Thursday, December 19, 2013

High-Speed Force Spectroscopy Unfolds Titin at the Velocity of Molecular Dynamics Simulations

Felix Rico, Laura Gonzalez, Ignacio Casuso, Manel Puig-Vidal, Simon Scheuring

The mechanical unfolding of the muscle protein titin by atomic force microscopy was a landmark in our understanding of single-biomolecule mechanics. Molecular dynamics simulations offered atomic-level descriptions of the forced unfolding. However, experiment and simulation could not be directly compared because they differed in pulling velocity by orders of magnitude. We have developed high-speed force spectroscopy to unfold titin at velocities reached by simulation (~4 millimeters per second). We found that a small β-strand pair of an immunoglobulin domain dynamically unfolds and refolds, buffering pulling forces up to ~100 piconewtons. The distance to the unfolding transition barrier is larger than previously estimated but is in better agreement with atomistic predictions. The ability to directly compare experiment and simulation is likely to be important in studies of biomechanical processes.


Particle tracking by full-field complex wavefront subtraction in digital holography microscopy

Lisa Miccio, Pasquale Memmolo , Francesco Merola, Sabato Fusco, Antonio Paciello, Maurizio Ventre, Paolo Netti and Pietro Ferraro

3D tracking of micro-objects, based on digital holography, is proposed through the analysis of the complex wavefront of the light scattered by the micro-samples. Exploiting the advantages of the off-axis full-field holographic interferometry, tracking of multiple objects is achieved by a direct wavefront analysis at the focal plane overcoming the limitation of the conventional Back Focal Plane interferometry in which one object at time can be tracked. Furthermore, the method proposed and demonstrated here is a step-forward also in respect to other holographic tracking tools. The approach is tested in two experiments, one for investigating Brownian motion of particles trapped by holographic optical tweezers, while second is related to cells motility in 3D collagen matric thus showing its usefulness for lab-on-chip systems in typical bio-assay testing.


Measurements of the Sensitivity of Aerosol Hygroscopicity and the κ Parameter to the O/C Ratio

Andrew M. J. Rickards , Rachael E. H. Miles , James F. Davies , Frances H. Marshall , and Jonathan P. Reid

We report measurements of the subsaturated hygroscopic growth of aerosol particles composed of single organic components of varying oxygen-to-carbon ratio up to relative humidities approaching saturation using the techniques of aerosol optical tweezers and an electrodynamic balance. The variation in the hygroscopicity parameter κ between compounds of even the same O/C ratio is found to be significant with, for example, a range in κ values from 0.12 to 0.38 for compounds with an O/C of 1. The measurements are compared with a review of all of the available literature data for which both the κ value and O/C ratio are reported, and a new parametrization is determined. Critical supersaturations predicted using this parametrization yield values that have associated uncertainties that are comparable to typical uncertainties in experimental measurements of critical supersaturations. However, the systematic variability between κ parametrizations determined from different studies remains large, consistent with the O/C ratio providing only an approximate guide to aerosol hygroscopicity and reflecting significant variations for aerosols of different chemical functionality, composition, and oxidation history.


Use of topological defects as templates to direct assembly of colloidal particles at nematic interfaces

Mohamed Amine Gharbi, Maurizio Nobili, Christophe Blanc

In this work, we experimentally investigate the ability of topological defects to guide interfacial assembly of spherical particles with homeotropic anchoring confined to nematic interfaces. We propose two different systems: In the first one, particles are trapped at an air/nematic interface where they spontaneously form various 2D patterns. We demonstrate that the phase transition between these patterns can be controlled by defects formed in the nematic bulk. In the second system, we explore the behavior of particles at the surface of bipolar nematic drops. We found that particles assemble into linear chains and interact with surface defects at the North and South poles of the drop, giving rise to the formation of star structures in a self-assembly process. We detail the mechanism that guides the behavior of particles and discuss the role of defects in the formation of the observed patterns.


Radiation pressure efficiency measurements of nanoparticle coated microspheres

Soo Y. Kim, Joseph D. Taylor, Harold D. Ladouceur Sean J. Hart and Alex Terray

Experimental measurements of the radiation pressure efficiency (Qpr ) for several microparticles have been compared to theoretical calculations extrapolated from the Bohren-Huffman code for Mie scattering of coated particles. An increased shift of the Qpr parameter was observed for 2 μm SiO2 core particles coated with nanoparticles of higher refractive indices. Coatings of 14 nm melamine particles were found to increase the Qpr parameter 135 times over similar coatings using SiO2 particles of the same size. While a coating of 100 nm polystyrene particles also showed a significant increase, they did not agree well with theoretical values. It is hypothesized that other factors such as increased scatter, drag, and finite coating coverage are no longer negligible for coatings using nanoparticles in this size regime.


Quantum noise in optical tweezers

Michael A Taylor and Warwick P Bowen

Quantum enhanced sensitivity in optical tweezers based particle tracking was recently demonstrated. This has provided the necessary tool for quantum metrology to play an important role in biological measurements. Here we introduce the basic theory relevant to such optical tweezers experiments, and overview the significance of sub-shot noise limited sensitivity to practical experiments. In particular, biophysical experiments are subject to optical power constraints, which therefore limits the absolute sensitivity which is classically achievable. Quantum enhanced particle tracking can overcome this limit, and is therefore likely to play an important role in such biophysical experiments in the near future.


