Thursday, November 28, 2013

Detecting the trapping of small metal nanoparticles in the gap of nanoantennas with optical second harmonic generation

Jérémy Butet, Andrea Lovera, and Olivier J. F. Martin

The second harmonic generation from gold nanoparticles trapped into realistic and idealized gold nanoantennas is numerically investigated using a surface integral equations technique. It is observed that the presence of a nanoparticle in the nanoantenna gap dramatically modifies the second harmonic intensity scattered into the far-field. These results clearly demonstrate that second harmonic generation is a promising alternative to the conventional linear optical methods for the detection of trapping events at the nanoscale.

Single-molecule force measurement via optical tweezers reveals different kinetic features of two BRaf mutants responsible for cardio-facial-cutaneous (CFC) syndrome

Cheng Wen and Anpei YeBRaf (B- Rapid Accelerated Fibrosarcoma) protein is an important serine/threonine-protein kinase. Two domains on BRaf can independently bind its upstream kinase, Ras (Rat Sarcoma) protein. These are the Ras binding domain (RBD) and cysteine-rich-domain (CRD). Herein we use customized optical tweezers to compare the Ras binding process in two pathological mutants of BRaf responsible for CFC syndrome, abbreviated BRaf (A246P) and BRaf (Q257R). The two mutants differ in their kinetics of Ras-binding, though both bind Ras with similar increased overall affinity. BRaf (A246P) exhibits a slightly higher Ras/CRD unbinding force and a significantly higher Ras/RBD unbinding force versus the wild type. The contrary phenomenon is observed in the Q257R mutation. Simulations of the unstressed-off rate, koff(0), yield results in accordance with the changes revealed by the mean unbinding force. Our approach can be applied to rapidly assess other mutated proteins to deduce the effects of mutation on their kinetics compared to wild type proteins and to each other.


Tuesday, November 26, 2013

Active contractions in single suspended epithelial cells

Markus Gyger, Roland Stange, Tobias R. Kießling, Anatol Fritsch, Katja B. Kostelnik, Annette G. Beck-Sickinger, Mareike Zink, Josef A. Käs

Investigations of active contractions in tissue cells to date have been focused on cells that exert forces via adhesion sites to substrates or to other cells. In this study we show that also suspended epithelial cells exhibit contractility, revealing that contractions can occur independently of focal adhesions. We employ the Optical Stretcher to measure adhesion-independent mechanical properties of an epithelial cell line transfected with a heat-sensitive cation channel. During stretching the heat transferred to the ion channel causes a pronounced Ca2+ influx through the plasma membrane that can be blocked by adequate drugs. This way the contractile forces in suspended cells are shown to be partially triggered by Ca2+ signaling. A phenomenological mathematical model is presented, incorporating a term accounting for the active stress exerted by the cell, which is both necessary and sufficient to describe the observed increase in strain when the Ca2+ influx is blocked. The median and the shape of the strain distributions depend on the activity of the cells. Hence, it is unlikely that they can be described by a simple Gaussian or log normal distribution, but depend on specific cellular properties such as active contractions. Our results underline the importance of considering activity when measuring cellular mechanical properties even in the absence of measurable contractions. Thus, the presented method to quantify active contractions of suspended cells offers new perspectives for a better understanding of cellular force generation with possible implications for medical diagnosis and therapy.


Wednesday, November 20, 2013

Investigations on rheological properties and gelation of tasar regenerated silk fibroin solution

