The glue proteins secreted by marine mussels bind strongly to virtually all inorganic and organic surfaces in aqueous environments in which most adhesives function poorly. Studies of these functionally unique proteins have revealed the presence of the unusual amino acid 3,4-dihydroxy-L-phenylalanine (dopa), which is formed by posttranslational modification of tyrosine. However, the detailed binding mechanisms of dopa remain unknown, and the chemical basis for mussels' ability to adhere to both inorganic and organic surfaces has never been fully explained. Herein, we report a single-molecule study of the substrate and oxidationdependent adhesive properties of dopa. Atomic force microscopy (AFM) measurements of a single dopa residue contacting a wet metal oxide surface reveal a surprisingly high strength yet fully reversible, noncovalent interaction. The magnitude of the bond dissociation energy as well as the inability to observe this interaction with tyrosine suggests that dopa is critical to adhesion and that the binding mechanism is not hydrogen bond formation. Oxidation of dopa, as occurs during curing of the secreted mussel glue, dramatically reduces the strength of the interaction to metal oxide but results in high strength irreversible covalent bond formation to an organic surface. A new picture of the interfacial adhesive role of dopa emerges from these studies, in which dopa exploits a remarkable combination of high strength and chemical multifunctionality to accomplish adhesion to substrates of widely varying composition from organic to metallic.3,4-dihydroxylphenylalanine ͉ atomic force microscopy ͉ mussel adhesive protein N umerous living creatures rely on physical adhesion to biotic and abiotic objects for essential activities, such as movement, protection, and self-defense (1-3). From a purely functional point of view, bioadhesion can be of two major types: temporary and permanent. A characteristic example of a temporary bioadhesive strategy is given by the specialized foot hairs used by geckos for climbing sheer surfaces (1). A classic example of permanent bioadhesion is exemplified by mussels, (4) which secrete holdfasts essential for stability within the tidal marine environment. The remarkable features of mussel adhesion include the ability to achieve long-lasting adhesion in a wet environment (3) and adherence to virtually all types of inorganic and organic surfaces (5). The adhesive apparatus of the mussel consists of a series of byssal threads that tether the organism to a substrate (Fig. 1A). At least five specialized adhesive protein subtypes known to contain 3,4-dihydroxy-L-phenylalanine (dopa) at concentrations ranging from a few mol % to 27 mol % (Fig. 1B) are found within the distal adhesive pad of the widely studied blue mussel, Mytilus edulis (6). The highest dopa content occurs in M. edulis foot protein (Mefp)-3 (21 mol %) and Mefp-5 (27 mol %) (7, 8), both of which are localized near the interface between the adhesive pad and the substrate (Fig. 1C).The role of dopa in mussel adhesi...

We quantitatively analyzed particle tracking data on insulin granules expressing fluorescent fusion proteins in MIN6 cells to better understand the motions contributing to intracellular transport and, more generally, the means for characterizing systems far from equilibrium. Care was taken to ensure that the statistics reflected intrinsic features of the individual granules rather than details of the measurement and overall cell state. We find anomalous diffusion. Interpreting such data conventionally requires assuming that a process is either ergodic with particles working against fluctuating obstacles (fractional Brownian motion) or nonergodic with a broad distribution of dwell times for traps (continuous-time random walk). However, we find that statistical tests based on these two models give conflicting results. We resolve this issue by introducing a subordinated scheme in which particles in cages with random dwell times undergo correlated motions owing to interactions with a fluctuating environment. We relate this picture to the underlying microtubule structure by imaging in the presence of vinblastine. Our results provide a simple physical picture for how diverse pools of insulin granules and, in turn, biphasic secretion could arise. Eukaryotic cells package proteins into vesicles for trafficking and spatially localized secretion. These essential functions are highly regulated, and defects in them can lead to disease (1, 2). Although optical microscopy, combined with molecular and cellular biology, can provide important insight into intracellular dynamics, in the past, most measurements detected variations in intensities from many molecular events and thus averaged in some way. These include fluorescence correlation spectroscopy (FCS) (3), fluorescence recovery after photobleaching (FRAP) (4), and image correlation spectroscopy (5). Recent advances in experimental methods now enable tracking single particles in cells (6). Although these measurements still involve a degree of time averaging (7), the resulting individual time trajectories contain more information than the mean values extracted from the aforementioned approaches.Qualitatively, the time trajectories reveal complex behaviors: combinations of random, directed, transiently stalled and constrained motions (e.g., refs. 7, 8). These different types of motion reflect the interplay of various molecular components in crowded environments. Quantifying their relative importance can constrain mechanisms, but extracting this information from the particle tracking data requires new theoretical tools. Operationally, one strategy is to classify segments of trajectories according to their motions (e.g., active and passive) (9, 10), but this requires long trajectories. A less data-demanding approach is to identify different types of anomalous diffusion (11).What features can give rise to the observed anomalous behavior? Simple crowding is insufficient, as it results in standard Brownian motion but with a reduced diffusion coefficient (12). Instead, anomalous b...

