Tracking of Single Quantum Dot Labeled EcoRV Sliding along DNA Manipulated by Double Optical Tweezers

Andreas Biebricher, Wolfgang Wende, Christophe Escudé, Alfred Pingoud and Pierre Desbiolles

Fluorescence microscopy provides a powerful method to directly observe single enzymes moving along a DNA held in an extended conformation. In this work, we present results from single EcoRV enzymes labeled with quantum dots which interact with DNA manipulated by double optical tweezers. The application of quantum dots facilitated accurate enzyme tracking without photobleaching whereas the tweezers allowed us to precisely control the DNA extension. The labeling did not affect the biochemical activity of EcoRV checked by directly observing DNA digestion on the single molecule level. We used this system to demonstrate that during sliding, the enzyme stays in close contact with the DNA. Additionally, slight overstretching of the DNA resulted in a significant decrease of the 1D diffusion constant, which suggests that the deformation changes the energy landscape of the sliding interaction. Together with the simplicity of the setup, these results demonstrate that the combination of optical tweezers with fluorescence tracking is a powerful tool for the study of enzyme translocation along DNA.

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Optical microassembly platform for constructing reconfigurable microenvironments for biomedical studies

Peter John Rodrigo, Lóránd Kelemen, Darwin Palima, Carlo Amadeo Alonzo, Pál Ormos, and Jesper Glückstad

Cellular development is highly influenced by the surrounding microenvironment. We propose user-reconfigurable microenvironments and bio-compatible scaffolds as an approach for understanding cellular development processes. We demonstrate a model platform for constructing versatile microenvironments by fabricating morphologically complex microstructures by two-photon polymerization (2PP) and then assembling these archetypal building blocks into various configurations using multiple, real-time configurable counterpropagating-beam (CB) traps. The demonstrated capacity for handling feature-rich microcomponents may be further developed into a generalized microassembly platform.

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Whispering gallery mode carousel – a photonic mechanism for enhanced nanoparticle detection in biosensing

S. Arnold, D. Keng, S. I. Shopova, S. Holler, W. Zurawsky, and F. Vollmer

Individual nanoparticles in aqueous solution are observed to be attracted to and orbit within the evanescent sensing ring of a Whispering Gallery Mode micro-sensor with only microwatts of driving power. This Carousel trap, caused by attractive optical gradient forces, interfacial interactions, and the circulating momentum flux, considerably enhances the rate of transport to the sensing region, thereby overcoming limitations posed by diffusion on such small area detectors. Resonance frequency fluctuations, caused by the radial Brownian motion of the nanoparticle, reveal the radial trapping potential and the nanoparticle size. Since the attractive forces draw particles to the highest evanescent intensity at the surface, binding steps are found to be uniform.

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Multiplexed force measurements on live cells with holographic optical tweezers

Cecile O. Mejean, Andrew W. Schaefer, Eleanor A. Millman, Paul Forscher, and Eric R. Dufresne

We describe open-loop and closed-loop multiplexed force measurements using holographic optical tweezers. We quantify the performance of our novel video-based control system in a driven suspension of colloidal particles. We demonstrate our system’s abilities with the measurement of the mechanical coupling between Aplysia bag cell growth cones and beads functionalized with the neuronal cell adhesion molecule, apCAM. We show that cells form linkages which couple beads to the underlying cytoskeleton. These linkages are intermittent, stochastic and heterogeneous across beads distributed near the leading edge of a single growth cone.

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Effect of hollow-core photonic crystal fiber microstructure on transverse optical trapping

P. Domachuk, N. Wolchover, M. Cronin-Golomb, and F. G. Omenetto

We investigate numerically and experimentally all-optical control of particles inside waterfilled, silica, hollow-core photonic crystal fiber (HC-PCF). We use an optical trapping beam focused outside the fiber, through its microstructure, perpendicular to the HC-PCF and independent of the guided fiber core mode. Finite difference time domain simulations model trapping through HC-PCF microstructure: trapping along the length of the HC-PCF is well maintained despite the significant effects due to scattering of the HC-PCF core structure. Trapped silica microspheres inside a HC-PCF is demonstrated experimentally as a reversible, reliable technique to control particles in fiber independent of the guided fiber mode. We observe a broadband attenuation of the HC-PCF transmission upon loading a silica microsphere into the fiber core.

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High bandwidth force estimation for optical tweezers

Hullas Sehgal, Tanuj Aggarwal, and Murti V. Salapaka

A prevalent mode of optical tweezers involves position clamping that regulates a constant position of a trapped bead. Traditional schemes employ the measured bead position in the open loop or the control signal in the position-clamp mode as an estimate of external force on the trapped bead. This article shows that traditional methods introduce fundamental limitations on bandwidth of the external force estimation. A method is presented that leads to an order of magnitude increase in the bandwidth of the external force estimation. Furthermore, a comprehensive modeling paradigm is introduced that facilitates estimation of forces on the bead.