Focused plasmonic trapping of metallic particles

Changjun Min, Zhe Shen, Junfeng Shen, Yuquan Zhang, Hui Fang, Guanghui Yuan, Luping Du, Siwei Zhu, Ting Lei & Xiaocong Yuan

Scattering forces in focused light beams push away metallic particles. Thus, trapping metallic particles with conventional optical tweezers, especially those of Mie particle size, is difficult. Here we investigate a mechanism by which metallic particles are attracted and trapped by plasmonic tweezers when surface plasmons are excited and focused by a radially polarized beam in a high-numerical-aperture microscopic configuration. This contrasts the repulsion exerted in optical tweezers with the same configuration. We believe that different types of forces exerted on particles are responsible for this contrary trapping behaviour. Further, trapping with plasmonic tweezers is found not to be due to a gradient force balancing an opposing scattering force but results from the sum of both gradient and scattering forces acting in the same direction established by the strong coupling between the metallic particle and the highly focused plasmonic field. Theoretical analysis and simulations yield good agreement with experimental results.


Wednesday, December 18, 2013

Single and dual fiber nano-tip optical tweezers: trapping and analysis

Jean-Baptiste Decombe, Serge Huant, and Jochen Fick

An original optical tweezers using one or two chemically etched fiber nano-tips is developed. We demonstrate optical trapping of 1 micrometer polystyrene spheres at optical powers down to 2 mW. Harmonic trap potentials were found in the case of dual fiber tweezers by analyzing the trapped particle position fluctuations. The trap stiffness was deduced using three different models. Consistent values of up to 1 fN/nm were found. The stiffness linearly decreases with decreasing light intensity and increasing fiber tip-to-tip distance.


Monday, December 16, 2013

Optimal Hydrodynamic Synchronization of Colloidal Rotors

Jurij Kotar, Luke Debono, Nicolas Bruot, Stuart Box, David Phillips, Stephen Simpson, Simon Hanna, and Pietro Cicuta

Synchronization of driven oscillators is a key aspect of flow generation in artificial and biological filaments such as cilia. Previous theoretical and numerical studies have considered the “rotor” model of a cilium in which the filament is coarse grained into a colloidal sphere driven with a given force law along a predefined trajectory to represent the oscillating motion of the cilium. These studies pointed to the importance of two factors in the emergence of synchronization: the modulation of the driving force around the orbit and the deformability of the trajectory. In this work it is shown via experiments, supported by numerical simulations and theory, that both of these factors are important and can be combined to produce strong synchronization (within a few cycles) even in the presence of thermal noise.


Timescales of water transport in viscous aerosol: measurements on sub-micron particles and dependence on conditioning history

Jessica W. Lu, Andrew M. J. Rickards, Jim S. Walker, Kerry J. Knox, Rachael E. H. Miles, Jonathan P. Reid and Ruth Signorell

Evaporation studies of single aqueous sucrose aerosol particles as a function of relative humidity (RH) are presented for coarse and fine mode particles down into the submicron size range (600 nm < r < 3.0 μm). These sucrose particles serve as a proxy for biogenic secondary organic aerosols that have been shown to exist, under ambient conditions, in an ultraviscous glassy state, which can affect the kinetics of water mass transport within the bulk phase and hinder particle response to changes in the gas phase water content. A counter-propagating Bessel beams (CPBBs) optical trapping setup is employed to monitor the real-time change in the particle radius with RH decreasing from 75% to 5%. The slow-down of the size change upon each RH step and the deviation from the theoretical equilibrium hygroscopic growth curve indicate the onset of glassy behavior in the RH range of 10–40%. Size-dependent effects were not observed within the uncertainty of the measurements. The influence of the drying time below the glass transition RH on the timescale of subsequent water condensation and re-equilibration for sucrose particles is explored by optical tweezers measurements of micron-sized particles (3 μm < r < 6 μm). The timescale for water condensation and re-equilibration is shown to increase with increasing drying time, i.e. the time over which a viscous particle is dried below 5% RH. These studies demonstrate the importance of the history of the particle conditioning on subsequent water condensation and re-equilibration dynamics of ultraviscous and glassy aerosol particles.


Differential contribution of basic residues to HIV-1 nucleocapsid protein’s nucleic acid chaperone function and retroviral replication

Hao Wu, Mithun Mitra, M. Nabuan Naufer, Micah J. McCauley, Robert J. Gorelick, Ioulia Rouzina, Karin Musier-Forsyth and Mark C. Williams

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC’s NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC’s aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity.


A high-speed vertical optical trap for the mechanical testing of living cells at piconewton forces

Kai Bodensiek, Weixing Li, Paula Sánchez, Schanila Nawaz and Iwan A. T. Schaap

Although atomic force microscopy is often the method of choice to probe the mechanical response of (sub)micrometer sized biomaterials, the lowest force that can be reliably controlled is limited to ≈0.1 nN. For soft biological samples, like cells, such forces can already lead to a strain large enough to enter the non-elastic deformation regime. To be able to investigate the response of single cells at lower forces we developed a vertical optical trap. The force can be controlled down to single piconewtons and most of the advantages of atomic force microscopy are maintained, such as the symmetrical application of forces at a wide range of loading rates. Typical consequences of moving the focus in the vertical direction, like the interferometric effect between the bead and the coverslip and a shift of focus, were quantified and found to have negligible effects on our measurements. With a fast responding force feedback loop we can achieve deformation rates as high as 50 μm/s, which allow the investigation of the elastic and viscous components of very soft samples. The potential of the vertical optical trap is demonstrated by measuring the linearity of the response of single cells at very low forces and a high bandwidth of deformation rates.