Yogesha Lakkegowda, Raghu Ammannappa, Sharath Ananthamurthy
Tasar silk is a variety of non-mulberry silk indigenous to the Indian subcontinent. We present the measured frequency-dependent viscoelastic moduli of Tasar regenerated silk fibroin (RSF) solution using optical tweezers at two concentrations (0.16% and 0.25% w/v) and extend these measurements to the low frequency regime using a video microscopy technique. We extend the investigation on the rheological behavior of Tasar RSF for four more RSF concentrations, viz., 0.50%, 1.00%, 2.50% and 5.00% using video microscopy. In all the RSF samples, both storage and loss moduli are found to increase with frequency. At lower frequencies the loss modulus is more than the storage modulus and exhibit similar behavior until a crossover frequency beyond which the storage modulus exceeds the loss modulus at all frequencies. The relaxation time which is inversely related to the crossover frequency is found to rise sharply at 5% w/v, indicating the onset of gelation in the sample. These results are examined in relation to the viscoelastic parameters of mulberry silk, wherein the larger crossover frequencies at the same higher concentrations indicate relaxation times that are an order of magnitude smaller than those measured for Tasar RSF.


Optical theorem for acoustic non-diffracting beams and application to radiation force and torque

Likun Zhang and Philip L. Marston
Acoustical and optical non-diffracting beams are potentially useful for manipulating particles and larger objects. An extended optical theorem for a non-diffracting beam was given recently in the context of acoustics. The theorem relates the extinction by an object to the scattering at the forward direction of the beam’s plane wave components. Here we use this theorem to examine the extinction cross section of a sphere centered on the axis of the beam, with a non-diffracting Bessel beam as an example. The results are applied to recover the axial radiation force and torque on the sphere by the Bessel beam.

Tuesday, November 12, 2013

Filopodial retraction force is generated by cortical actin dynamics and controlled by reversible tethering at the tip

Thomas Bornschlögl, Stéphane Romero, Christian L. Vestergaard, Jean-François Joanny, Guy Tran Van Nhieu, and Patricia Bassereau
Filopodia are dynamic, finger-like plasma membrane protrusions that sense the mechanical and chemical surroundings of the cell. Here, we show in epithelial cells that the dynamics of filopodial extension and retraction are determined by the difference between the actin polymerization rate at the tip and the retrograde flow at the base of the filopodium. Adhesion of a bead to the filopodial tip locally reduces actin polymerization and leads to retraction via retrograde flow, reminiscent of a process used by pathogens to invade cells. Using optical tweezers, we show that filopodial retraction occurs at a constant speed against counteracting forces up to 50 pN. Our measurements point toward retrograde flow in the cortex together with frictional coupling between the filopodial and cortical actin networks as the main retraction-force generator for filopodia. The force exerted by filopodial retraction, however, is limited by the connection between filopodial actin filaments and the membrane at the tip. Upon mechanical rupture of the tip connection, filopodia exert a passive retraction force of 15 pN via their plasma membrane. Transient reconnection at the tip allows filopodia to continuously probe their surroundings in a load-and-fail manner within a well-defined force range.


Monday, November 11, 2013

Dynamical analysis of an optical rocking ratchet: Theory and experiment

Alejandro V. Arzola, Karen Volke-Sepúlveda, José L. Mateos
A thorough analysis of the dynamics in a deterministic optical rocking ratchet [ A. V. Arzola et al. Phys. Rev. Lett. 106 168104 (2011)] and a comparison with experimental results are presented. The studied system consists of a microscopic particle interacting with a periodic and asymmetric light pattern, which is driven away from equilibrium by means of an unbiased time-periodic external force. It is shown that the asymmetry of the effective optical potential depends on the relative size of the particle with respect to the spatial period, and this is analyzed as an effective mechanism for particle fractionation. The necessary conditions to obtain current reversals in the deterministic regime are discussed in detail.

Self-Assembly of Mesoscopic Materials to form Controlled and Continuous Patterns by Thermo-Optically Manipulated Laser Induced Microbubbles