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Optical force model based on sequential ray tracing

Eric Aspnes, Tom D. Milster, and Koen Visscher

We discuss how information available from ray-tracing techniques can be used to calculate optical forces and torques on particles. A general ray-trace computer code is augmented with the polarization and irradiance distributions of the illumination and Fresnel surface coefficients to give a reasonably accurate prediction of interaction with large particles out of the focal plane. Calculations of trapping location versus nonuniform illumination conditions are compared with an experiment. Other example calculations include trapping a hemispherical lens and a two-particle trap.

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Magnetic Tweezers Measurement of Single Molecule Torque

Alfredo Celedon, Ilana M. Nodelman, Bridget Wildt, Rohit Dewan, Peter Searson, Denis Wirtz, Gregory D. Bowman and Sean X. Sun

Torsional stress in linear biopolymers such as DNA and chromatin has important consequences for nanoscale biological processes. We have developed a new method to directly measure torque on single molecules. Using a cylindrical magnet, we manipulate a novel probe consisting of a nanorod with a 0.1 μm ferromagnetic segment coupled to a magnetic bead. We achieve controlled introduction of turns into the molecule and precise measurement of torque and molecule extension as a function of the number of turns at low pulling force. We show torque measurement of single DNA molecules and demonstrate for the first time measurements of single chromatin fibers.

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Trapping and manipulating gas bubbles in water with ultrashort laser pulses at a high repetition rate

S. V. Oshemkov, L. P. Dvorkin and V. Yu. Dmitriev

We have experimentally observed the trapping of a gas bubble in water by focused laser radiation. The optical trap was provided by 200-fs pulses of a Ti-sapphire laser operating at a repetition rate of 100 kHz. The laser radiation was focused in water by an objective with a numerical aperture of 0.5. The trapping force in water is estimated as ∼200 pN at an average laser power of 20 mW, which is by two orders of magnitude greater than the efficiency of a traditional laser tweezers. The trapping force arises upon local heating of gas inside a bubble due to nonlinear absorption in the focal region.

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Measuring the optical properties of a trapped ZnO tetrapod

R. Sharma, J.P. Mondia, J. Schäfer, W. Smith, S.-H. Li, Y.-P. Zhao, Z.H. Lu and L.J. Wang

We report the onset of lasing from an electrodynamically trapped ZnO tetrapod. Each of the four legs of such an isolated tetrapod behaves as a single nanowire, where light is guided along the length of the wire and the necessary resonant feedback for lasing is provided at the two end facets. A diluted solution of ZnO tetrapods in methanol was sprayed in the form of a charged mist into a chamber containing the electrodynamic endcap trap. The quick evaporation of methanol assisted in trapping a single charged ZnO tetrapod. The trapped tetrapod is optically pumped with pulses from a Q-switched laser at a wavelength of 355 nm and emits light at 390 nm. For increasing pump fluences above 15 mJ/cm2, a superlinear increase in intensity and a narrowing in the spectral width of the photoluminescence were observed, indicating lasing.

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INDEPENDENT TRAPPING, MANIPULATION AND CHARACTERIZATION BY AN ALL-OPTICAL BIOPHOTONICS WORKSTATION

H.-U. Ulriksen, J. Thøgersen, S. Keiding, I. Perch-Nielsen, J. Dam, D. Z. Palima, H. Stapelfeldt, J. Glückstad

Optical trapping has enabled a multitude of applications focusing, in particular, on non-invasive studies of cellular material. The full potential of optical trapping has, however, not yet been exploited due to restricted access to the trapped samples, caused by high numerical aperture objectives needed to focus the trapping laser beams. Here, we use our recently developed biophotonics workstation to overcome this limitation by introducing probing and spectroscopic characterization of optically trapped particles in a side-view geometry perpendicular to the trapping beams rather than in the traditional top-view geometry parallel to the trapping beams. Our method is illustrated by CARS and fluorescence spectroscopy of trapped polystyrene beads. The side-view geometry opens intriguing possibilities for accessing trapped particles with optical as well as other types of probe methods independent from the trapping process.

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Focal depth and focal splitting of truncated hyperbolic-cosine-Gaussian beam induced by phase plate

Jinsong Li, Songlin Zhuang, Xiumin Gao and Yinzhong Xie

Focal depth and focal splitting of hyperbolic-cosine-Gaussian beams induced by a phase plate were investigated. The pure phase plate consists of three concentric zones: a center circle zone, an inner annular zone and an outer annular zone. The phase variance of the inner annular zone is adjustable. Simulation results show that the focal depth can be adjusted by changing the radii of zones. With the increase of the inner radius of the outer annular zone, the focal spot broadens along the optical axis and splits into two peaks. Then the two peaks combine back into one peak. There are two critical values for the inner radius of the outer annular zone, at which focal spot changes sharply. The tunable range of the focal depth varies considerably. The phase variance of the inner annular zone affects focal depth also; when the phase variance is π, the effect attains maximum. The parameters of cosh parts of the beam affect both focal splitting and focal depth evidently; focal splitting disappears with increasing parameters of cosh parts, and focal depth increases with increasing the parameters of cosh parts in both the low and the high numerical-aperture optical systems.