We introduce a novel spectroscopic technique which utilizes a two-pulse sequence of femtosecond duration phase-locked optical laser pulses to resonantly excite vibronic transitions of a molecule. In contrast with other ultrafast pump-probe methods, in this experiment a definite optical phase angle between the pulses is maintained while varying the interpulse delay with interferometric precision. For the cases of in-phase, in-quadrature, and out-of-phase pulse pairs, respectively, the optical delay is controlled to positions that are integer, integer plus one quarter, and integer plus one half multiples of the wavelength of a selected Fourier component. In analogy with a double slit optical interference experiment, the two pulse experiments reported herein involve the preparation and quantum interference of two nuclear wave packet amplitudes in an excited electronic state of a molecule. These experiments are designed to be sensitive to the total phase evolution of the wave packet prepared by the initial pulse. The direct determination of wave packet phase evolution is possible because phase locking effectively transforms the interferogram to a frame which is referenced to the optical carrier frequency, thereby eliminating the high (optical) frequency modulations. This has the effect of isolating the rovibrational molecular dynamics. The phase locking scheme is demonstrated for molecular iodine. The excited state population following the passage of both pulses is detected as the resultant two-beam dependent fluorescence emission from the B state. The observed signals have periodically recurring features that result from the vibrational dynamics of the molecule on the electronically excited potential energy surface. In addition, coherent interference effects cause the magnitUde and sign of the periodic features to be strongly modulated. The two-pulse phase-locked interferograms are interpreted herein by use of a simple analytic model, by first order perturbation theory and by quantum mechanical wave packet calculations. We find the form of the interferogram to be determined by the ground state level from which the amplitude originates, the deviation from impulsive preparation of the wave packet due to nonzero pulse duration, the frequency and anharmonicity of the target vibrational levels in the B state, and the detuning of the phase-locked frequency from resonance. The dependence of the interferogram on the phase-locked frequency and phase angle is investigated in detail.

Uncovering the quantitative laws that govern the growth and division of single cells remains a major challenge. Using a unique combination of technologies that yields unprecedented statistical precision, we find that the sizes of individual Caulobacter crescentus cells increase exponentially in time. We also establish that they divide upon reaching a critical multiple (≈1.8) of their initial sizes, rather than an absolute size. We show that when the temperature is varied, the growth and division timescales scale proportionally with each other over the physiological temperature range. Strikingly, the cell-size and division-time distributions can both be rescaled by their mean values such that the conditionspecific distributions collapse to universal curves. We account for these observations with a minimal stochastic model that is based on an autocatalytic cycle. It predicts the scalings, as well as specific functional forms for the universal curves. Our experimental and theoretical analysis reveals a simple physical principle governing these complex biological processes: a single temperature-dependent scale of cellular time governs the stochastic dynamics of growth and division in balanced growth conditions. single-cell dynamics | cell-to-cell variability | exponential growth | Hinshelwood cycle | Arrhenius law

Off-resonant transient birefringence measurements are analyzed using a reduced equation of motion for the ground state density matrix, which is expanded using an effective Hamiltonian. Assuming that the pump field is weak, we express the polarization relevant for the birefringence signal in terms of a convolution of the tensorial polarizability response function with the external fields. The homodyne-detected birefringence signal is directly compared with the coherent Raman signal. The relationship between off-resonant birefringence and spontaneous Raman experiments is discussed. By expanding the polarizability in powers of the nuclear coordinates and applying the Brownian oscillator model to the coordinate response function, we separate the birefringence signal into intra-and intermolecular coordinate response functions. Off-resonant transient birefiingences of acetonitrile, chloroform, dimethylsulfoxide, and a series of alcohols were measured. The data are transformed to the frequency domain by using a model independent analysis method. The spectra are discussed in the context of various models for the distribution of intermolecular modes (spectral density) in liquids.

Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale ''rulers'' to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate singlemolecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated. It is well known that the spatial resolution of an optical system is limited by the Rayleigh criterion (1),where is the wavelength of the collected photons and N.A. is the numerical aperture of the system. Using visible light (i.e., Ϸ500 nm) and a high N.A. objective (i.e. Ͼ1) allows achieving Ϸ250-nm lateral resolution. However, most biological macromolecules (DNA, RNA, proteins, etc.) and molecular machines (e.g., ribosome and spliceosome) are much smaller in two or all three dimensions, with important features or subassemblies lying within 10 nm of each other (2). Fluorescence resonance energy transfer (FRET) is widely used for studying biological systems on the nanometer scale (i.e., Ͻ10 nm; ref. 3). FRET occurs over distances similar to the Förster radius, which is Ϸ5 nm for common fluorophores (4). However, FRET only gives the relative distance between two probes, whereas their absolute positions remain unknown. Centroid localization, which has been used for single-particle tracking, allows determination of the centroid position of the particle to a much better precision than the length scale defined by the Rayleigh criterion (5-7). Recently, this approach to localizing fluorophores has been applied to single dye molecules (Cy3 and Rhodamine dyes) bound to molecular motors, and 2-nm precision in localization has been achieved when an oxygen scavenging buffer was used to suppress photobleaching (8, 9). However, centroid localization can only be used for isolated single molecules and it does not improve the resolution of the imaging system. It has been shown that two probes with different fluorescent wavelengths can be resolved within 10 nm because they are spectrally distinguishable (10). This method does not help when two or more objects with the same emission spectrum are within a diffraction-limited spot. Imaging applications beyond even the capabilities of ''su...

Fluorescence blinking in nanocrystal quantum dots is known to exhibit power-law dynamics, and several different mechanisms have been proposed to explain this behavior. We have extended the measurement of quantum-dot blinking by characterizing fluctuations in the fluorescence of single dots over time scales from microseconds to seconds. The power spectral density of these fluctuations indicates a change in the power-law statistics that occurs at a time scale of several milliseconds, providing an important constraint on possible mechanisms for the blinking. In particular, the observations are consistent with the predictions of models wherein blinking is controlled by diffusion of the energies of electron or hole trap states.fluorescence intermittency ͉ power spectrum ͉ nanocrystals H igh-quality, monodisperse semiconductor nanocrystals can be produced in large quantities by colloidal-synthesis techniques (1). These nanocrystals, known as quantum dots (QDs), can exhibit bright luminescence, whose wavelength is controlled by the size of the nanocrystals (2). This property makes colloidal QDs attractive candidates for several applications, including light-emitting diodes (3), solid-state lasers (4), and biological labeling (5, 6). Such applications, however, may be compromised by fluctuations in the QD luminescence. In particular, individual QDs emit light intermittently, switching irregularly between bright (''on'') and dark (''off'') states (7). Widespread interest in this blinking phenomenon was stimulated by the surprising observation that the durations of bright and dark periods follow power-law statistics (8, 9). Specifically, the blinking periods are described by probability densities of the formwith a value of between 0.4 and 1.0. The power-law behavior holds regardless of sample temperature (9), QD size or composition (10), nanoparticle shape (11), or excitation intensity (12). So far, though, experimental studies of QD blinking have been limited in their temporal resolution. A resolution of 200 s was achieved in one of the earliest measurements (8), and a small number of later experimental studies have included analysis of submicrosecond blinking dynamics (14,16,17,41). The remainder of the quantitative characterizations have been restricted to time scales of several milliseconds or longer. In this paper, we report measurements of fluctuations in QD fluorescence on time scales from microseconds to tens of seconds. We observe a change in the fluctuation dynamics for time scales less than several milliseconds.This observation is consistent with the predictions of a class of models where blinking is controlled by slow diffusion of the energies of electron or hole trap states. In these models, the fluorescence of a QD is quenched through the trapping of a carrier, which occurs when the energy of the trap state fulfills a resonance condition. The duration of the blinking periods is determined by the diffusion of the energy of the QD-trap system about this resonance condition. This diffusion-controlled mechanism ...

We demonstrate three-dimensional trapping and orientation of individual Au nanorods by using laser light slightly detuned from their longitudinal plasmon mode. Detuning to the long-wavelength side of the resonance allows stable trapping for several minutes, with an exponential dependence of trapping time on laser power (consistent with a Kramer's escape process). Detuning to the short-wavelength side causes repulsion of the rods from the laser focus. Alignment of the long axis of the rods with the trapping laser polarization is observed as a suppression of rotational diffusion about the short axis.