Basudev Roy , Manish Arya , Preethi Thomas , Julius Jurgschat , K. Venkata Rao , Ayan Banerjee , Chilla Malla Reddy , and Soumyajit Roy
The formation of continuous patterns of nano-structured material using directed self assembly under external fields has generated considerable current research interest. We demonstrate for the first time such continuous patterning by inducing irreversible self-assembly leading to nucleation in mesocopic materials (inorganic, organic, and nano-particles) using a tightly focused laser beam in an optical tweezers apparatus. A dense aqueous dispersion or solution of the material which has high absorption at the laser wavelength is taken in a sample holder so that some material is adsorbed on the top surface. A hot spot is created on the top surface when the adsorbed material absorbs the high intensity at the focus of the laser beam (a sub-micron sized spot), due to which a water vapour bubble is formed. This causes self assembly of material around the bubble due to Gibbs-Marangoni convection and capillary flow after which the material eventually nucleates into a crystalline state. The bubble is ‘trapped’ at the hot spot due to the temperature gradient around it, and can be manipulated by thermal forces generated optically, so that the system may be described as a thermo-optic tweezers. We translate the trapped bubble using the microscope sample holder stage of the apparatus so that the nucleation site of the material is simultaneously translated generating continuous patterns. We have demonstrated the technique using exotic inorganic materials such as soft oxometalates, an organic material such as glycine, a fluorescent dye such as perylene, as well as with carbon nano-tubes. We have written patterns over lengths of nearly 1 mm at the rate of 1 Hz, with best resolution of about 4 μm. The technique has potential for a wide range of applications ranging from solution processed printable electronics to controlled catalysis.

Robust measurement of membrane bending moduli using light sheet fluorescence imaging of vesicle fluctuations

Andrew F. Loftus , Sigrid Noreng , Vivian L. Hsieh , and Raghuveer Parthasarathy
The mechanical rigidity of lipid membranes is a key determinant of the energetics of cellular membrane deformation. Measurements of membrane bending moduli remain rare, however, and show a large variance, a situation that can be addressed by the development of improved techniques and by comparisons between disparate techniques applied to the same systems. We introduce here the use of selective plane illumination microscopy (SPIM, also known as light sheet fluorescence microscopy) to image thermal fluctuations of giant vesicles. The optical sectioning of SPIM enables high-speed fluorescence imaging of freely suspended vesicles and quantification of edge localization precision, yielding robust fluctuation spectra and rigidity estimates. For both lipid-only membranes and membranes bound by the intracellular trafficking protein Sar1p, which lowers membrane rigidity in a concentration-dependent manner, we show that the resulting bending modulus values are in close agreement with those derived from an independent assay based on membrane tether pulling. We also show that the fluctuation spectra of vesicles bound by the mammalian Sar1A protein, which stiffens membranes at high concentrations, are not well fit by a model of homogeneous quasi-spherical vesicles, suggesting that SPIM-based analysis can offer insights into spatially inhomogeneous properties induced by protein assemblies.

Optical forces induced by metal nanoparticle clusters

Jordi Sancho-Parramon, Salvador Bosch
The strong field localization generated between closely placed metal particles excited by electromagnetic radiation induces intense forces on small polarizable objects. In this study we investigate the optical forces that can be generated in the vicinity of metal nanoparticle clusters using fully electrodynamic numerical simulations. The influence of the cluster configuration as well as of the excitation parameters is analyzed.

Friday, November 8, 2013

Introduction: Optical trapping and applications feature issue

Carlos López-Mariscal and David McGloin

The editors introduce the Biomedical Optics Express feature issue on “Optical Trapping and Applications.” The works presented in the papers within this issue include were the focus of the third OTA Topical Meeting that was held on April 14–18, 2013, in Waikoloa, Hawaii.

Non-Processive Force Generation by Mammalian Axonemal Dynein In Situ on Doublet Microtubules

David P. Lorch, Kathleen A. Lesich, Charles B. Lindemann, Alan J. Hunt
We utilize optical tweezers to examine displacements produced by small numbers of dynein motors located in situ on doublet microtubules from disintegrated mammalian sperm axonemes. In contrast with cytoplasmic dynein, we find that axonemal dynein is not processive, and the duration of individual force-generating interactions with a microtubule are longer than predicted from the velocity of movements generated by large ensembles of motors. These findings suggest that tension is required for rapid release of dynein following a power stroke and may explain how axonemal dynein is adapted to work in arrays within an axoneme, where cyclical bending patterns require motors to function over a range of sliding velocities.