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Analysis of the optical force dependency on beam polarization: dielectric/metallic spherical shell in a Gaussian beam

Juntao Xi and Malin Premaratne

We carry out a detailed numerical simulation study to investigate the dependency of optical force on beam polarization. Using the well-known finite-difference time domain method and Maxwell's stress tensor, we consider general dielectric (e.g., glass) or metallic (e.g., gold) spherical shells immersed in a Gaussian optical beam. Our results show that TE and TM polarized Gaussian beams exert different amounts of optical force depending on the shell dimensions and material properties. We specifically show that purely dielectric shells do not experience different optical forces due to polarization differences but TM polarized beams exert higher optical force on metallic shells than equivalent TM polarized beams.

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Persistent correlation of constrained colloidal motion

Thomas Franosch and Sylvia Jeney

We have investigated the motion of a single optically trapped colloidal particle close to a limiting wall at time scales where the inertia of the surrounding fluid plays a significant role. The velocity autocorrelation function exhibits a complex interplay due to the momentum relaxation of the particle, the vortex diffusion in the fluid, the obstruction of flow close to the interface, and the harmonic restoring forces due to the optical trap. We show that already a weak trapping force has a significant impact on the velocity autocorrelation function C(t)= at times where the hydrodynamic memory leads to an algebraic decay. The long-time behavior for the motion parallel and perpendicular to the wall is derived analytically and compared to numerical results. Then, we discuss the power spectral densities of the displacement and provide simple interpolation formulas. The theoretical predictions are finally compared to recent experimental observations.

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Measurement of Mechanical Forces Acting on Optically Trapped Dielectric Spheres Induced by Surface-Enhanced Raman Scattering

Satish Rao, Štefan Bálint, Pål Løvhaugen,  Mark Kreuzer, and Dmitri Petrov

Surface enhanced Raman scattering (SERS) is studied from optically trapped dielectric spheres partially covered with silver colloids in a solution with SERS active molecules. The Raman scattering and Brownian motion of the sphere are simultaneously measured to reveal correlations between the enhancement of the Raman signal and average position of the sphere. The correlations are due to the momenta transfer of the emitted Raman photons from the probe molecules. The addition of a mechanical force measurement provides a different dimension to the study of Raman processes.

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Scattering Forces from the Curl of the Spin Angular Momentum of a Light Field

Silvia Albaladejo, Manuel I. Marqués, Marine Laroche, and Juan José Sáenz

Light forces on small (Rayleigh) particles are usually described as the sum of two terms: the dipolar or gradient force and the scattering or radiation pressure force. The scattering force is traditionally considered proportional to the Poynting vector, which gives the direction and magnitude of the momentum flow. However, as wewill show, there is an additional nonconservative contribution to the scattering force arising in a light field with nonuniform helicity. This force is shown to be proportional to the curl of the spin angular momentum of the light field. The relevance of the spin force is illustrated in the simple case of a 2D field geometry arising in the intersection region of two standing waves.

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Precision Surface-Coupled Optical-Trapping Assay with One-Base pair Resolution

Ashley R. Carter,Yeonee Seol and Thomas T. Perkins

The most commonly used optical-trapping assays are coupled to surfaces, yet such assays lack atomic-scale (0.1nm) spatial resolution due to drift between the surface and trap. We used active stabilization techniques to minimize surface motion to 0.1 nm in three dimensions and decrease multiple types of trap laser noise (pointing, intensity, mode, and polarization). As a result, we achieved nearly the thermal limit (kT = 0.05 - 0.5pN/nm) and frequency (f = 0.03-100 Hz). We next demonstrated sensitivity to one-basepair (0.34-nm) steps along DNA in a surface-coupled assay at moderate force (6 pN). Moreover, basepair stability was achieved immediately after substantial (3.4 pN) changes in force. Active intensity stabilization also led to enhanced force precision (0.01%) that resolved 0.1-pN force-induced changes in DNA hairpin unfolding dynamics. This work brings the benefit of atomic-scale resolution, currently limited to dual-beam trapping assays, along with enhanced force precision to the widely used, surface-coupled optical-trapping assay.

