical analysis of the on- and off-times renders a characteristic off-time of 1.6 +/- 0.2 s and allows us to calculate a transition yield of approximately 0.5 x 10(-5) from the emissive to the nonemissive state. The saturation excitation intensity at which on- and off-times are equal is approximately 1.5 kW/cm(2). On the basis of the single-molecule data we calculate an absorption cross section of 6.5 x 10(-17) cm(2) for the S65T mutant. These results have important implications for the use of the GFP to follow dynamic processes in time at the single-molecular level.

Influencing the angular emission of a single molecule.

We present the first experimental proof for the influence of a nearby nanosized metal object on the angular photon emission by a single molecule. Using a novel angular sensitive detection scheme, we directly quantify the redirection of angular emission for different molecular dipole orientations as an object is scanned laterally over the molecule at different heights. An excellent agreement between experiments and 2D-numerical simulations is found for molecules oriented perpendicular to the sample, whereas, for parallel orientations, the observed behavior contradicts the calculated behavior.

Visualising individual green fluorescent proteins with a near field optical microscope.

The use of the green fluorescence protein (GFP) as an individual marker for applications in molecular biology requires detailed understanding of its photophysical and photodynamical properties. We investigated individual S65T mutants of GFP both on a glass surface and embedded in a water-pore gel. An aperture-type near field scanning optical microscope (NSOM) with two polarisation detection channels was applied to afford high spatial (approximately 70 nm) and temporal (0.5 ms) resolution. Shear-force and near field fluorescence imaging were performed simultaneously, allowing direct correlation between topographic and optical features. Polarisation data showed that the emission dipole moment of the proteins is fixed in space within both the barrel structure of the protein and the gel matrix used for spatial confinement of the proteins. The photophysical behaviour of the S65T-GFP mutants was monitored in time, with 500-micros real-time resolution and continuous imaging for periods of more than 2 h. Our results show the reversible on-off behaviour on a time scale that spans from 10(-4) to 10(3) s. Even a process generally identified as "bleaching" turns out to be reversible if a sufficient long observation time is allowed. As such, the photodynamics of individual GFPs appear to be much more complex than the properties deduced from ensemble-averaged measurements.