Holographic optical tweezers combined with back-focal-plane displacement detection

Ferran Marsà, Arnau Farré, Estela Martín-Badosa, and Mario Montes-Usategui

A major problem with holographic optical tweezers (HOTs) is their incompatibility with laser-based position detection methods, such as back-focal-plane interferometry (BFPI). The alternatives generally used with HOTs, like high-speed video tracking, do not offer the same spatial and temporal bandwidths. This has limited the use of this technique in precise quantitative experiments. In this paper, we present an optical trap design that combines digital holography and back-focal-plane displacement detection. We show that, with a particularly simple setup, it is possible to generate a set of multiple holographic traps and an additional static non-holographic trap with orthogonal polarizations and that they can be, therefore, easily separated for measuring positions and forces with the high positional and temporal resolutions of laser-based detection. We prove that measurements from both polarizations contain less than 1% crosstalk and that traps in our setup are harmonic within the typical range. We further tested the instrument in a DNA stretching experiment and we discuss an interesting property of this configuration: the small drift of the differential signal between traps.


Single Molecule Applications of Quantum Dots

Thomas E. Rasmussen, Liselotte Jauffred, Jonathan Brewer, Stefan Vogel, Esben R. Torbensen, B. Christoffer Lagerholm, Lene Oddershede, Eva C. Arnspang

Fluorescent nanocrystals composed of semiconductor materials were first introduced for biological applications in the late 1990s. The focus of this review is to give a brief survey of biological applications of quantum dots (QDs) at the single QD sensitivity level. These are described as follows: 1) QD blinking and bleaching statistics, 2) the use of QDs in high speed single particle tracking with a special focus on how to design the biofunctional coatings of QDs which enable specific targeting to single proteins or lipids of interest, 3) a hybrid lipid-DNA analogue binding QDs which allows for tracking single lipids in lipid bilayers, 4) two-photon fluorescence correlation spectroscopy of QDs and 5) optical trapping and excitation of single QDs. In all of these applications, the focus is on the single particle sensitivity level of QDs. The high applicability of QDs in live cell imaging experiments held together with the prospects in localization microscopy and single molecule manipulation experiments gave QDs a promising future in single molecule research.


Determining the Specificity of Monoclonal Antibody HPT-101 to Tau-Peptides with Optical Tweezers

Tim Stangner, Carolin Wagner, David Singer, Stefano Angioletti-Uberti, Christof Gutsche, Joachim Dzubiella, Ralf Hoffmann, and Friedrich Kremer

Optical tweezers-assisted dynamic force spectroscopy is employed to investigate specific receptor–ligand interactions on the level of single binding events. In particular, we analyze binding of the phosphorylation-specific monoclonal antibody (mAb) HPT-101 to synthetic tau-peptides with two potential phosphorylation sites (Thr231 and Ser235), being the most probable markers for Alzheimer’s disease. Whereas the typical interpretation of enzyme-linked immunosorbent assay (ELISA) suggests that this monoclonal antibody binds exclusively to the double-phosphorylated tau-peptide, we show here by DFS that the specificity of only mAb HPT-101 is apparent. In fact, binding occurs also to each sort of monophosphorylated peptide. Therefore, we characterize the unbinding process by analyzing the measured rupture force distributions, from which the lifetime of the bond without force τ0, its characteristic length xts, and the free energy of activation ΔG are extracted for the three mAb/peptide combinations. This information is used to build a simple theoretical model to predict features of the unbinding process for the double-phosphorylated peptide purely based on data on the monophosphorylated ones. Finally, we introduce a method to combine binding and unbinding measurements to estimate the relative affinity of the bonds. The values obtained for this quantity are in accordance with ELISA, showing how DFS can offer important insights about the dynamic binding process that are not accessible with this common and widespread assay.


Friday, December 13, 2013

Theory and experiment on particle trapping and manipulation via optothermally generated bubbles

Chenglong Zhao, Yuliang Xie, Zhangming Mao, Yanhui Zhao, Joseph Rufo, Shikuan Yang, Feng Guo, John D. Mai and Tony Jun Huang

We present a theoretical analysis and experimental demonstration of particle trapping and manipulation around optothermally generated bubbles. We show that a particle located within 500 μm of a surface bubble can be attracted towards a bubble by drag force resulting from a convective flow. Once the particle comes in contact with the bubble's surface, a balance between surface tension forces and pressure forces traps the particle on the bubble surface, allowing the particle to move with the bubble without detaching. The proposed mechanism is confirmed by computational fluid dynamics simulations, force calculations, and experiments. Based on this mechanism, we experimentally demonstrated a novel approach for manipulating microparticles via optothermally generated bubbles. Using this approach, randomly distributed microparticles were effectively collected and carried to predefined locations. Single particles were also manipulated along prescribed trajectories. This bubble-based particle trapping and manipulation technique can be useful in applications such as micro assembly, particle concentration, and high-precision particle separation.


Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein

Kathy R. Chaurasiya, Micah J. McCauley, Wei Wang, Dominic F. Qualley, Tiyun Wu, Shingo Kitamura, Hylkje Geertsema, Denise S. B. Chan, Amber Hertz, Yasumasa Iwatani, Judith G. Levin, Karin Musier-Forsyth, Ioulia Rouzina & Mark C. Williams

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalysed DNA elongation. Here, we show that APOBEC3G initially binds ssDNA with rapid on–off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription.


