Quantitative optical trapping on single organelles in cell extract

Pradeep Barak, Ashim Rai, Priyanka Rai & Roop Mallik

We have developed an optical trapping method to precisely measure the force generated by motor proteins on single organelles of unknown size in cell extract. This approach, termed VMatch, permits the functional interrogation of native motor complexes. We apply VMatch to measure the force, number and activity of kinesin-1 on motile lipid droplets isolated from the liver of normally fed and food-deprived rats.
DOI

PilT2 enhances the speed of gonococcal type IV pilus retraction and of twitching motility

Rainer Kurre, Andrea Höne, Martin Clausen, Claudia Meel, Berenike Maier

Type IV pilus (T4P) dynamics is important for various bacterial functions including host cell interaction, surface motility, and horizontal gene transfer. T4P retract rapidly by depolymerization, generating large mechanical force. The gene that encodes the pilus retraction ATPase PilT has multiple paralogues, whose number varies between different bacterial species, but their role in regulating physical parameters of T4P dynamics remains unclear. Here, we address this question in the human pathogen Neisseria gonorrhoeae, which possesses two pilT paralogues, namelypilT2 and pilU. We show that the speed of twitching motility is strongly reduced in a pilT2 deletion mutant, while directional persistence time and sensitivity of speed to oxygen are unaffected. Using laser tweezers, we found that the speed of single T4P retraction was reduced by a factor of ≈ 2 in a pilT2 deletion strain, whereas pilU deletion showed a minor effect. The maximum force and the probability for switching from retraction to elongation under application of high force were not significantly affected. We conclude that the physical parameters of T4P are fine-tuned through PilT2.
DOI

An optically actuated surface scanning probe

D. B. Phillips, G. M. Gibson, R. Bowman, M. J. Padgett, S. Hanna, D. M. Carberry, M. J. Miles, and S. H. Simpson

We demonstrate the use of an extended, optically trapped probe that is capable of imaging surface topography with nanometre precision, whilst applying ultra-low, femto-Newton sized forces. This degree of precision and sensitivity is acquired through three distinct strategies. First, the probe itself is shaped in such a way as to soften the trap along the sensing axis and stiffen it in transverse directions. Next, these characteristics are enhanced by selectively position clamping independent motions of the probe. Finally, force clamping is used to refine the surface contact response. Detailed analyses are presented for each of these mechanisms. To test our sensor, we scan it laterally over a calibration sample consisting of a series of graduated steps, and demonstrate a height resolution of ∼ 11 nm. Using equipartition theory, we estimate that an average force of only ∼ 140 fN is exerted on the sample during the scan, making this technique ideal for the investigation of delicate biological samples.

DOI

Mapping the local dielectric response at the nanoscale by means of plasmonic force spectroscopy

Francesco De Angelis, Remo Proietti Zaccaria, and Enzo Di Fabrizio
At the present, the local optical properties of nanostructured materials are difficult to be measured by available instrumentation. We investigated the capability of plasmonic force spectroscopy of measuring the optical response at the nanoscale. The proposed technique is based on force measurements performed by combining Atomic Force Microscopy, or optical tweezers, and adiabatic compression of surface plasmon polaritons. We show that the optical forces, caused by the plasmonic field, depend on the local response of the substrates and, in principle, allow probing both the real and the imaginary part of the local permittivity with a spatial resolution of few nanometers.
DOI

Angular orientation of nanorods using nanophotonic tweezers

Pilgyu Kang, Xavier Serey, Yih-Fan Chen, and David Erickson

Near-field optical techniques have enabled the trapping, transport, and handling of nanoscopic materials much smaller than what can be manipulated with traditional optical tweezers. Here we extend the scope of what is possible by demonstrating angular orientation and rotational control of both biological and nonbiological nanoscale rods using photonic crystal nanotweezers. In our experiments, single microtubules (diameter 25 nm, length 8 μm) and multiwalled carbon nanotubes (outer diameter 110–170 nm, length 5 μm) are rotated by the optical torque resulting from their interaction with the evanescent field emanating from these devices. An angular trap stiffness of κ = 92.8 pN·nm/rad2·mW is demonstrated for the microtubules, and a torsional spring constant of 22.8 pN·nm/rad2·mW is measured for the nanotubes. We expect that this new capability will facilitate the development of high precision nanoassembly schemes and biophysical studies of bending strains of biomolecules.
DOI

Trapping volume control in optical tweezers using cylindrical vector beams

S. E. Skelton, M. Sergides, R. Saija, M. A. Iatì, O. M. Maragó, and P. H. Jones
We present the result of an investigation into the optical trapping of spherical microparticles using laser beams with a spatially inhomogeneous polarization direction [cylindrical vector beams (CVBs)]. We perform three-dimensional tracking of the Brownian fluctuations in the position of a trapped particle and extract the trap spring constants. We characterize the trap geometry by the aspect ratio of spring constants in the directions transverse and parallel to the beam propagation direction and evaluate this figure of merit as a function of polarization angle. We show that the additional degree of freedom present in CVBs allows us to control the optical trap strength and geometry by adjusting only the polarization of the trapping beam. Experimental results are compared with a theoretical model of optical trapping using CVBs derived from electromagnetic scattering theory in the T-matrix framework.

DOI

Flow-dependent double-nanohole optical trapping of 20 nm polystyrene nanospheres

Ana Zehtabi-Oskuie, Jarrah Gerald Bergeron, and Reuven Gordon

We study the influence of fluid flow on the ability to trap optically a 20 nm polystyrene particle from a stationary microfluidic environment and then hold it against flow. Increased laser power is required to hold nanoparticles as the flow rate is increased, with an empirical linear dependence of 1 μl/(min×mW). This is promising for the delivery of additional nanoparticles to interact with a trapped nanoparticle; for example, to study protein-protein interactions, and for the ability to move the trapped particle in solution from one location to another.
DOI

Mapping 3D Focal Intensity Exposes the Stable Trapping Positions of Single Nanoparticles

Anders Kyrsting , Poul M. Bendix , and Lene B. Oddershede
The photonic interactions between a focused Gaussian laser beam and a nanoscopic particle are highly dependent on exact particle location and focal intensity distribution. So far, the 3D focal intensity distribution and the preferred position of a nanoparticle confined within the focal region were only theoretically predicted. Here, we directly map the three-dimensional focal intensity distribution, quantify stable trapping positions, and prove that certain sizes of nanoparticles stably trap in front of the focus.

DOI

Antibody Binding to Cryptococcus neoformans Impairs Budding by Altering Capsular Mechanical Properties

Radames J. B. Cordero, Bruno Pontes, Susana Frases, Antonio S. Nakouzi, Leonardo Nimrichter, Marcio L. Rodrigues, Nathan B. Viana and Arturo Casadevall

Abs to microbial capsules are critical for host defense against encapsulated pathogens, but very little is known about the effects of Ab binding on the capsule, apart from producing qualitative capsular reactions (“quellung” effects). A problem in studying Ab–capsule interactions is the lack of experimental methodology, given that capsules are fragile, highly hydrated structures. In this study, we pioneered the use of optical tweezers microscopy to study Ab–capsule interactions. Binding of protective mAbs to the capsule of the fungal pathogen Cryptococcus neoformans impaired yeast budding by trapping newly emerging buds inside the parental capsule. This effect is due to profound mAb-mediated changes in capsular mechanical properties, demonstrated by a concentration-dependent increase in capsule stiffness. This increase involved mAb-mediated cross-linking of capsular polysaccharide molecules. These results provide new insights into Ab-mediated immunity, while suggesting a new nonclassical mechanism of Ab function, which may apply to other encapsulated pathogens. Our findings add to the growing body of evidence that Abs have direct antimicrobial functions independent of other components of the immune system.
DOI

Lateral migration of an elastic capsule by optical force in a uniform flow

Cheong Bong Chang, Wei-Xi Huang, and Hyung Jin Sung

The lateral migration of an elastic capsule under an optical force in a uniform flow was studied to show the separation characteristics according to the elastic properties in the cross-type optical separator. The initially spherical capsule was moved through the fluid flow using a laser beam with a Gaussian distribution focused along the direction normal to the flow device surface. To simulate such a system, a penalty immersed boundary method was adopted to enable fluid-membrane coupling, and a dynamic ray tracing method was applied to the optical force calculation. The effects of the elastic properties of the capsule membrane (the surface Young's modulus and the bending modulus) on the lateral migration were studied. By increasing the surface Young's modulus, the capsule deformed less and the migration distance increased; however, buckling occurred in the capsule with a high surface Young's modulus. Buckling could be suppressed by increasing the bending rigidity. The effects of the flow velocity and the laser beam power were also examined. In the simulation, the S number, i.e., the ratio of the optical force to the viscous force, was adjusted by decreasing the flow velocity or increasing the laser beam power. The migration distance increased as the S number increased, and a constant lateral migration distance was obtained for a rigid particle for a given S number. An elastic capsule under conditions intermediate between a fixed flow velocity and a fixed laser beam power, however, did not yield a constant lateral migration distance due to the extent of the deformation in the different situations. To predict the lateral migration distance of an elastic capsule, a nondimensional parameter, Se, was defined to include the effects of the optical force, the elastic force, and the fluid viscous force. A unified tendency of the lateral migration distance with Se was obtained for capsules with intermediate elasticity, by varying either the flow velocity or the laser beam power.
DOI

Quantitative Assessment of the Interplay Between DNA Elasticity and Cooperative Binding of Ligands

L. Siman, I. S. S. Carrasco, J. K. L. da Silva, M. C. de Oliveira, M. S. Rocha, and O. N. Mesquita
Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We propose a methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation. We obtain an expression for the effective persistence length as a function of the total ligand concentration, which we apply to our data of the DNA-cationic β-cyclodextrin and to the DNA-HU protein data available in the literature, determining the values of the local persistence lengths, the dissociation constant, and the degree of cooperativity for each set of data. In both cases the persistence length behaves nonmonotonically as a function of ligand concentration and based on the results obtained we discuss some physical aspects of the interplay between DNA elasticity and cooperative binding of ligands.