Keratins significantly contribute to cell stiffness and impact invasive behavior

Kristin Seltmann, Anatol W. Fritsch, Josef A. Käs, and Thomas M. Magina
Cell motility and cell shape adaptations are crucial during wound healing, inflammation, and malignant progression. These processes require the remodeling of the keratin cytoskeleton to facilitate cell–cell and cell–matrix adhesion. However, the role of keratins for biomechanical properties and invasion of epithelial cells is only partially understood. In this study, we address this issue in murine keratinocytes lacking all keratins on genome engineering. In contrast to predictions, keratin-free cells show about 60% higher cell deformability even for small deformations. This response is compared with the less pronounced softening effects for actin depolymerization induced via latrunculin A. To relate these findings with functional consequences, we use invasion and 3D growth assays. These experiments reveal higher invasiveness of keratin-free cells. Reexpression of a small amount of the keratin pair K5/K14 in keratin-free cells reverses the above phenotype for the invasion but does not with respect to cell deformability. Our data show a unique role of keratins as major players of cell stiffness, influencing invasion with implications for epidermal homeostasis and pathogenesis. This study supports the view that down-regulation of keratins observed during epithelial–mesenchymal transition directly contributes to the migratory and invasive behavior of tumor cells.

Optical trapping and manipulation of nanostructures

Onofrio M. Maragò, Philip H. Jones, Pietro G. Gucciardi, Giovanni Volpe & Andrea C. Ferrari
Optical trapping and manipulation of micrometre-sized particles was first reported in 1970. Since then, it has been successfully implemented in two size ranges: the subnanometre scale, where light–matter mechanical coupling enables cooling of atoms, ions and molecules, and the micrometre scale, where the momentum transfer resulting from light scattering allows manipulation of microscopic objects such as cells. But it has been difficult to apply these techniques to the intermediate — nanoscale — range that includes structures such as quantum dots, nanowires, nanotubes, graphene and two-dimensional crystals, all of crucial importance for nanomaterials-based applications. Recently, however, several new approaches have been developed and demonstrated for trapping plasmonic nanoparticles, semiconductor nanowires and carbon nanostructures. Here we review the state-of-the-art in optical trapping at the nanoscale, with an emphasis on some of the most promising advances, such as controlled manipulation and assembly of individual and multiple nanostructures, force measurement with femtonewton resolution, and biosensors.

Thursday, November 7, 2013

Numerical study of the properties of optical vortex array laser tweezers

Chun-Fu Kuo and Shu-Chun Chu
Chu et al. constructed a kind of Ince-Gaussian modes (IGM)-based vortex array laser beams consisting of p x p embedded optical vortexes from Ince-Gaussian modes, IGep,p modes [Opt. Express 16, 19934 (2008)]. Such an IGM-based vortex array laser beams maintains its vortex array profile during both propagation and focusing, and is applicable to optical tweezers. This study uses the discrete dipole approximation (DDA) method to study the properties of the IGM-based vortex array laser tweezers while it traps dielectric particles. This study calculates the resultant force exerted on the spherical dielectric particles of different sizes situated at the IGM-based vortex array laser beam waist. Numerical results show that the number of trapping spots of a structure light (i.e. IGM-based vortex laser beam), is depended on the relation between the trapped particle size and the structure light beam size. While the trapped particle is small comparing to the beam size of the IGM-based vortex array laser beams, the IGM-based vortex array laser beams tweezers are suitable for multiple traps. Conversely, the tweezers is suitable for single traps. The results of this study is useful to the future development of the vortex array laser tweezers applications.