Raman spectroscopic investigations on optical trap induced deoxygenation of red blood cells

Sunita Ahlawat, Nitin Kumar, Raktim Dasgupta, Ravi Shanker Verma, Abha Uppal and Pradeep Kumar Gupta

Raman spectroscopic investigations on the oxygenation status of optically trapped red blood cells show that the cellular site in the trap beam is more deoxygenated compared to the rest of the cell, and the level of deoxygenation increases with an increase in the trap beam power. These observations and the changes in the Raman spectrum of hemoglobin solution as a function of the trapping beam power suggest that observed deoxygenation may be due to photodissociation of oxygen from hemoglobin at increased trapping power.


White Light Trapping Using Supercontinuum Generation Spectra in a Lead-Silicate Fibre Taper

Pengfei Wang; Lee, T. ; Ming Ding ; Zhenggang Lian ; Xian Feng ; Youqiao Ma ; Lin Bo ; Qiang Wu ; Semenova, Y. ; Wei Loh ; Farrell, G. ; Brambilla, G.

We experimentally investigate white light optical trapping by generating a supercontinuum in a lead silicate fibre pumped by femtosecond pulses from a Ti:Sapphire laser near the zero-dispersion wavelength of 1030 nm, before confining the light using a microfibre half taper with a final tip diameter of 75 nm. Due to the high intensity gradient at the output, robust optical trapping is possible, as demonstrated for individual yeast cells using an average pumping power of 100 mW.


Observer-Based Optical Manipulation of Biological Cells With Robotic Tweezers

Cheah, C.C. ; Li, X. ; Yan, X. ; Sun, D.

While several automatic manipulation techniques have recently been developed for optical tweezer systems, the measurement of the velocity of cell is required and the interaction between the cell and the manipulator of laser source is usually ignored in these formulations. Although the position of cell can be measured by using a camera, the velocity of cell is not measurable and usually estimated by differentiating the position of cell, which amplifies noises and may induce chattering of the system. In addition, it is also assumed in existing methods that the image Jacobian matrix from the Cartesian space to image space of the camera is exactly known. In the presence of estimation errors or variations of depth information between the camera and the cell, it is not certain whether the stability of the system could still be ensured. In this paper, vision-based observer techniques are proposed for optical manipulation to estimate the velocity of cell. Using the proposed observer techniques, tracking control strategies are developed to manipulate biological cells with different Reynolds numbers, which do not require camera calibration and measurement of the velocity of cell. The control methods are based on the dynamic formulation where the laser source is controlled by the closed-loop robotic manipulation technique. The stability is analyzed using Lyapunov-like analysis. Simulation and experimental results are presented to illustrate the performance of the proposed cell manipulation methods.


Living cell manipulation in a microfluidic device by femtosecond optical tweezers

Yan Li, Zhongyi Guo, Shiliang Qu

We have realized cell sorting and manipulation in a fabricated microfluidic device by a self-constructed femtosecond optical tweezer efficaciously. The used microfluidic device with two micro-pools inside silica glass is fabricated by water-assisted femtosecond laser ablation and subsequent heat treatment. After the heat treatment, the diameter of the fabricated microchannels could be reduced significantly and the internal surface of the device could also be made much smoother comparatively, which was crucial for the subsequent experiments of living cell manipulation and cell sorting by using femtosecond optical tweezers because of the optical beam quality required. Our experimental results show that we can manipulate cells very easily by our self-constructed femtosecond optical tweezer, which demonstrates that the incorporation of the microfluidic device and the femtosecond optical tweezer is accessible and practical for the micromanipulation experiments, such as cell sorting and manipulation.


Thursday, December 12, 2013

Hydrodynamic effects in the measurement of interparticle forces in nematic colloids

Kuniyoshi Izaki and Yasuyuki Kimura

We propose improved measurement methods of interparticle force between nematic colloids. Although various methods have been utilized for the force measurement, the comparison between the forces obtained by different methods has not been reported. In the frequently used method called the “free-release” method, the hydrodynamic interaction between moving particles has a serious influence on the measurement. In this study we modified those measurement methods by taking the long-ranged hydrodynamic interaction into account. The evaluated forces have been compared with that obtained by the dual beam “optical trap” method, which is free from the hydrodynamic effect. The agreement between them is quantitatively fairly good.


Optical Nonlinearities and Enhanced Light Transmission in Soft-Matter Systems with Tunable Polarizabilities

Weining Man, Shima Fardad, Ze Zhang, Jai Prakash, Michael Lau, Peng Zhang, Matthias Heinrich, Demetrios N. Christodoulides, and Zhigang Chen

We demonstrate a new class of synthetic colloidal suspensions capable of exhibiting negative polarizabilities, and observe for the first time robust propagation and enhanced transmission of self-trapped light over long distances that would have been otherwise impossible in conventional suspensions with positive polarizabilities. Such light penetration through the strong scattering environment is attributed to the interplay between optical forces and self-activated transparency effects while no thermal effect is involved. By judiciously mixing colloidal particles of both negative and positive polarizabilities, we show that the resulting nonlinear response of these systems can be fine-tuned. Our experimental observations are in agreement with theoretical analysis based on a thermodynamic model that takes into account particle-particle interactions. These results may open up new opportunities in developing soft-matter systems with engineered optical nonlinearities.