DOI

Dynamical instabilities of a Brownian particle in weak adhesion

Deepak Kumar, Shankar Ghosh, and S. Bhattacharya

Dynamical processes involved in weak adhesion are explored through a single cycle of an optically trapped Brownian colloidal silica particle detaching from, and reattaching to, a glass substrate immersed in a fluid in the presence of an externally applied force. Micro-rheology, video-microscopy, and Nyquist noise measurements reveal both stochastic and deterministic dynamics of the process. When analyzed in terms of the viscoelastic response of the stress coupling medium between the objects, the unsticking instability shows remarkable similarities with yielding and fracture-mechanics of macro-scale solids. The resticking dynamics demonstrates stochastic instabilities through a spatiotemporally punctuated descent of the particle down an energy landscape with a hierarchy of metastable minima.

DOI

Future Challenges in Single-Molecule Fluorescence and Laser Trap Approaches to Studies of Molecular Motors

Mary Williard Elting, James A. Spudich

Single-molecule analysis is a powerful modern form of biochemistry, in which individual kinetic steps of a catalytic cycle of an enzyme can be explored in exquisite detail. Both single-molecule fluorescence and single-molecule force techniques have been widely used to characterize a number of protein systems. We focus here on molecular motors as a paradigm. We describe two areas where we expect to see exciting developments in the near future: first, characterizing the coupling of force production to chemical and mechanical changes in motors, and second, understanding how multiple motors work together in the environment of the cell.
DOI

Mechanical Characteristics of Human Red Blood Cell Membrane Changing due to C60 Nano-particles Infiltration

Yue Zheng

The mechanical characteristics of human red blood cell (RBC) membrane changing due to C60 nano-particles (NPs) infiltration have been investigated in present work. Using experimental approaches including optical tweezers (OT) stretching and atomic force microscopy (AFM) indentation, we find that RBCs with C60 NPs presence are softer than normal RBCs. The stress-strain relations of both normal and C60 infiltrated RBC membrane are extracted from the data of AFM indentation, from which we proved that C60 NPs infiltration can affect the mechanical properties of RBC membrane and tend to weaken the tensile resistance of lipids bilayers. In order to explain experimental phenomenon, a mechanical model has been developed. Based on this model, the stress-strain relations of both normal and C60 infiltrated lipids bilayers are calculated with considering intermolecular interaction. The theoretical results are in great agreement with the experimental results. The influence of C60 NP’s concentration on mechanical properties of RBC membrane is successfully predicted. Higher concentrations of C60 NPs in the lipids bilayers will bring more damage to the cell membrane, implying that the dosage of C60 NPs should be controlled in medical applications.
DOI

Mechanical identities of RNA and DNA double helices unveiled at the single-molecule level

Elías Herrero-Galán , Maria Eugenia Fuentes-Perez ,Carolina Carrasco , Jose M Valpuesta , Jose L Carrascosa , Fernando Moreno-Herrero , and J. Ricardo Arias-Gonzalez
Double-stranded (ds) RNA is the genetic material of a variety of viruses and has been recently recognized as a relevant molecule in cells for its regulatory role. Despite the elastic response of dsDNA has been thoroughly characterized in recent years in single-molecule stretching experiments, an equivalent study with dsRNA is still lacking. Here, we have engineered long dsRNA molecules for their individual characterization contrasting information with dsDNA molecules of the same sequence. It is known that dsRNA is an A-form molecule unlike dsDNA which exhibits B-form in physiological conditions. These structural types are distinguished at the single-molecule level with Atomic Force Microscopy (AFM) and are the basis to understand their different elastic response. Force-extension curves of dsRNA with optical and magnetic tweezers manifest two main regimes of elasticity, an entropic regime whose end is marked by the A-form contour-length and an intrinsic regime that ends in a low-cooperative overstretching transition in which the molecule extends to 1.7 times its A-form contour-length. DsRNA does not switch between the A and B conformations in the presence of force. Finally, dsRNA presents both a lower stretch modulus and overstretching transition force than dsDNA, whereas the electrostatic and intrinsic contributions to the persistence length are larger.
DOI

Cytoskeletal Strains in Modeled Optohydrodynamically Stressed Healthy and Diseased Biological Cells

Sean S. Kohles, Yu Liang, and Asit K. Saha

Controlled external chemomechanical stimuli have been shown to influence cellular and tissue regeneration/degeneration, especially with regards to distinct disease sequelae or health maintenance. Recently, a unique three-dimensional stress state was mathematically derived to describe the experimental stresses applied to isolated living cells suspended in an optohydrodynamic trap (optical tweezers combined with microfluidics). These formulae were previously developed in two and three dimensions from the fundamental equations describing creeping flows past a suspended sphere. The objective of the current study is to determine the full-field cellular strain response due to the applied three-dimensional stress environment through a multiphysics computational simulation. In this investigation, the multiscale cytoskeletal structures are modeled as homogeneous, isotropic, and linearly elastic. The resulting computational biophysics can be directly compared with experimental strain measurements, other modeling interpretations of cellular mechanics including the liquid drop theory, and biokinetic models of biomolecule dynamics. The described multiphysics computational framework will facilitate more realistic cytoskeletal model interpretations, whose intracellular structures can be distinctly defined, including the cellular membrane substructures, nucleus, and organelles.

DOI

Acousto-optically generated potential energy landscapes: Potential mapping using colloids under flow

Michael P. N. Juniper, Rut Besseling, Dirk G. A. L. Aarts, and Roel P. A. Dullens
Optical potential energy landscapes created using acousto-optical deflectors are characterized via solvent-driven colloidal particles. The full potential energy of both single optical traps and complex landscapes composed of multiple overlapping traps are determined using a simple force balance argument. The potential of a single trap is shown to be well described by a Gaussian trap with stiffness found to be consistent with those obtained by a thermal equilibrium method. We also obtain directly the depth of the well, which (as with stiffness) varies with laser power. Finally, various complex systems ranging from double-well potentials to random landscapes are generated from individually controlled optical traps. Predictions of these landscapes as a sum of single Gaussian wells are shown to be a good description of experimental results, offering the potential for fully controlled design of optical landscapes, constructed from single optical traps.
DOI

Attractive-repulsive dynamics on light-responsive chiral microparticles induced by polarized tweezers

Raul Josue Hernandez , Alfredo Mazzulla , Alfredo Pane , Karen Volke Sepulveda and Gabriella Cipparrone
Multifunctional colloidal micro and nano-particles with controlled architectures have very promising properties for applications in bio and nanotechnologies. Here we report on the unique dichotomous dynamical behaviour of chiral spherical microparticles, either fluid or solid, manipulated by polarized optical tweezers. The particles are created using a reactive mesogen mixed with a chiral dopant to form cholesteric droplets in water emulsion. The photopolymerization enables to freeze the chiral supramolecular configurations in solid particles. Different internal architectures in the supramolecular structures, guided by the interfacial chemistry, enable to obtain global-optically isotropic or anisotropic spherical objects. Their optical manipulation reveals particular features. We show that the light can exert a repulsive or attractive force on the same particle depending on the handedness of its circular polarization, for particles having radial configuration of the cholesteric helices. We demonstrate that the unique selective reflection property of the cholesteric phase is responsible of the observed effect. On the other hand very exotic dynamics is observed in case of anisotropic chiral particles. Depending on the light handedness, they behave like Janus spherical particles with dissimilar optical properties that mean dielectric particles having a surface that is in part transparent and in part reflecting. The combination of optical manipulation with other control parameters (electric or magnetic), as well as the integration of other functionalities suggest exciting developments for applications of these micro-devices in the micro and optofluidics, microphotonics and materials science.
DOI