Laser trapping-induced crystallization of L-phenylalanine through its high-concentration domain formation

Ken-ichi Yuyama, Chi-Shiun Wu, Teruki Sugiyama and Hiroshi Masuhara
We present the laser trapping-induced crystallization of L-phenylalanine through high-concentration domain formation in H2O and D2O solutions which is achieved by focusing a continuous-wave (CW) near-infrared laser beam at the solution surface. Upon laser irradiation into the H2O solution, laser trapping of the liquid-like clusters increases the local concentration, accompanying laser heating, and a single plate-like crystal is eventually prepared at the focal spot. On the other hand, in the D2O solution, a lot of the monohydrate needle-like crystals are observed, not at the focal spot where the concentration is high enough to trigger crystal nucleation, but in the 0.5–1.5 mm range from the focal spot. The dynamics and mechanism of the amazing crystallization behaviour induced by laser trapping are discussed from the viewpoints of the concentration increase due to laser heating depending on solvent, the large high-concentration domain formation by laser trapping of liquid-like clusters, and the orientational disorder of molecules/clusters at the domain edge.

DNA Interaction With Diaminobenzidine Studied With Optical Tweezers and Dynamic Light Scattering

Luana Reis , Esio B. Ramos , and Marcio S. Rocha
We have studied the interaction of the DNA molecule with the ligand 3,3'-Diaminobenzidine (DAB) by performing single molecule stretching experiments with optical tweezers and dynamic light scattering (DLS) on the DNA-DAB complexes. In the stretching experiments, the persistence and contour lengths of the complexes were measured as a function of DAB concentration, allowing one to infer the main binding mechanism and also to determine the physicochemical parameters of the interaction. In the DLS experiments, the effective size of the complexes, measured as the hydrodynamic radius, was monitored as a function of DAB concentration. We found a qualitative agreement between the results obtained from the two techniques by comparing the behaviors of the hydrodynamics radius and the radius of gyration, since this last one can be expressed as a function of the persistence and contour lengths.

Memory effects for a trapped Brownian particle in viscoelastic shear flows

Romi Mankin, Katrin Laas, and Neeme Lumi
The long-time limit behavior of the positional distribution for an underdamped Brownian particle in a fluctuating harmonic potential well, which is simultaneously exposed to an oscillatory viscoelastic shear flow is investigated using the generalized Langevin equation with a power-law-type memory kernel. The influence of a fluctuating environment is modeled by a multiplicative white noise (fluctuations of the stiffness of the trapping potential) and by an additive internal fractional Gaussian noise. The exact expressions of the second-order moments of the fluctuating position for the Brownian particle in the shear plane have been calculated. Also, shear-induced cross correlation between particle fluctuations along orthogonal directions as well as the angular momentum are found. It is shown that interplay of shear flow, memory, and multiplicative noise can generate a variety of cooperation effects, such as energetic instability, multiresonance versus the shear frequency, and memory-induced anomalous diffusion in the direction of the shear flow. Particularly, two different critical memory exponents have been found, which mark dynamical transitions from a stationary regime to a subdiffusive (or superdiffusive) regime of the system. Similarities and differences between the behaviors of the models with oscillatory and nonoscillatory shear flow are also discussed.

Determining the unique refractive index properties of solid polystyrene aerosol using broadband Mie scattering from optically trapped beads

Stephanie H. Jones, Martin D. King and Andrew D. Ward
A method is described to measure the refractive index dispersion with wavelength of optically trapped solid particles in air. Knowledge of the refraction properties of solid particles is critical for the study of aerosol; both in the laboratory and in the atmosphere for climate studies. Single micron-sized polystyrene beads were optically trapped in air using a vertically aligned counter-propagating configuration of focussed laser beams. Each bead was illuminated using white light from a broadband light emitting diode (LED) and elastic scattering within the bead was collected onto a spectrograph. The resulting Mie spectra were analysed to accurately determine polystyrene bead radii to ±0.4 nm and values of the refractive index to ±0.0005 over a wavelength range of 480–700 nm. We demonstrate that optical trapping combined with elastic scattering can be used to both accurately size polystyrene beads suspended in air and determine their wavelength dependent refractive index. The refractive index dispersions are in close agreement with reported values for polystyrene beads in aqueous dispersion. Our results also demonstrate a variation in the refractive index of polystyrene, from bead to bead, in a commercial sample. The measured variation highlights that care must be taken when using polystyrene beads as a calibration aerosol.