Bacterial nucleoid structure probed by active drag and resistive pulse sensing

Vivek V. Thacker, Krystyna Bromek, Benoit Meijer, Jurij Kotar, Bianca Sclavi, Marco Cosentino Lagomarsino, Ulrich F. Keyser and Pietro Cicuta

Recent biophysical approaches have provided key insights into the enthalpic and entropic forces that compact the nucleoid in the cell. Our biophysical approach combines two complementary, non-invasive and label-free techniques: a precisely timed steerable optical trap and a high throughput microcapillary Coulter counter. We demonstrate the ability of the latter technique to probe the physical properties and size of many purified nucleoids, at the individual nucleoid level. The DNA-binding protein H-NS is central to the organization of the bacterial genome. Our results show that nucleoids purified from the Δhns strain in the stationary phase expand approximately five fold more than the form observed in WT bacteria. This compaction is consistent with the role played by H-NS in regulating the nucleoid structure and the significant organizational changes that occur as the cell adapts to the stationary phase. We also study the permeability to the flow of ions and find that in the experiment nucleoids behave as solid colloids.


Optical trapping of red blood cells in living animals with a water immersion objective

Min-Cheng Zhong, Lei Gong, Jin-Hua Zhou, Zi-Qiang Wang, and Yin-Mei Li

We demonstrate optical trapping of red blood cells (RBCs) in living animals by using a water immersion objective. First, the cells within biological tissue are mimicked by the particles immersed in aqueous solutions of glycerol. The optical forces depending on trapping depth are investigated when a parallel laser beam enters the water immersion objective. The results show that the optical forces vary with trapping depth, and the optimal trapping depth in aqueous solutions of glycerol (n=1.39) is 50 μm. Second, the optimal trapping depth in aqueous solutions of glycerol can be changed by altering the actual tube length of the water immersion objective. Finally, we achieved optical trapping and manipulation of RBCs in living mice.


Monday, December 9, 2013

Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

Tobias Sawetzki, Charles D. Eggleton, Sanjay A. Desai, David W.M. Marr

The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods.


Direct Observation of Phosphate Inhibiting the Force-Generating Capacity of a Miniensemble of Myosin Molecules

Edward P. Debold, Sam Walcott, Mike Woodward, Matthew A. Turner

Elevated levels of phosphate (Pi) reduce isometric force, providing support for the notion that the release of Pi from myosin is closely associated with the generation of muscular force. Pi is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of Pi on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of Pi on the force-generating capacity of a small ensemble of myosin (∼12 myosin heads) using a three-bead laser trap assay. In the absence of Pi, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM Pi, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated Pi also caused a >65% reduction in binding-event lifetime, suggesting that Pi induces premature detachment from a strongly bound state. Definitive evidence of a Pi-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which Pi induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of Pi on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that Pi-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.


Mode-based microparticle conveyor belt in air-filled hollow-core photonic crystal fiber

Oliver A. Schmidt, Tijmen G. Euser, and Philip St.J. Russell

We show how microparticles can be moved over long distances and precisely positioned in a low-loss air-filled hollow-core photonic crystal fiber using a coherent superposition of two co-propagating spatial modes, balanced by a backward-propagating fundamental mode. This creates a series of trapping positions spaced by half the beat-length between the forward-propagating modes (typically a fraction of a millimeter). The system allows a trapped microparticle to be moved along the fiber by continuously tuning the relative phase between the two forward-propagating modes. This mode-based optical conveyor belt combines long-range transport of microparticles with a positional accuracy of 1 µm. The technique also has potential uses in waveguide-based optofluidic systems.


An Optical Trap Combined with Three-Color FRET

Sanghwa Lee and Sungchul Hohng

We developed a hybrid technique combining optical tweezers and single-molecule three-color fluorescence resonance energy transfer (FRET). In demonstrative experiments, we observed the force-sensitive correlated motion of three helical arms of a Holliday junction and identified the independent unfolding/folding dynamics of two DNA hairpins of the same length. With 3 times the number of observable elements of single-molecule FRET, this new instrument will enable the measurement of the complex, multidimensional effects of mechanical forces in various biomolecular systems, such as RNA and proteins.


Mechanosensitive channels and bacterial cell wall integrity: does life end with a bang or a whimper?

Marcel Reuter, Nicholas J. Hayward, Susan S. Black, Samantha Miller, David T. F. Dryden and Ian R. Booth

Mechanogated channels are fundamental components of bacterial cells that enable retention of physical integrity during extreme increases in cell turgor. Optical tweezers combined with microfluidics have been used to study the fate of individual Escherichia coli cells lacking such channels when subjected to a bursting stress caused by increased turgor. Fluorescence-activated cell sorting and electron microscopy complement these studies. These analyses show that lysis occurs with a high probability, but the precise path differs between individual cells. By monitoring the loss of cytoplasmic green fluorescent protein, we have determined that some cells release this protein but remain phase dark (granular) consistent with the retention of the majority of large proteins. By contrast, most cells suffer cataclysmic wall failure leading to loss of granularity but with the retention of DNA and overall cell shape (protein-depleted ghosts). The time span of these events induced by hypo-osmotic shock varies but is of the order of milliseconds. The data are interpreted in terms of the timing of mechanosensitive channel gating relative to osmotically induced water influx.