Cell elongation via intrinsic antipodal stretching forces

T. Sawetzki, C. D. Eggleton, and D. W. M. Marr
To probe the mechanical properties of cells, we investigate a technique to perform deformability-based cytometry that inherently induces normal antipodal surface forces using a single line-shaped optical trap. We show theoretically that these opposing forces are generated simultaneously over curved microscopic object surfaces with optimal magnitude at low numerical apertures, allowing the directed stretching of elastic cells with a single, weakly focused laser source. Matching these findings with concomitant experimental observations, we elongate red blood cells, effectively stretching them within the narrow confines of a steep, optically induced potential well.
DOI

Confocal Raman Microscopy Probing of Temperature-Controlled Release from Individual, Optically-Trapped Phospholipid Vesiclesan

Jonathan J. Schaefer , Chaoxiong Ma , and Joel M. Harris
Control of permeability of phospholipid vesicle (liposome) membranes is critical to their applications in analytical sensing, in fundamental studies of chemistry in small volumes, and in encapsulation and release of payloads for site-directed drug delivery. Applications of liposome formulations in drug delivery often take advantage of the enhanced permeability of phospholipid membranes at their gel-to-fluid phase transition, where the release of encapsulated molecules can be initiated by an increase in temperature. Despite numerous successful liposome formulations for encapsulation and release methods to study the kinetics, this process has been limited to investigations of bulk vesicle dispersions, which provide little or no information about the vesicle membrane structure and its relationship to the kinetics of trans-membrane transport. In this work, confocal Raman microscopy is adapted to study temperature-dependent release of a model compound, 3-nitrobenzene sulfonate (3-NBS), from individual optically trapped phospholipid vesicles, while simultaneously monitoring structural changes in the vesicle membrane reported by vibrational modes of phospholipid acyl chains and the local environment of the encapsulated compound. The confocal geometry allows efficient excitation and collection of Raman scattering from a single vesicle, while optical trapping allows more than hour-long observations of the same vesicle. With window factor analysis to resolve component spectra, temperature-controlled release of 3-NBS through vesicle membranes composed of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was measured and compared to transport through a lysolipid-containing membrane specifically formulated for efficient drug delivery.

DOI

Dynamic of the Dielectric Nano-Particle in Optical Tweezer Using Counter-Propagating Pulsed Laser Beams

Quang Quy Ho and Dinh Hai Hoang

In this article, the dynamical process of the dielectric particle in the optical tweezer using the counter-propagating Gaussian pulses is investigated by the Langevin equation concerning the Brownian motion. The temporal stabilities of particle is simulated. The influence of the duration, repetition period and delay time between pulses on stability is discussed.

DOI

Computation of Optical Force on Nanoparticles Using Locally Non-Orthogonal Overlapping Yee FDTD Method

Jinjie Liu, Moysey Brio, Jerome V. Moloney

In this paper, a locally non-orthogonal overlapping Yee (OY) FDTD method is proposed in order to accurately calculates the optical force on dielectric and dispersive nanoparticles. It extends our previous work to geometries with sharp corners and dispersive materials. In addition to consistently achieving the smallest errors in comparison to the standard FDTD method, the OY approach is a stable non-orthogonal FDTD method that attains second-order convergence when sharp corners are present.

DOI

Torturing Proteins–Useful Science or Just a Clever Experiment?

Philip Micheal Williams

Unfolding individual proteins by mechanical force has occupied biophysicists for the past 15 years or so. From the initial studies of the muscle protein titin, researchers have studied the effect of force on biomolecules ranging from small enzymes to large ribosomes. The majority of experiments use the atomic force microscope (AFM) as a means to stress proteins tethered between a surface and the probe, and elegant molecular biology approaches are used to produce constructs of repeating protein units which, when stretched and unfolded, produce a characteristic saw-toothed pattern that can be used to confirm complete or partial unfolding. In 2005 my colleague Jane Clarke and I noted that much of the work that had been done by then was to demonstrate the power of the AFM as a tool and that forced unfolding was beginning to move beyond a descriptive science towards a quantitative analysis of protein structure, stability and unfolding landscapes. We noted that the study of proteins that experience force in vivo was obvious but necessitating the use of new techniques such as optical tweezers (OT), and in discussing experiments to study the effects of solvent conditions, the direction of force, the marrying with computational simulations and the like, we were implying that the theoretical knowledge underpinning such quantification and necessary technological developments were in place. Some seven years later, it is interesting to see how the field has advanced and whether such quantitative analysis of our ‘obvious’ proteins has occurred.

DOI

Fiber-optic spanner

Bryan J. Black and Samarendra K. Mohanty

Methods of controllable, noncontact rotation of optically trapped microscopic objects have garnered significant attention for tomographic imaging and microfluidic actuation. Here, we report development of a fiber-optic spanner and demonstrate controlled rotation of smooth muscle cells. The rotation is realized by introducing a transverse offset between two counterpropagating beams emanating from single-mode optical fibers. The rotation speed and surrounding microfluidic flow could be controlled by varying balanced laser beam powers. Further, we demonstrate simultaneous translation and rotation of the fiber-optically trapped cell by varying the laser power of one fiber-optic arm.

DOI

Radiation pressure makes ellipsoidal particles tumble

B. M. Mihiretie, P. Snabre, J. C. Loudet and B. Pouligny

We report on optical levitation of dielectric particles, of prolate ellipsoidal shape, a few tens of micrometers in length, in a low-aperture laser beam. Ellipsoids of moderate aspect ratio (k < 3) are observed to be trapped on the axis of the laser beam, similarly to simple spheres. Conversely, elongated particles (k > 3) cannot be kept immobile, and rather undergo sustained oscillating motions, comprising both lateral and angular excursions around the beam axis; hence the name "tumble". The observed tumbling motion, a straightforward manifestation of the non-conservative character of radiation pressure forces, is explained through a 2-dimensional ray optics model of the interaction of light with an ellipsoid.

DOI

Modeling highly focused laser beam in optical tweezers with the vector Gaussian beam in the T-matrix method

Paul B. Bareil and Yunlong Sheng

The vector Gaussian beam with high-order corrections is used to describe accurately the laser beam up to numerical aperture NA=1.20 in the optical tweezers for trapping nanoparticles. The beam is then expanded in the T-matrix method into the vector spherical wave function (VSWF) series using the point matching method with a new selection of the matching points. The errors in the beam description and in the VSWF expansion are much lower than those that occur in the paraxial Gaussian beam model.

DOI

A compact holographic optical tweezers instrument

G. M. Gibson, R. W. Bowman, A. Linnenberger, M. Dienerowitz, D. B. Phillips, D. M. Carberry, M. J. Miles, and M. J. Padgett
Holographic optical tweezers have found many applications including the construction of complex micron-scale 3D structures and the control of tools and probes for position, force, and viscosity measurement. We have developed a compact, stable, holographic optical tweezers instrument which can be easily transported and is compatible with a wide range of microscopy techniques, making it a valuable tool for collaborative research. The instrument measures approximately 30×30×35 cm and is designed around a custom inverted microscope, incorporating a fibre laser operating at 1070 nm. We designed the control software to be easily accessible for the non-specialist, and have further improved its ease of use with a multi-touch iPad interface. A high-speed camera allows multiple trapped objects to be tracked simultaneously. We demonstrate that the compact instrument is stable to 0.5 nm for a 10 s measurement time by plotting the Allan variance of the measured position of a trapped 2 μm silica bead. We also present a range of objects that have been successfully manipulated.

DOI

Improvement of Laser Trapping Based Microprobe in Laser Shaded Condition

Masaki MICHIHATA, Yasuhiro TAKAYA, Terutake HAYASHI

A micro-probe based on laser trapping has been proposed for the probe system of a nano-coordinate measuring machine. This study focused on the “shadow effect,” which is the case wherein a partial beam for laser trapping is shaded by the specimen. A radially polarized beam was introduced to improve the shadow effect. As the result of increasing the trapping efficiency by 3.2 times, this high-efficient laser trapping made it possible to extend the measurable range of the vertical surface under the shadow effect.