Monday, November 4, 2013

Optical manipulation of charged microparticles in polar fluids

Giuseppe Pesce, Vincenzo Lisbino, Giulia Rusciano, Antonio Sasso

In this study, we report a systematic study of the response of a charged microparticle confined in an optical trap and driven by electric fields. The particle is embedded in a polar fluid, hence, the role of ions and counterions forming a double layer around the electrodes and the particle surface itself has been taken into account. We analyze two different cases: (i) electrodes energized by a step-wise voltage (DC mode) and (ii) electrodes driven by a sinusoidal voltage (AC mode). The experimental outcomes are analyzed in terms of a model that combines the electric response of the electrolytic cell and the motion of the trapped particle. In particular, for the DC mode we analyze the transient particle motion and correlate it with the electric current flowing in the cell. For the AC mode, the stochastic and deterministic motion of the trapped particle is analyzed either in the frequency domain (power spectral density, PSD) or in the time domain (autocorrelation function). Moreover, we will show how these different approaches (DC and AC modes) allow us, assuming predictable the applied electric field (here generated by plane parallel electrodes), to provide accurate estimation (3%) of the net charge carried by the microparticle. Vice versa, we also demonstrate how, once predetermined the charge, the trapped particle acts as a sensitive probe to reveal locally electric fields generated by arbitrary electrode geometries (in this work, wire-tip geometry).

Optical vault: A reconfigurable bottle beam based on conical refraction of light

A. Turpin, V. Shvedov, C. Hnatovsky, Yu. V. Loiko, J. Mompart, and W. Krolikowski
We employ conical refraction of light in a biaxial crystal to create an optical bottle for photophoretic trapping and manipulation of particles in gaseous media. We show that by only varying the polarization state of the input light beam the optical bottle can be opened and closed in order to load and unload particles in a highly controllable manner.

Selective Optical Assembly of Highly Uniform Nanoparticles by Doughnut-Shaped Beams

Syoji Ito, Hiroaki Yamauchi, Mamoru Tamura, Shimpei Hidaka, Hironori Hattori, Taichi Hamada, Keisuke Nishida, Shiho Tokonami, Tamitake Itoh, Hiroshi Miyasaka & Takuya Iida

A highly efficient natural light-harvesting antenna has a ring-like structure consisting of dye molecules whose absorption band changes through selective evolutionary processes driven by external stimuli, i.e., sunlight depending on its territory and thermal fluctuations. Inspired by this fact, here, we experimentally and theoretically demonstrate the selective assembling of ring-like arrangements of many silver nanorods with particular shapes and orientations onto a substrate by the light-induced force of doughnut beams with different colours (wavelengths) and polarizations in conjunction with thermal fluctuations at room temperature. Furthermore, the majority of nanorods are electromagnetically coupled to form a prominent red-shifted collective mode of localized surface plasmons resonant with the wavelength of the irradiated light, where a spectral broadening also appears for the efficient broadband optical response. The discovered principle is a promising route for "bio-inspired selective optical assembly" of various nanomaterials that can be used in the wide field of nanotechnology.

The ClpXP Protease Unfolds Substrates Using a Constant Rate of Pulling but Different Gears

Maya Sen, Rodrigo A. Maillard, Kristofor Nyquist, Piere Rodriguez-Aliaga, Steve Presse Andreas Martin, and Carlos Bustamante
ATP-dependent proteases are vital to maintain cellular protein homeostasis. Here, we study the mechanisms of force generation and intersubunit coordination in the ClpXP protease from E. coli to understand how these machines couple ATP hydrolysis to mechanical protein unfolding. Single-molecule analyses reveal that phosphate release is the force generating step in the ATP-hydrolysis cycle and that ClpXP translocates substrate polypeptides in bursts resulting from highly coordinated conformational changes in two to four ATPase subunits. ClpXP must use its maximum successive firing capacity of four subunits to unfold stable substrates like GFP. The average dwell duration between individual bursts of translocation is constant, regardless of the number of translocating subunits, implying that ClpXP operates with constant ‘‘rpm’’ but uses different ‘‘gears.’’