Optical tweezers technique and its applications

HongLian Guo, ZhiYuan Li

Since their advent in the 1980s, optical tweezers have attracted more and more attention due to their unique non-contact and non-invasion characteristics and their wide applications in physics, biology, chemistry, medical science and nanoscience. In this paper, we introduce the basic principle, the history and typical applications of optical tweezers and review our recent experimental works on the development and application of optical tweezers technique. We will discuss in detail several technological issues, including high precision displacement and force measurement in single-trap and dual-trap optical tweezers, multi-trap optical tweezers with each trap independently and freely controlled by means of space light modulator, and incorporation of cylindrical vector optical beams to build diversified optical tweezers beyond the conventional Gaussian-beam optical tweezers. We will address the application of these optical tweezers techniques to study biophysical problems such as mechanical deformation of cell membrane and binding energy between plant microtubule and microtubule associated proteins. Finally we present application of the optical tweezers technique for trapping, transporting, and patterning of metallic nanoparticles, which can be harnessed to manipulate surface plasmon resonance properties of these nanoparticles.


Crystal Growth of Lysozyme Controlled by Laser Trapping

Jing-Ru Tu, Atsushi Miura, Ken-ichi Yuyama, Hiroshi Masuhara, and Teruki Sugiyama

We demonstrate the growth of a tetragonal crystal of hen egg-white lysozyme (HEWL) in D2O buffer solution controlled by laser trapping with a focused continuous-wave (CW) near-infrared (NIR) laser beam. The focal spot was located at 10 μm away from the edge of the target crystal that was generated spontaneously, and the crystal growth was observed although the focal spot size was much smaller than the distance. The growth rate of (101) and {110} faces of the tetragonal crystal was examined with various laser powers and polarizations. The rate observed under the irradiation was much different from those in spontaneous growth, namely, the growth rate of the {110} face showed a large decrease or increase depending on the irradiation time. The dynamics and mechanism of this unusual crystal growth behavior is discussed from the viewpoint of a large stable domain formation of the HEWL liquidlike clusters through liquid nucleation and growth and by considering the anisotropy of the cluster domain.


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.


Thursday, October 24, 2013

Particle laden fluid interfaces: Dynamics and Interfacial rheology

Alma J. Mendoza, Eduardo Guzman, Fernando Martinez-Pedrero, Herman Ritacco, Ramon G. Rubio, Francisco Ortega, Victor M. Starov, Reinhard Miller
We review the dynamics of particle laden interfaces, both particle monolayers and particle + surfactant monolayers. We also discuss the use of the Brownian motion of microparticles trapped at fluid interfaces for measuring the shear rheology of surfactant and polymer monolayers. We describe the basic concepts of interfacial rheology and the different experimental methods for measuring both dilational and shear surface complex moduli over a broad range of frequencies, with emphasis in the micro-rheology methods. In the case of particles trapped at interfaces the calculation of the diffusion coefficient from the Brownian trajectories of the particles is calculated as a function of particle surface concentration. We describe in detail the calculation in the case of subdiffusive particle dynamics. A comprehensive review of dilational and shear rheology of particle monolayers and particle + surfactant monolayers is presented. Finally the advantages and current open problems of the use of the Brownian motion of microparticles for calculating the shear complex modulus of monolayers are described in detail.


Tuesday, October 22, 2013

Dislocation reactions, grain boundaries, and irreversibility in two-dimensional lattices using topological tweezers

William T. M. Irvine, Andrew D. Hollingsworth, David G. Grier, and Paul M. Chaikin

Dislocations, disclinations, and grain boundaries are topological excitations of crystals that play a key role in determining out-of-equilibrium material properties. In this article we study the kinetics, creation, and annihilation processes of these defects in a controllable way by applying “topological tweezers,” an array of weak optical tweezers which strain the lattice by weakly pulling on a collection of particles without grabbing them individually. We use topological tweezers to deterministically control individual dislocations and grain boundaries, and reversibly create and destroy dislocation pairs in a 2D crystal of charged colloids. Starting from a perfect lattice, we exert a torque on a finite region and follow the complete step-by-step creation of a disoriented grain, from the creation of dislocation pairs through their reactions to form a grain boundary and their reduction of elastic energy. However, when the grain is rotated back to its original orientation the dislocation reactions do not retrace. Rather, the process is irreversible; the grain boundary expands instead of collapsing.

Monday, October 21, 2013

Lipid Droplets Purified from Drosophila Embryos as an Endogenous Handle for Precise Motor Transport Measurements

Tobias F. Bartsch, Rafael A. Longoria, Ernst-Ludwig Florin, and George T. Shubeita
Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport.

Optical trapping of microparticles from a stream in vacuum

D. A. Plutenko, O. M. Sarkisov, V. A. Nadtochenko
The feasibility of trapping microparticles from a stream in vacuum using dynamic optical tweezers is proven theoretically. A new approach to stabilizing particles and cooling the translational degrees of freedom by modulating power in the laser trap is presented.

Thursday, October 17, 2013

High bandwidth optical force clamp for investigation of molecular motor motion

Subhrajit Roychowdhury, Tanuj Aggarwal, Srinivasa Salapaka and Murti V. Salapaka
Use of optical tweezers for load force regulation on processive motors has yielded significant insights into intracellular transport mechanisms. The methodology developed in this letter circumvents the limitations of existing active force clamps with the use of experimentally determined models for various components of the optical tweezing system, thus making it possible to probe motor proteins at higher speeds. This paradigm also allows for real-time step estimation for step sizes as small as 8 nm with dwell time of 5 ms or higher without sacrificing force regulation.