DOI

Microrheology with optical tweezers: data analysis

Manlio Tassieri, R M L Evans, Rebecca L Warren, Nicholas J Bailey and Jonathan M Cooper
We present a data analysis procedure that provides the solution to a long-standing issue in microrheology studies, i.e. the evaluation of the fluids' linear viscoelastic properties from the analysis of a finite set of experimental data, describing (for instance) the time-dependent mean-square displacement of suspended probe particles experiencing Brownian fluctuations. We report, for the first time in the literature, the linear viscoelastic response of an optically trapped bead suspended in a Newtonian fluid, over the entire range of experimentally accessible frequencies. The general validity of the proposed method makes it transferable to the majority of microrheology and rheology techniques.
DOI

High Degree of Coordination and Division of Labor among Subunits in a Homomeric Ring ATPase

Gheorghe Chistol, Shixin Liu, Craig L. Hetherington, Jeffrey R. Moffitt, Shelley Grimes, Paul J. Jardine, Carlos Bustamante

Ring NTPases of the ASCE superfamily perform a variety of cellular functions. An important question about the operation of these molecular machines is how the ring subunits coordinate their chemical and mechanical transitions. Here, we present a comprehensive mechanochemical characterization of a homomeric ring ATPase—the bacteriophage φ29 packaging motor—a homopentamer that translocates double-stranded DNA in cycles composed of alternating dwells and bursts. We use high-resolution optical tweezers to determine the effect of nucleotide analogs on the cycle. We find that ATP hydrolysis occurs sequentially during the burst and that ADP release is interlaced with ATP binding during the dwell, revealing a high degree of coordination among ring subunits. Moreover, we show that the motor displays an unexpected division of labor: although all subunits of the homopentamer bind and hydrolyze ATP during each cycle, only four participate in translocation, whereas the remaining subunit plays an ATP-dependent regulatory role.
DOI

Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy

Irène Tatischeff, Eric Larquet Juan M. Falcón-Pérez, Pierre-Yves Turpin and Sergei G. Kruglik

The joint use of 3 complementary techniques, namely, nanoparticle tracking analysis (NTA), cryo-electron microscopy (Cryo-EM) and Raman tweezers microspectroscopy (RTM), is proposed for a rapid characterisation of extracellular vesicles (EVs) of various origins. NTA is valuable for studying the size distribution and concentration, Cryo-EM is outstanding for the morphological characterisation, including observation of vesicle heterogeneity, while RTM provides the global chemical composition without using any exogenous label. The capabilities of this approach are evaluated on the example of cell-derived vesicles of Dictyostelium discoideum, a convenient general model for eukaryotic EVs. At least 2 separate species differing in chemical composition (relative amounts of DNA, lipids and proteins, presence of carotenoids) were found for each of the 2 physiological states of this non-pathogenic microorganism, that is, cell growth and starvation-induced aggregation. These findings demonstrate the specific potency of RTM. In addition, the first Raman spectra of human urinary exosomes are reported, presumably constituting the primary step towards Raman characterisation of EVs for the purpose of human diseases diagnoses.
DOI

Mechanics: The forces of cancer

Erika Jonietz

Hippocrates first used the term 'carcinoma' to describe cancerous tumours more than 2,000 years ago. But until the end of the nineteenth century, doctors knew little about solid tumours other than how much stiffer they were than the surrounding tissue. Even now, one of the most common ways people detect cancer is by discovering a lump. Yet despite the obvious contrast in hardness between a tumour and the surrounding tissue, the differences between their physical properties have largely been overlooked.
DOI

Three-Dimensional Exploration and Mechano-Biophysical Analysis of the Inner Structure of Living Cells

Álvaro Barroso, Mike Woerdemann, Angelika Vollmer, Gert von Bally, Björn Kemper, Cornelia Denz

A novel mechanobiological method is presented to explore qualitatively and quantitatively the inside of living biological cells in three dimensions, paving the way to sense intracellular changes during dynamic cellular processes. For this purpose, holographic optical tweezers, which allow the versatile manipulation of nanoscopic and microscopic particles by means of tailored light fields, are combined with self-interference digital holographic microscopy. This biophotonic holographic workstation enables non-contact, minimally invasive, flexible, high-precision optical manipulation and accurate 3D tracking of probe particles that are incorporated by phagocytosis in cells, while simultaneously quantitatively phase imaging the cell morphology. In a first model experiment, internalized polystyrene microspheres with 1 μm diameter are three-dimensionally moved and tracked in order to quantify distances within the intracellular volume with submicrometer accuracy. Results from investigations on cell swelling provoked by osmotic stimulation demonstrate the homogeneous stretching of the cytoskeleton network, and thus that the proposed method provides a new way for the quantitative 3D analysis of the dynamic intracellular morphology.

DOI

Absorption spectroscopy of single red blood cells in the presence of mechanical deformations induced by optical traps

Michal Wojdyla; Saurabh Raj; Dmitri Petrov
The electronic properties of single human red blood cells under mechanical deformations were investigated using a combination of dual beam optical tweezers and UV-vis absorption spectroscopy. The mechanical deformations were induced by two near-infrared optical traps with different trapping powers and trap configurations. The deformations were applied in two ways: locally, due to the mechanical forces around the traps, and by stretching the cell by moving the traps in opposite directions. In the presence of local deformations, the single cell undergoes a transition from an oxygenated state to a partially deoxygenated state. This process was found to be reversible and strongly power-dependent. Stretching the cell caused an opposite effect, indicating that the electronic response of the whole cell is dominated by the local interaction with the trapping beams. Results are discussed considering light-induced local heating, the Stark effect, and biochemical alterations due to mechanical forces, and are compared with reports of previous Raman spectroscopy studies. The information gained by the analysis of a single red blood cell’s electronic response facilitates the understanding of fundamental physiological processes and sheds further light on the cell’s mechanochemistry. This information may offer new opportunities for the diagnosis and treatment of blood diseases.
DOI

Three-dimensional subpixel estimation in holographic position measurement of an optically trapped nanoparticle

Akira Sato, Quang Duc Pham, Satoshi Hasegawa, and Yoshio Hayasaki

We propose three-dimensional (3D) subpixel estimation in the position measurement of a nanoparticle held in optical tweezers in water by using an in-line, low-coherence digital holographic microscope. The 3D subpixel estimation was performed with the addition of axial subpixel estimation to the lateral subpixel estimation introduced in our previous work [Appl. Opt. 50, H183 (2011)]. The axial subpixel estimation allowed the step length in the diffraction calculation of a hologram to be increased to ∼20  nm while keeping the axial resolution of ∼3  nm. This drastically decreased the computation time of the diffraction calculation to less than 10% of the two-dimensional subpixel estimation.
DOI

Recent advances in laser tweezers Raman spectroscopy (LTRS) for label-free analysis of single cells

James W. Chan

Laser tweezers Raman spectroscopy (LTRS), a technique that integrates optical tweezers with confocal Raman spectroscopy, is a variation of micro-Raman spectroscopy that enables the manipulation and biochemical analysis of single biological particles in suspension. This article provides an overview of the LTRS method, with an emphasis on highlighting recent advances over the past several years in the development of the technology and several new biological and biomedical applications that have been demonstrated. A perspective on the future developments of this powerful cytometric technology will also be presented.
DOI

Chromatin Decondensation and Nuclear Softening Accompany Nanog Downregulation in Embryonic Stem Cells

Kevin J. Chalut, Markus Höpfler, Franziska Lautenschläger, Lars Boyde, Chii Jou Chan, Andrew Ekpenyong, Alfonso Martinez-Arias, Jochen Guck

The interplay between epigenetic modification and chromatin compaction is implicated in the regulation of gene expression, and it comprises one of the most fascinating frontiers in cell biology. Although a complete picture is still lacking, it is generally accepted that the differentiation of embryonic stem (ES) cells is accompanied by a selective condensation into heterochromatin with concomitant gene silencing, leaving access only to lineage-specific genes in the euchromatin. ES cells have been reported to have less condensed chromatin, as they are capable of differentiating into any cell type. However, pluripotency itself—even prior to differentiation—is a split state comprising a naïve state and a state in which ES cells prime for differentiation. Here, we show that naïve ES cells decondense their chromatin in the course of downregulating the pluripotency marker Nanog before they initiate lineage commitment. We used fluorescence recovery after photobleaching, and histone modification analysis paired with a novel, to our knowledge, optical stretching method, to show that ES cells in the naïve state have a significantly stiffer nucleus that is coupled to a globally more condensed chromatin state. We link this biophysical phenotype to coinciding epigenetic differences, including histone methylation, and show a strong correlation of chromatin condensation and nuclear stiffness with the expression of Nanog. Besides having implications for transcriptional regulation and embryonic cell sorting and suggesting a putative mechanosensing mechanism, the physical differences point to a system-level regulatory role of chromatin in maintaining pluripotency in embryonic development.