Following the Mechanisms of Bacteriostatic versus Bactericidal Action Using Raman Spectroscopy

Silvie Bernatová, Ota Samek, Zdeněk Pilát, Mojmír Šerý, Jan Ježek, Petr Jákl, Martin Šiler, Vladislav Krzyžánek, Pavel Zemánek, Veronika Holá, Milada Dvořáčková and Filip Růžička
Antibiotics cure infections by influencing bacterial growth or viability. Antibiotics can be divided to two groups on the basis of their effect on microbial cells through two main mechanisms, which are either bactericidal or bacteriostatic. Bactericidal antibiotics kill the bacteria and bacteriostatic antibiotics suppress the growth of bacteria (keep them in the stationary phase of growth). One of many factors to predict a favorable clinical outcome of the potential action of antimicrobial chemicals may be provided using in vitro bactericidal/bacteriostatic data (e.g., minimum inhibitory concentrations—MICs). Consequently, MICs are used in clinical situations mainly to confirm resistance, and to determine the in vitro activities of new antimicrobials. We report on the combination of data obtained from MICs with information on microorganisms’ “fingerprint” (e.g., DNA/RNA, and proteins) provided by Raman spectroscopy. Thus, we could follow mechanisms of the bacteriostatic versus bactericidal action simply by detecting the Raman bands corresponding to DNA. The Raman spectra of Staphylococcus epidermidis treated with clindamycin (a bacteriostatic agent) indeed show little effect on DNA which is in contrast with the action of ciprofloxacin (a bactericidal agent), where the Raman spectra show a decrease in strength of the signal assigned to DNA, suggesting DNA fragmentation.


Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy

Gabriel Žoldák, Johannes Stigler, Benjamin Pelz, Hongbin Li, and Matthias Rief
In this study we expand the accessible dynamic range of single-molecule force spectroscopy by optical tweezers to the microsecond range by fast sampling. We are able to investigate a single molecule for up to 15 min and with 300-kHz bandwidth as the protein undergoes tens of millions of folding/unfolding transitions. Using equilibrium analysis and autocorrelation analysis of the time traces, the full energetics as well as real-time kinetics of the ultrafast folding of villin headpiece 35 and a stable asparagine 68 alanine/lysine 70 methionine variant can be measured directly. We also performed Brownian dynamics simulations of the response of the bead-DNA system to protein-folding fluctuations. All key features of the force-dependent deflection fluctuations could be reproduced: SD, skewness, and autocorrelation function. Our measurements reveal a difference in folding pathway and cooperativity between wild-type and stable variant of headpiece 35. Autocorrelation force spectroscopy pushes the time resolution of single-molecule force spectroscopy to ∼10 µs thus approaching the timescales accessible for all atom molecular dynamics simulations.


Hydrodynamic and subdiffusive motion of tracers in a viscoelastic medium

Denis S. Grebenkov, Mahsa Vahabi, Elena Bertseva, László Forró, and Sylvia Jeney
We investigate the diffusive motion of micron-sized spherical tracers in a viscoelastic actin filament network over the time span of 8 orders of magnitude using optical-tweezers single-particle tracking. The hydrodynamic interactions of a tracer with the surrounding fluid are shown to dominate at microsecond time scales, while subdiffusive scaling due to viscoelastic properties of the medium emerges at millisecond time scales. The transition between these two regimes is analyzed in the frame of a minimal phenomenological model which combines the Basset force and the generalized Stokes force. The resulting Langevin equation accounts for various dynamical features of the thermal motion of endogenous or exogenous tracers in viscoelastic media such as inertial and hydrodynamic effects at short times, subdiffusive scaling at intermediate times, and eventual optical trapping at long times. Simple analytical formulas for the mean-square displacement and velocity autocorrelation function are derived.