A Landau-Squire nanojet

Nadanai Laohakunakorn , Benjamin Gollnick , Fernando Moreno-Herrero , Dirk Aarts , Roel P.A. Dullens , Sandip Ghosal , and Ulrich F Keyser
Fluid jets are found in nature at all length scales – microscopic to cosmological. Here we report on an electroosmotically driven jet from a single glass nanopore about 75 nm in radius with a maximum flow rate ~ 30 pL/s. A novel anemometry technique allows us to map out the vorticity and velocity fields which show excellent agreement with the classical Landau-Squire solution of the Navier Stokes equations for a point jet. We observe a phenomenon that we call flow rectification: an asymmetry in the flow rate with respect to voltage reversal. Such a nanojet could potentially find applications in micro manipulation, nano patterning, and as a diode in microfluidic circuits.

Enhanced Optical Forces by Hybrid Long-Range Plasmonic Waveguides

Lin Chen, Tian Zhang, and Xun Li
Compared with optical resonant structures, current plasmonic waveguides have the advantage of enhancing optical forces in a broad range of wavelengths, but the enhancement can only be maintained for several dozens of microns at 1.55 μm. Here, a hybrid long-range plasmonic waveguide, consisting of two identical dielectric nanowires symmetrically placed on each side of a thin metal film, is proposed for optical forces. Strong optical coupling between the dielectric waveguide mode and long-range plasmonic mode leads to enhanced optical forces on the dielectric nanowire at low input optical power due to the deep subwavelength optical energy confinement. The enhancement can be maintained for distances of 1∼2 orders of magnitude larger than that of previous plasmonic waveguides. The deep subwavelength optical confinement as well as enhanced field gradient also allows eff icient trapping of single nanoscale particle, while the smaller propagation loss ensures a much larger trapping region at the same input optical power. The present results enable the potential applications of precisely controlling the positions of dielectric nanowires as well as manipulating a single nanoparticle such as a biomolecule and one quantum dot.

Tailoring photonic forces on a magnetodielectric nanoparticle with a fluctuating optical source

Juan Miguel Auñón, Cheng Wei Qiu, and Manuel Nieto-VesperinasWe address the forces exerted by the random electromagnetic field emitted by a fluctuating optical source on a kind of dielectric nanoparticles that have arisen much interest because of their recently shown magnetodielectric behavior. The illumination with light, or other electromagnetic wave, of a given state of coherence allows us to create photonic forces, a particular case of which are optical analogous to the Casimir-Polder and van der Waals forces, as well as of thermal forces out of thermodynamic equilibrium. This leads to a deeper understanding of the conditions and limitations under which some theories of these forces were established. We also study the effects of the coherence length and of sharp changes in the particle differential scattering cross section due to Kerker minimum forward or zero backward conditions. We show how the nanoparticle Mie resonances, constituted by the induced electric and magnetic dipoles, lead to long distance attractions to the source, as well as to the possible predominance of magnetic forces. In addition, it is shown how, by manipulating the fluctuating source, either pushing or tractor beams are obtained, even in the far zone. These effects are specially relevant when quasimonochromatic emission is employed, and manifest the possibility of performing a monitoring of these mechanical interactions, in particular by a photonic analogy of those aforementioned classical thermal forces. This opens paths to nanoparticle ensembling and manipulation. The influence of the excitation of surface waves of the source is also considered.

Friday, October 11, 2013

Direct measurement of osmotic pressure via adaptive confinement of quasi hard disc colloids

Ian Williams, Erdal C. Oguz, Paul Bartlett, Hartmut Lowen and C. Patrick Royall
Confining a system in a small volume profoundly alters its behaviour. Hitherto, attention has focused on static confinement where the confining wall is fixed such as in porous media. However, adaptive confinement where the wall responds to the interior has clear relevance in biological systems. Here we investigate this phenomenon with a colloidal system of quasi hard discs confined by a ring of particles trapped in holographic optical tweezers, which form a flexible elastic wall. This elasticity leads to quasi-isobaric conditions within the confined region. By measuring the displacement of the tweezed particles, we obtain the radial osmotic pressure. We further find a novel bistable state of a hexagonal structure and concentrically layered fluid mimicking the shape of the confinement. The hexagonal configurations are found at lower pressure than those of the fluid, thus the bistability is driven by the higher entropy of disordered arrangements, unlike bulk hard systems.


Radiation force of abruptly autofocusing Airy beams on a Rayleigh particle

Yunfeng Jiang, Kaikai Huang, and Xuanhui Lu
The radiation force of circular Airy beams (CAB) on a dielectric Rayleigh particle is investigated in this paper. Our results show that the CAB can be used to trap the particle whose refractive index is larger than the ambient at different positions along the beam axis. Comparing with the Gaussian beam under the same conditions, the longitudinal and the transverse gradient force of CAB on the Rayleigh particle are increased, and the particle can be trapped more stable. Our analyses also demonstrate that the trapping properties of CAB can be modulated by controlling corresponding parameters of CAB.

Thursday, October 10, 2013

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 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 the laser irradiation into the H2O solution, laser trapping of the liquid-like clusters increases 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 concentration is high enough to trigger crystal nucleation, but in 0.5–1.5 millimetres range from the focal spot. The dynamics and mechanism of the amazing crystallization behaviour induced by laser trapping are discussed from the viewpoints of concentration increase due to laser heating depending on solvent, large high concentration domain formation by laser trapping of liquid-like clusters, and orientational disorder of molecules/clusters at the domain edge.