DOI

The dynamic pause-unpackaging state, an off-translocation recovery state of a DNA packaging motor from bacteriophage T4

Vishal I. Kottadiel, Venigalla B. Rao, and Yann R. Chemla

Tailed bacteriophages and herpes viruses use powerful ATP-driven molecular motors to translocate their viral genomes into a preformed capsid shell. The bacteriophage T4 motor, a pentamer of the large terminase protein (gp17) assembled at the portal vertex of the prohead, is the fastest and most powerful known, consistent with the need to package a ∼170-kb viral genome in approximately 5 min. Although much is known about the mechanism of DNA translocation, very little is known about how ATP modulates motor–DNA interactions. Here, we report single-molecule measurements of the phage T4 gp17 motor by using dual-trap optical tweezers under different conditions of perturbation. Unexpectedly, the motor pauses randomly when ATP is limiting, for an average of 1 s, and then resumes translocation. During pausing, DNA is unpackaged, a phenomenon so far observed only in T4, where some of the packaged DNA is slowly released. We propose that the motor pauses whenever it encounters a subunit in the apo state with the DNA bound weakly and incorrectly. Pausing allows the subunit to capture ATP, whereas unpackaging allows scanning of DNA until a correct registry is established. Thus, the “pause-unpackaging” state is an off-translocation recovery state wherein the motor, sometimes by taking a few steps backward, can bypass the impediments encountered along the translocation path. These results lead to a four-state mechanochemical model that provides insights into the mechanisms of translocation of an intricately branched concatemeric viral genome.

DOI

Superdiffusion in optically controlled active media

Kyle M. Douglass, Sergey Sukhov & Aristide Dogariu

Active media are complex systems driven by both thermal fluctuations and additional energy sources and are encountered in a variety of phenomena including mobile bacteria, protein diffusion or turbulent flows. However, studying the non-equilibrium dynamics of active media is often difficult because of their size and complexity. Here, we demonstrate that an active medium can be realized and controlled optically through dynamic coupling between multiply scattered light and colloidal particles. As a result of a strong light–matter interaction, the particles undergo diffusion upon a spatiotemporal random potential that leads to an apparent superdiffusion over timescales controlled by, among other things, both the input power and particle size. This model could serve as a convenient tool for exploring the intricacies of non-equilibrium thermodynamics of soft matter while also offering new possibilities for the coherent control of strongly coupled, complex systems.
DOI

Electromagnetic plane-wave force on a slab having various constitutive parameters and embedded in a background material

Shivanand and Kevin J. Webb
An exact theory describing the electromagnetic plane-wave force density on a scattering slab having various constitutive parameters and embedded in a background material with complex impedance is presented. It is shown that the constitutive parameters of the background medium contribute to the force density only through the impedance and not the refractive index. Asymptotic expressions show that the total force per unit area for sufficiently thick slabs having overall loss or gain remains positive, irrespective of the refractive index sign in the slab. However, example material responses indicate that for thin slabs the total force per unit area can be negative for both positive and negative refractive index slabs with gain.
DOI

Studying genomic processes at the single-molecule level: introducing the tools and applications

David Dulin, Jan Lipfert, M. Charl Moolman & Nynke H. Dekker

To understand genomic processes such as transcription, translation or splicing, we need to be able to study their spatial and temporal organization at the molecular level. Single-molecule approaches provide this opportunity, allowing researchers to monitor molecular conformations, interactions or diffusion quantitatively and in real time in purified systems and in the context of the living cell. This Review introduces the types of application of single-molecule approaches that can enhance our understanding of genome function.

DOI

Disease Detection and Management via Single Nanopore-Based Sensors

Joseph E. Reiner, Arvind Balijepalli, Joseph W. F. Robertson, Jason Campbell, John Suehle, and John J. Kasianowicz
As nanometer-scale portals in biological membranes, protein ionic channels act as gatekeepers, controlling the traffic of ions and macromolecules into and out of cells, organelles, and the nucleus. Because of their ubiquitous nature, proper channel function is critical to all aspects of life. One might suppose that the most obvious feature of these transmembrane proteins, a nanometer-scale hole in a ca. 4 nm thick phospholipid bilayer membrane, renders channels as the simplest of biological machines. However, channels have evolved in rather sophisticated ways to control a wide range of biological function. We briefly discuss below some of the roles channels play in biology, as well as why they and mimics of them are emerging as effective biosensors for characterizing and quantifying many types of molecules. We then describe some examples of how such a novel measurement capability could prove useful for detecting disease states, assessing the efficacy of therapeutic agents, and managing the treatment of human disease.
DOI

Optimizing Diffusive Transport Through a Synthetic Membrane Channel

Stefano Pagliara, Christian Schwall, Ulrich F. Keyser

Channel-facilitated transport is investigated by introducing a novel, synthetic model system at the microscale. Colloidal particle flux is increased beyond the limit of free diffusion by introducing an attractive and tunable binding potential created by holographic optical tweezers. The optimal potential depth enhances the diffusive current by a factor of three.
DOI

Radiation pressure in stratified moving media

S. A. R. Horsley, M. Artoni, and G. C. La Rocca
A general theory of optical forces on moving bodies is here developed in terms of generalized 4×4 transfer and scattering matrices. Results are presented for a planar dielectric of arbitrary refractive index placed in an otherwise empty space and moving parallel and perpendicular to the slab-vacuum interface. In both regimes of motion the resulting force comprises lateral and normal velocity-dependent components, which may depend in a subtle way on the Doppler effect and s-p-polarization mixing. For lateral displacements in particular, polarization mixing, which is here interpreted as an effective magnetoelectric effect due to the reduced symmetry induced by the motion of the slab, gives rise to a velocity-dependent force contribution that is sensitive to the phase difference between the two polarization amplitudes. This term gives rise to a rather peculiar optical response on the moving body, and specific cases are illustrated for incident radiation of arbitrarily directed linear polarization. The additional force due to polarization mixing may cancel to first order in V/c with the first order Doppler contribution yielding an overall vanishing of the velocity-dependent component of the force on the body. The above findings bear some relevance to modern developments of nano-optomechanics and to the problem of the frictional component of the Casimir force.
DOI

Moving Groups of Microparticles Into Array With a Robot–Tweezers Manipulation System

Haoyao Chen, Dong Sun
Significant demand for both accuracy and productivity in batch manipulation of microparticles highlights the need to develop an automatic arraying approach to placing groups of particles into a predefined array with right pairs. This paper presents our latest effort to achieve this objective using integrated robotics and holographic optical tweezers technologies, where holographic optical tweezers function as special robot end-effectors to manipulate the microparticles. Based on the physical dynamics of trapping, a potential-field-based controller is developed to drive every pair of particles to the assigned array, while preventing collisions between particles. The significance of the proposed controller lies in the capability of driving two groups of particles into a common array in right pair and controlling the interdistances between the particles in pairs. Experiments are performed to demonstrate the effectiveness of the proposed approach.

DOI

Comparison of stresses on homogeneous spheroids in the optical stretcher computed with geometrical optics and generalized Lorenz–Mie theory

Lars Boyde, Andrew Ekpenyong, Graeme Whyte, and Jochen Guck
We present two electromagnetic frameworks to compare the surface stresses on spheroidal particles in the optical stretcher (a dual-beam laser trap that can be used to capture and deform biological cells). The first model is based on geometrical optics (GO) and limited in its applicability to particles that are much greater than the incident wavelength. The second framework is more sophisticated and hinges on the generalized Lorenz–Mie theory (GLMT). Despite the difference in complexity between both theories, the stress profiles computed with GO and GLMT are in good agreement with each other (relative errors are on the order of 1–10%). Both models predict a diminishing of the stresses for larger wavelengths and a strong increase of the stresses for shorter laser-cell distances. Results indicate that surface stresses on a spheroid with an aspect ratio of 1.2 hardly differ from the stresses on a sphere of similar size. Knowledge of the surface stresses and whether or not they redistribute during the stretching process is of crucial importance in real-time applications of the stretcher that aim to discern the viscoelastic properties of cells for purposes of cell characterization, sorting, and medical diagnostics.

DOI

Controlled formation and manipulation of colloidal lattices by dynamically reconfigurable three dimensional interferometric optical traps

Jolly Xavier, Raktim Dasgupta, Sunita Ahlawat, Joby Joseph, and Pradeep Kumar Gupta
We present real-time controlled manipulation of microparticles optically trapped in three dimensional (3D)-trap array lattices generated by dynamically reconfigurable n + 1 non-coplanar multiple plane wave interference in an umbrella-like configuration. Through a programmable spatial light modulator-assisted approach, reconfigurable stable 3D interferometric optical traps belonging to diverse transverse rotational symmetry are realized and used to trap micro beads in multi-layers. Dynamically controlled translation as well as rotation of trapped array of particles are also demonstrated using presented approach. Further, the optical stacking of microparticles in an array of 3D single-stranded chiral traps portrays the versatility in tailoring axially tunable trap arrays.