Phase-separation and photoresponse in binary azobenzene-containing polymer vesicles

Guosheng Xue, Kun Chen, Guangyong Shen, Ziqiang Wang, Qijin Zhang, Jun Cai , Yinmei Li

Understanding the photoresponse of azobenzene polymer in different conditions is essential for the potential application of azobenzene-based technologies. Herein, the microscale, photoresponsive hybrid polymersomes (polymer vesicles) composed of binary blends of azobenzene-containing block copolymers is prepared. The Janus morphology which presents phase-separation within the surface of hybrid polymersomes is observed. The composition and photoisomerization characteristic time of different domains are studied with Laser Trapping Raman Spectroscope (LTRS) system. The results indicated that the morphology of polymersomes can be tuned by the ratio of azobenzene-containing copolymer contents. We find the photoisomerization rate of azobenzene in hybrid vesicles is marginally slower than those in pure vesicles. These experiments provide a quantitative measurement method for dynamic photoresponse of azobenzene hybrid polymersomes.


The Role of Vimentin Intermediate Filaments in Cortical and Cytoplasmic Mechanics

Ming Guo, Allen J. Ehrlicher, Saleemulla Mahammad|, Hilary Fabich, Mikkel H. Jensen, Jeffrey R. Moore, Jeffrey J. Fredberg, Robert D. Goldman, David A. Weitz
The mechanical properties of a cell determine many aspects of its behavior, and these mechanics are largely determined by the cytoskeleton. Although the contribution of actin filaments and microtubules to the mechanics of cells has been investigated in great detail, relatively little is known about the contribution of the third major cytoskeletal component, intermediate filaments (IFs). To determine the role of vimentin IF (VIF) in modulating intracellular and cortical mechanics, we carried out studies using mouse embryonic fibroblasts (mEFs) derived from wild-type or vimentin−/− mice. The VIFs contribute little to cortical stiffness but are critical for regulating intracellular mechanics. Active microrheology measurements using optical tweezers in living cells reveal that the presence of VIFs doubles the value of the cytoplasmic shear modulus to ∼10 Pa. The higher levels of cytoplasmic stiffness appear to stabilize organelles in the cell, as measured by tracking endogenous vesicle movement. These studies show that VIFs both increase the mechanical integrity of cells and localize intracellular components.

A microengineered cell fusion approach with combined optical tweezers and microwell array technologies

Xiaolin Wang, Shuxun Chen, Yu Ting Chow, Chi-Wing Kong, Ronald A Li and Dong Sun

Cell fusion in vitro can be artificially achieved through microengineering technology. This paper presents a laser-induced cell fusion methodology on the microwell array-based microfluidic chip, with high selectivity and controllability at the single cell level. Optical tweezers and optical scissors are employed to achieve cell pairing and fusion, respectively. The specific cells are first characterized with high spatio-temporal resolution and preselected from the mixture through an on-chip isolation method prior to pairing. The paired cells are then transported, deposited, fused, and released at the desired location with high controllability. Biophysical analysis on the fused cells shows that the fusion efficiency of the homotypical pairs is higher than that of the heterogenic pairs obtained based on different combinations of sample cells, such as human embryonic stem cells and Jurkat cells. This laser-induced cell fusion technique offers a new opportunity to explore specific targeted therapy in stem cell research for the treatment of human diseases.

Friday, October 4, 2013

Invited Article: A review of haptic optical tweezers for an interactive microworld exploration

Cécile Pacoret and Stéphane Régnier
This paper is the first review of haptic optical tweezers, a new technique which associates force feedback teleoperation with optical tweezers. This technique allows users to explore the microworld by sensing and exerting picoNewton-scale forces with trapped microspheres. Haptic optical tweezers also allow improved dexterity of micromanipulation and micro-assembly. One of the challenges of this technique is to sense and magnify picoNewton-scale forces by a factor of 10^12 to enable human operators to perceive interactions that they have never experienced before, such as adhesion phenomena, extremely low inertia, and high frequency dynamics of extremely small objects. The design of optical tweezers for high quality haptic feedback is challenging, given the requirements for very high sensitivity and dynamic stability. The concept, design process, and specification of optical tweezers reviewed here are focused on those intended for haptic teleoperation. In this paper, two new specific designs as well as the current state-of-the-art are presented. Moreover, the remaining important issues are identified for further developments. The initial results obtained are promising and demonstrate that optical tweezers have a significant potential for haptic exploration of the microworld. Haptic optical tweezers will become an invaluable tool for force feedback micromanipulation of biological samples and nano- and micro-assembly parts.

The molecular yo-yo method: Live jump detection improves throughput of single-molecule force spectroscopy for out-of-equilibrium transitions

A. H. Mack, D. J. Schlingman, M. Kamenetska, R. Collins, L. Regan, and S. G. J. Mochrie

By monitoring multiple molecular transitions, force-clamp, and trap-position-clamp methods have led to precise determinations of the free energies and free energy landscapes for molecular states populated in equilibrium at the same or similar forces. Here, we present a powerful new elaboration of the force-clamp and force-jump methods, applicable to transitions far from equilibrium. Specifically, we have implemented a live jump detection and force-clamp algorithm that intelligently adjusts and maintains the force on a single molecule in response to the measured state of that molecule. We are able to collect hundreds of individual molecular transitions at different forces, many times faster than previously, permitting us to accurately determine force-dependent lifetime distributions and reaction rates. Application of our method to unwinding and rewinding the nucleosome inner turn, using optical tweezers reveals experimental lifetime distributions that comprise a statistically meaningful number of transitions, and that are accurately single exponential. These measurements significantly reduce the error in the previously measured rates, and demonstrate the existence of a single, dominant free energy barrier at each force studied. A key benefit of the molecular yo-yo method for nucleosomes is that it reduces as far as possible the time spent in the tangentially bound state, which minimizes the loss of nucleosomes by dissociation.