DOI

Dynamic force sensing of filamin revealed in single-molecule experiments

Lorenz Rognoni, Johannes Stigler, Benjamin Pelz, Jari Ylänne, and Matthias Rief

Mechanical forces are important signals for cell response and development, but detailed molecular mechanisms of force sensing are largely unexplored. The cytoskeletal protein filamin is a key connecting element between the cytoskeleton and transmembrane complexes such as integrins or the von Willebrand receptor glycoprotein Ib. Here, we show using single-molecule mechanical measurements that the recently reported Ig domain pair 20–21 of human filamin A acts as an autoinhibited force-activatable mechanosensor. We developed a mechanical single-molecule competition assay that allows online observation of binding events of target peptides in solution to the strained domain pair. We find that filamin force sensing is a highly dynamic process occurring in rapid equilibrium that increases the affinity to the target peptides by up to a factor of 17 between 2 and 5 pN. The equilibrium mechanism we find here can offer a general scheme for cellular force sensing.

DOI

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level

Attila Nagy, Yasuharu Takagi, Neil Billington, Sara A. Sun, Davin K. T. Hong, Earl Homsher, Aibing Wang and James R. Sellers

Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ~14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (~20-25%) motor. The ADP release step (~0.35 s-1), of NMIIB is only ~3 times faster than the rate-limiting phosphate release (0.13 +/- 0.01 s-1). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of single- and double-headed myosin fragments, and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished by the use of TIRF using FIONA and dual-beam optical trapping. At a physiological ATP concentration (1 mM), the rate of detachment of the single-headed and double-headed molecules was similar (~0.4 s-1). Using optical tweezers we found that the power-stroke sizes of single- and double-headed HMM were each ~6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments.

DOI

Generating Nanostructures with Multiphoton Absorption Polymerization using Optical Trap Assisted Nanopatterning

Yu-Cheng Tsai, Karl-Heinz Leitz, Romain Fardel, Michael Schmidt, Craig B. Arnold

The need to generate sub 100 nm features is of interest for a variety of applications including optics, optoelectronics, and plasmonics. To address this requirement, several advanced optical lithography techniques have been developed based on either multiphoton absorption polymerization or near-field effects. In this paper, we combine strengths from multiphoton absorption and near field using optical trap assisted nanopatterning (OTAN). A Gaussian beam is used to position a microsphere in a polymer precursor fluid near a substrate. An ultrafast laser is focused by that microsphere to induce multiphoton polymerization in the near field, leading additive direct-write nanoscale processing.

DOI

Optical trapping, driving, and arrangement of particles using a tapered fibre probe

Hongbao Xin, Rui Xu & Baojun Li

The ability of manipulating mesoscopic objects with high precision and flexibility is extremely important for a wide variety of fields from physics, biochemistry, to biomedicine. Particularly, the ability of arranging particles/cells into desired patterns precisely is a challenge for numerous physical and biological applications. Here, we report a strategy of realizing highly flexible trapping, driving, and precise arrangement of particles using a tapered fibre probe. Using randomly distributed 3-µm-diameter silica particles as an example, we demonstrate that the strategy is able to stably trap the particles and drive them to targeted regions, subsequently arrange the particles into desired patterns. To further demonstrate the ability of this strategy, experiments were done using sub-micron sized particles and biological samples (bacteria and cells). This strategy provides a new approach to manipulate mesoscopic objects precisely and flexibly, and hopefully can be used in future fundamental and applied researches of interdiscipline.

DOI

Cell palpation with an optically trapped particle

Tadao Sugiura, Hideaki Miyoshi, Tetsu Nishio, Ayae Honda

We have developed a cell palpation system to investigate cell stiffness from the reaction force generated on a particle that is fixed on a cell. In this method, a particle is used as a probe, and is manipulated towards a cell using optical tweezers. Using this method, we can obtain information of local stiffness of a cell. We investigate focal adhesion formation of a cell probed by a particle and we report different particle coating utilized for attaching certain protein in cell membrane. Also we discuss the effects of endocytosis.

DOI

Hydrodynamic interactions in active colloidal crystal microrheology

R. Weeber and J. Harting

In dense colloids it is commonly assumed that hydrodynamic interactions do not play a role. However, a found theoretical quantification is often missing. We present computer simulations that are motivated by experiments where a large colloidal particle is dragged through a colloidal crystal. To qualify the influence of long-ranged hydrodynamics, we model the setup by conventional Langevin dynamics simulations and by an improved scheme with limited hydrodynamic interactions. This scheme significantly improves our results and allows to show that hydrodynamics strongly impacts the development of defects, the crystal regeneration, as well as the jamming behavior.

DOI

Many-body effects in optically-trapped metallic nanoparticles under thermal fluctuations

Takuya Iida, Mamoru Tamura

We have theoretically studied light-induced many-body dynamics of metallic nanoparticles (NPs) under the focused laser beam and the thermal fluctuations in liquid medium at room temperature. The effect of surface ionization is also taken into account. The random motion of NPs by collisions of medium molecules can be suppressed with increasing the laser intensity, and the transition from a diffusive state to metastable states of metallic NPs is possible. Obtained results will provide a novel way to control the phase transitions in assembled structure of NPs under the balance of optically-modulated interparticle forces and thermal fluctuations. Such a mechanism is significantly different from a conventional gas-liquid-solid transition based on spontaneous interparticle forces because it is based on the transient anisotropic force controlled by light.

DOI

Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility

Micah J. McCauley, Emily M. Rueter, Ioulia Rouzina, L. James Maher III and Mark C. Williams

Eukaryotic High-Mobility Group B (HMGB) proteins alter DNA elasticity while facilitating transcription, replication and DNA repair. We developed a new single-molecule method to probe non-specific DNA interactions for two HMGB homologs: the human HMGB2 box A domain and yeast Nhp6Ap, along with chimeric mutants replacing neutral N-terminal residues of the HMGB2 protein with cationic sequences from Nhp6Ap. Surprisingly, HMGB proteins constrain DNA winding, and this torsional constraint is released over short timescales. These measurements reveal the microscopic dissociation rates of HMGB from DNA. Separate microscopic and macroscopic (or local and non-local) unbinding rates have been previously proposed, but never independently observed. Microscopic dissociation rates for the chimeric mutants (∼10 s−1) are higher than those observed for wild-type proteins (∼0.1–1.0 s−1), reflecting their reduced ability to bend DNA through short-range interactions, despite their increased DNA-binding affinity. Therefore, transient local HMGB–DNA contacts dominate the DNA-bending mechanism used by these important architectural proteins to increase DNA flexibility.

DOI

Nucleosomal Elements that Control the Topography of the Barrier to Transcription

Lacramioara Bintu, Toyotaka Ishibashi, Manchuta Dangkulwanich, Yueh-Yi Wu, Lucyna Lubkowska, Mikhail Kashlev, Carlos Bustamante
The nucleosome represents a mechanical barrier to transcription that operates as a general regulator of gene expression. We investigate how each nucleosomal component—the histone tails, the specific histone-DNA contacts, and the DNA sequence—contributes to the strength of the barrier. Removal of the tails favors progression of RNA polymerase II into the entry region of the nucleosome by locally increasing the wrapping-unwrapping rates of the DNA around histones. In contrast, point mutations that affect histone-DNA contacts at the dyad abolish the barrier to transcription in the central region by decreasing the local wrapping rate. Moreover, we show that the nucleosome amplifies sequence-dependent transcriptional pausing, an effect mediated through the structure of the nascent RNA. Each of these nucleosomal elements controls transcription elongation by affecting distinctly the density and duration of polymerase pauses, thus providing multiple and alternative mechanisms for control of gene expression by chromatin remodeling and transcription factors.

DOI

Force Spectroscopy with Dual-Trap Optical Tweezers: Molecular Stiffness Measurements and Coupled Fluctuations Analysis

M. Ribezzi-Crivellari, F. Ritort

Dual-trap optical tweezers are often used in high-resolution measurements in single-molecule biophysics. Such measurements can be hindered by the presence of extraneous noise sources, the most prominent of which is the coupling of fluctuations along different spatial directions, which may affect any optical tweezers setup. In this article, we analyze, both from the theoretical and the experimental points of view, the most common source for these couplings in dual-trap optical-tweezers setups: the misalignment of traps and tether. We give criteria to distinguish different kinds of misalignment, to estimate their quantitative relevance and to include them in the data analysis. The experimental data is obtained in a, to our knowledge, novel dual-trap optical-tweezers setup that directly measures forces. In the case in which misalignment is negligible, we provide a method to measure the stiffness of traps and tether based on variance analysis. This method can be seen as a calibration technique valid beyond the linear trap region. Our analysis is then employed to measure the persistence length of dsDNA tethers of three different lengths spanning two orders of magnitude. The effective persistence length of such tethers is shown to decrease with the contour length, in accordance with previous studies.

DOI

Lasing in optically manipulated, dye-doped emulsion microdroplets

M. Aas, A. Jonáš, A. Kiraz

We introduce a portable, all-liquid microlaser based on optically pumped dye-doped emulsion microdroplets held in a single beam optical trap. We show high stability of the laser emission spectra during prolonged optical manipulation of the droplets within an immiscible host liquid. We investigate the effects of droplet size and dye concentration on the spectral position of lasing wavelength and show how these parameters can be used for the emission wavelength tuning. We also study shifting of the average lasing wavelength to the blue side of the spectrum due to dye photobleaching. The presented optically manipulated fluidic microlasers are disposable and can be easily combined with microfluidic chip technology. This makes them especially attractive for on-chip applications in chemical and biological analysis and sensing.

DOI

Direct Observation of Cotranscriptional Folding in an Adenine Riboswitch

Kirsten L. Frieda, Steven M. Block

Growing RNA chains fold cotranscriptionally as they are synthesized by RNA polymerase. Riboswitches, which regulate gene expression by adopting alternative RNA folds, are sensitive to cotranscriptional events. We developed an optical-trapping assay to follow the cotranscriptional folding of a nascent RNA and used it to monitor individual transcripts of the pbuE adenine riboswitch, visualizing distinct folding transitions. We report a particular folding signature for the riboswitch aptamer whose presence directs the gene-regulatory transcription outcome, and we measured the termination frequency as a function of adenine level and tension applied to the RNA. Our results demonstrate that the outcome is kinetically controlled. These experiments furnish a means to observe conformational switching in real time and enable the precise mapping of events during cotranscriptional folding.

DOI

Improving Single-Molecule Experiments With Feedback Control of Optical Traps

D. G. Cole
This article explores various types of feedback control—position feedback, which was shown to be equivalent to force feedback, rate feedback, andintegral feedback—for the purpose of improving instrument performance for single-molecule experiments. The ability of each of each types of feedbackto lower the measurement signal-to-noise ratio (SNR) is evaluated andcompared to the open-loop case. While position feedback does not result in any improvement in the SNR, the cases of rate feedback and integral feedback both resulted in improvements in the measurement's SNR. Rate feedback is shown to effectively “cool” the beads held in the optical trap, thereby limiting the effect that Brownian disturbances have on the beads' motion. Integral feedback is shown to improve the SNR of the measuredsignal of interest and is robust and easy to implement. It is also shown that integral feedback acts as an exogenous force estimator. Ultimately, feedback does not provide better resolution as measured by SNR than an open-loop filtering approach can but does provide other advantages, including the ability to control other variables and to make a more robust instrument that can be easily adapted to changes in experimental conditions or the environment.
DOI

Measuring Thermal Rupture Force Distributions from an Ensemble of Trajectories

J. W. Swan, M. M. Shindel, and E. M. FurstRupture, bond breaking, or extraction from a deep and narrow potential well requires considerable force while producing minimal displacement. In thermally fluctuating systems, there is not a single force required to achieve rupture, but a spectrum, as thermal forces can both augment and inhibit the bond breaking. We demonstrate measurement and interpretation of the distribution of rupture forces between pairs of colloidal particles bonded via the van der Waals attraction. The otherwise irreversible bond is broken by pulling the particles apart with optical tweezers. We show that an ensemble of the particle trajectories before, during and after the rupture event may be used to produce a high fidelity description of the distribution of rupture forces. This analysis is equally suitable for describing rupture forces in molecular and biomolecular contexts with a number of measurement techniques.
DOI

Real-space studies of the structure and dynamics of self-assembled colloidal clusters

Rebecca W. Perry , Guangnan Meng , Thomas G. Dimiduk , Jerome Fung and Vinothan N. Manoharan

The energetics and assembly pathways of small clusters may yield insights into processes occurring at the earliest stages of nucleation. We use a model system consisting of micrometer-sized, spherical colloidal particles to study the structure and dynamics of small clusters, where the number of particles is small (N ≤ 10). The particles interact through a short-range depletion attraction with a depth of a few kBT. We describe two methods to form colloidal clusters, one based on isolating the particles in microwells and another based on directly assembling clusters in the gas phase using optical tweezers. We use the first technique to obtain ensemble-averaged probabilities of cluster structures as a function of N. These experiments show that clusters with symmetries compatible with crystalline order are rarely formed under equilibrium conditions. We use the second technique to study the dynamics of the clusters, and in particular how they transition between free-energy minima. To monitor the clusters we use a fast three-dimensional imaging technique, digital holographic microscopy, that can resolve the positions of each particle in the cluster with 30–45 nm precision on millisecond timescales. The real-space measurements allow us to obtain estimates for the lifetimes of the energy minima and the transition states. It is not yet clear whether the observed dynamics are relevant for small nuclei, which may not have sufficient time to transition between states before other particles or clusters attach to them. However, the measurements do provide some glimpses into how systems containing a small number of particles traverse their free-energy landscape.
DOI

An interacting dipole model to explore broadband transverse optical binding

Michael Mazilu, Andrew Rudhall, Ewan M Wright and Kishan Dholakia
The demonstration of optical binding of micro-particles placed in intense optical fields has resulted in unique and exciting prospects for studying new forms of condensed matter. The ability to tailor optical fields in the spatial and temporal domains elicits the possibility of creating novel condensed matter with the structure controlled by tailoring the optical field. Here, we theoretically calculate the transverse optical binding forces for nanoparticles within monochromatic and broadband optical fields. We demonstrate the decrease in amplitude of the optical binding forces for broadband fields as a function of inter-particle separation and attribute the effect to the averaging effect of spectrally dependent optical forces. We also examine multiple particle optically bound systems and use the interacting dipole method to find self-organized positions for six and ten particles illuminated by a monochromatic plane wave.
DOI

Counting unfolding events in stretched helices with induced oscillation by optical tweezers

ROMMEL GAUD BACABAC, ROLAND OTADOY

Correlation measures based on embedded probe fluctuations, single or paired, are now widely used for characterizing the viscoelastic properties of biological samples. However, more robust applications using this technique are still lacking. Considering that the study of living matter routinely demonstrates new and complex phenomena, mathematical and experimental tools for analysis have to catch up in order to arrive at newer insights. Therefore, we derive ways of probing non-equilibrium events in helical biopolymers provided by stretching beyond thermal forces. We generalize, for the first time, calculations for winding turn probabilities to account for unfolding events in single fibrous biopolymers and globular proteins under tensile stretching using twin optical traps. The approach is based on approximating the ensuing probe fluctuations as originating from a damped harmonic oscillator under oscillatory forcing.
DOI

Periodic Modulations of Optical Tweezers Near Solid-State Membranes

Gautam V. Soni, Magnus P. Jonsson, Cees Dekker

Optical tweezers coupled to surfaces and thin solid-state membranes are very useful in a wide range of nanophotonics applications and open up new ways of measuring surface adhesion and molecular forces. A recent example is the coupling of optical tweezers to solid-state nanopore sensors for accurate control and biophysical investigation of single DNA molecules. Such membrane-integrated optical traps do, however, show a variety of optical effects that are not well understood. A major limitation in these experiments comes from periodic modulations of the bead position from the trapping plane when the optical trap is axially moved towards the membrane. While previously considered detection artifacts, it is shown here that these modulations correspond to real movements of the optical trap position that results from interference between the incident trapping laser and reflections from the thin solid-state membrane. An experimental study of these oscillations is presented, as well as optical simulations based on the finite-difference time-domain method, providing insight into the underlying interference phenomenon. Finally, an alternate measurement geometry is presented that eliminates these oscillations, specifically useful for performing optical-trap-coupled nanopore force spectroscopy.

DOI

Bespoke optical springs and passive force clamps from shaped dielectric particles

S.H. Simpson, D.B. Phillips, D.M. Carberry, S. Hanna

By moulding optical fields, holographic optical tweezers are able to generate structured force fields with magnitudes and length scales of great utility for experiments in soft matter and biological physics. It has recently been noted that optically induced force fields are determined not only by the incident optical field, but by the shape and composition of the particles involved [Gluckstad J. Optical manipulation: sculpting the object. Nat Photonics 2011;5:7–8]. Indeed, there are desirable but simple attributes of a force field, such as orientational control, that cannot be introduced by sculpting optical fields alone. With this insight in mind, we show, theoretically, how relationships between force and displacement can be controlled by optimizing particle shapes. We exhibit a constant force optical spring, made from a tapered microrod and discuss methods by which it could be fabricated. In addition, we investigate the optical analogue of streamlining, and show how objects can be shaped so as to reduce the effects of radiation pressure, and hence switch from non-trapping to trapping regimes.
DOI

Review on recent advances in the analysis of isolated organelles

Chad P. Satori, Vratislav Kostal, Edgar A. Arriaga

The analysis of isolated organelles is one of the pillars of modern bioanalytical chemistry. This review describes recent developments on the isolation and characterization of isolated organelles both from living organisms and cell cultures. Salient reports on methods to release organelles focused on reproducibility and yield, membrane isolation, and integrated devices for organelle release. New developments on organelle fractionation after their isolation were on the topics of centrifugation, immunocapture, free flow electrophoresis, flow field-flow fractionation, fluorescence activated organelle sorting, laser capture microdissection, and dielectrophoresis. New concepts on characterization of isolated organelles included atomic force microscopy, optical tweezers combined with Raman spectroscopy, organelle sensors, flow cytometry, capillary electrophoresis, and microfluidic devices.

DOI

A Highly Compliant Protein Native State with a Spontaneous-like Mechanical Unfolding Pathway

Pétur O. Heidarsson, Immanuel Valpapuram, Carlo Camilloni, Alberto Imparato, Guido Tiana, Flemming M. Poulsen, Birthe B. Kragelund, and Ciro Cecconi
The mechanical properties of proteins and their force-induced structural changes play key roles in many biological processes. Previous studies have shown that natively folded proteins are brittle under tension, unfolding after small mechanical deformations, while partially folded intermediate states, such as molten globules, are compliant and can deform elastically a great amount before crossing the transition state barrier. Moreover, under tension proteins appear to unfold through a different sequence of events than during spontaneous unfolding. Here, we describe the response to force of the four-α-helix acyl-CoA binding protein (ACBP) in the low-force regime using optical tweezers and ratcheted molecular dynamics simulations. The results of our studies reveal an unprecedented mechanical behavior of a natively folded protein. ACBP displays an atypical compliance along two nearly orthogonal pulling axes, with transition states located almost halfway between the unfolded and folded states. Surprisingly, the deformability of ACBP is greater than that observed for the highly pliant molten globule intermediate states. Furthermore, when manipulated from the N- and C-termini, ACBP unfolds by populating a transition state that resembles that observed during chemical denaturation, both for structure and position along the reaction coordinate. Our data provide the first experimental evidence of a spontaneous-like mechanical unfolding pathway of a protein. The mechanical behavior of ACBP is discussed in terms of topology and helix propensity.

DOI

Programmed −1 frameshifting efficiency correlates with RNA pseudoknot conformational plasticity, not resistance to mechanical unfolding

Dustin B. Ritchie, Daniel A. N. Foster, and Michael T. Woodside

Programmed −1 frameshifting, whereby the reading frame of a ribosome on messenger RNA is shifted in order to generate an alternate gene product, is often triggered by a pseudoknot structure in the mRNA in combination with an upstream slippery sequence. The efficiency of frameshifting varies widely for different sites, but the factors that determine frameshifting efficiency are not yet fully understood. Previous work has suggested that frameshifting efficiency is related to the resistance of the pseudoknot against mechanical unfolding. We tested this hypothesis by studying the mechanical properties of a panel of pseudoknots with frameshifting efficiencies ranging from 2% to 30%: four pseudoknots from retroviruses, two from luteoviruses, one from a coronavirus, and a nonframeshifting bacteriophage pseudoknot. Using optical tweezers to apply tension across the RNA, we measured the distribution of forces required to unfold each pseudoknot. We found that neither the average unfolding force, nor the unfolding kinetics, nor the parameters describing the energy landscape for mechanical unfolding of the pseudoknot (energy barrier height and distance to the transition state) could be correlated to frameshifting efficiency. These results indicate that the resistance of pseudoknots to mechanical unfolding is not a primary determinant of frameshifting efficiency. However, increased frameshifting efficiency was correlated with an increased tendency to form alternate, incompletely folded structures, suggesting a more complex picture of the role of the pseudoknot involving the conformational dynamics.

DOI

Optomechanical deformation and strain in elastic dielectrics

M Sonnleitner, M Ritsch-Marte and H Ritsch
Light forces induced by scattering and absorption in elastic dielectrics lead to local density modulations and deformations. These perturbations in turn modify light propagation in the medium and generate an intricate nonlinear response. We generalize an analytic approach where light propagation in one-dimensional media of inhomogeneous density is modelled as a result of multiple scattering between polarizable slices. Using the Maxwell stress tensor formalism we compute the local optical forces and iteratively approach self-consistent density distributions where the elastic back-action balances gradient- and scattering forces. For an optically trapped dielectric we derive the nonlinear dependence of trap position, stiffness and total deformation on the object's size and field configuration. Generally trapping is enhanced by deformation, which exhibits a periodic change between stretching and compression. This strongly deviates from qualitative expectations based on the change of photon momentum of light crossing the surface of a dielectric. We conclude that optical forces have to be treated as volumetric forces and that a description using the change of photon momentum at the surface of a medium is inappropriate.
DOI

Collective synchronization states in arrays of driven colloidal oscillators

Romain Lhermerout, Nicolas Bruot, Giovanni M Cicuta, Jurij Kotar and Pietro Cicuta
The phenomenon of metachronal waves in cilia carpets has been well known for decades; these waves are widespread in biology, and have fundamental physiological importance. While it is accepted that in many cases cilia are mainly coupled together by the hydrodynamic velocity field, a clear understanding of which aspects determine the collective wave properties is lacking. It is a difficult problem, because both the behavior of the individual cilia and their coupling together are nonlinear. In this work, we coarse-grain the degrees of freedom of each cilium into a minimal description in terms of a configuration-based phase oscillator. Driving colloidal particles with optical tweezers, we then experimentally investigate the coupling through hydrodynamics in systems of many oscillators, showing that a collective dynamics emerges. This work generalizes to a wider class of systems our recent finding that the non-equilibrium steady state can be understood based on the equilibrium properties of the system, i.e. the positions and orientations of the active oscillators. In this model system, it is possible to design configurations of oscillators with the desired collective dynamics. The other face of this problem is to relate the collective patterns found in biology to the architecture and behavior of individual active elements.

DOI

Measuring Nanoscale Forces with Living Probes

S. N. Olof, J. A. Grieve, D. B. Phillips, H. Rosenkranz, M. L. Yallop, M. J. Miles, A. J. Patil, S. Mann, and D. M. Carberry

Optical trapping techniques have been used to investigate fundamental biological processes ranging from the identification of the processive mechanisms of kinesin and myosin to understanding the mechanics of DNA. To date, these investigations have relied almost exclusively on the use of isotropic probes based on colloidal microspheres. However, there are many potential advantages in utilizing more complex probe morphologies: use of multiple trapping points enables control of the interaction volume; increasing the distance between the optical trap and the sample minimizes photodamage in sensitive biological materials; and geometric anisotropy introduces the potential for asymmetric surface chemistry and multifunctional probes. Here we demonstrate that living cells of the freshwater diatomNitzschia subacicularis Hustedt can be exploited as advanced probes for holographic optical tweezing applications. We characterize the optical and material properties associated with the high shape anisotropy of the silica frustule, examine the trapping behavior of the living algal cells, and demonstrate how the diatoms can be calibrated for use as force sensors and as force probes in the presence of rat B-cell hybridoma (11B11) cells.
DOI

Optical forces on Mie particles in an Airy evanescent field

Yang Yang, Wei-Ping Zang, Zhi-Yu Zhao, and Jian-Guo Tian
Using vector potential and spectrum representation, we derive the expressions of the Airy evanescent field existed at the interface. Utilizing these expressions and the Arbitrary Beam Theory, the optical forces exerted on a Mie dielectric particle in the Airy evanescent field were theoretically investigated in detail. Numerical results show that the optical forces exhibit strong oscillations which are corresponding to the distributions of the evanescent field. With the increasing the size of particle radius, Morphology Dependent Resonance occurs for the particle with specific refractive index.
DOI

Optical sorting in holographic trap arrays by tuning the inter-trap separation

S Ahlawat, R Dasgupta, R S Verma, V N Kumar and P K Gupta
Particle motion through a holographic trap array has been investigated theoretically and experimentally, and it is shown that a change in inter-trap separation can be used to selectively control the motion of particles of different sizes. By an appropriate choice of inter-trap separation in a holographically generated two-dimensional trap array, optical potential channels can be created in orthogonal directions such that, from a suspension having a mixture of two different particle sizes, the particles can be sorted in the two orthogonal channels. The use of the approach to sort 3 and 5 μm silica spherical particles in the two orthogonal channels, from a mixed suspension of these, has also been demonstrated.
DOI

Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA

Jason C. Bell, Jody L. Plank, Christopher C. Dombrowski & Stephen C. Kowalczykowski

Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA family compared to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts. Previous single-molecule assays have measured the nucleation and growth of RecA—and its eukaryotic homologue RAD51—on naked double-stranded DNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. Using single-molecule microscopy, here we directly visualize RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5′3′ direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism in which RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (DNA unwrapping) and then the RecA filament grows. We further demonstrate that the recombination mediator protein pair, RecOR (RecO and RecR), accelerates both RecA nucleation and filament growth, and that the introduction of RecF further stimulates RecA nucleation.

DOI