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Atom trapping in an interferometrically generated bottle beam trap

L. Isenhower, W. Williams, A. Dally, and M. Saffman

We demonstrate an optical bottle beam trap created by interfering two fundamental Gaussian beams with different waists. The beams are derived from a single laser source using a Mach-Zehnder interferometer whose arms have unequal magnifications. Destructive interference of the two beams from the Mach-Zehnder leads to a three-dimensional intensity null at the mutual focus of the beams. We demonstrate trapping of cold cesium atoms in a blue detuned bottle beam trap.

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Grab a Golgi: Laser Trapping of Golgi Bodies Reveals in vivo Interactions with the Endoplasmic Reticulum

Imogen A. Sparkes, Tijs Ketelaar, Norbert C.A. de Ruijter and Chris Hawes

In many vacuolate plant cells, individual Golgi bodies appear to be attached to tubules of the pleiomorphic cortical endoplasmic reticulum (ER) network. Such observations culminated in the controversial mobile secretory unit hypothesis to explain transport of cargo from the ER to Golgi via Golgi attached export sites. This proposes that individual Golgi bodies and an attached-ER exit machinery move over or with the surface of the ER whilst collecting cargo for secretion. By the application of infrared laser optical traps to individual Golgi bodies within living leaf cells, we show that individual Golgi bodies can be micromanipulated to reveal their association with the ER. Golgi bodies are physically attached to ER tubules and lateral displacement of individual Golgi bodies results in the rapid growth of the attached ER tubule. Remarkably, the ER network can be remodelled in living cells simply by movement of laser trapped Golgi dragging new ER tubules through the cytoplasm and new ER anchor sites can be established. Finally, we show that trapped Golgi ripped off the ER are 'sticky' and can be docked on to and attached to ER tubules, which will again show rapid growth whilst pulled by moving Golgi.

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Integration of plasmonic trapping in a microfluidic environment

Lina Huang, Sebastian J. Maerkl, and Olivier J. Martin

Near field generated by plasmonic structures has recently been proposed to trap small objects. We report the first integration of plasmonic trapping with microfluidics for lab–on–a–chip applications. A three–layer plasmo–microfluidic chip is used to demonstrate the trapping of polystyrene spheres and yeast cells. This technique enables cell immobilization without the complex optics required for conventional optical tweezers. The benefits of such devices are optical simplicity, low power consumption and compactness; they have great potential for implementing novel functionalities for advanced manipulations and analytics in lab–on–a–chip applications.

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Leveraging Single Protein Polymers To Measure Flexural Rigidity

Joost van Mameren, Karen C. Vermeulen, Fred Gittes and Christoph F. Schmidt

The micrometer-scale length of some protein polymers allows them to be mechanically manipulated in single-molecule experiments. This provides a direct way to measure persistence length. We have used a double optical trap to elastically deform single microtubules and actin filaments. Axial extensional force was exerted on beads attached laterally to the filaments. Because the attachments are off the line of force, pulling the beads apart couples to local bending of the filament. We present a simple mechanical model for the resulting highly nonlinear elastic response of the dumbbell construct. The flexural rigidities of the microfilaments that were found by fitting the model to the experimentally observed force−distance curves are (7.1 ± 0.8) × 104 pN·nm2 (persistence length Lp = 17.2 μm) for F-actin and (6.1 ± 1.3) × 106 pN·nm2 (Lp = 1.4 mm) for microtubules.

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Passive and Active Microrheology of Hard-sphere Colloids

L. G. Wilson, A. W. Harrison, A. B. Schofield, J. Arlt and W. C. K. Poon

We performed passive and active microrheology using probe particles in a bath of well-characterized, model hard-sphere colloids in the fluid state over the whole range of volume fractions below the glass transition. The probe and bath particles have nearly the same size. Passive tracking of probe particles yields short-time self-diffusion coefficients. Comparison with literature data demonstrates that the interaction between probe and bath particles is hard-sphere-like. The short-time diffusivities yield one set of microviscosities as a function of volume fraction, which agrees with previous macrorheological measurements of the high-frequency viscosity of hard-sphere colloids. Using optical tweezers, we measure the force on a trapped probe particle as the rest of the sample is translated at constant velocity. This yields a second set of microviscosities at high Pclet numbers. These agree with previous macrorheological measurements of the high-shear viscosity of similar colloids, at shear-rates below the onset of shear-thickening.

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Optical traps for single molecule biophysics: a primer

T.T. Perkins

Optical trapping experiments of different complexities are making a significant impact in biology. This review seeks to highlight design choices for scientists entering the field or building new instruments and to discuss making calibrated measurements with optical traps. For specificity, this review focuses on nucleic acid-based assays, but the discussion reflects the general experimental design considerations of developing a biological assay and an optical trap to measure it.

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Mechanical Unfolding of Two DIS RNA Kissing Complexes from HIV-1

Pan T.X. Li and Ignacio Tinoco Jr

An RNA kissing complex formed by the dimerization initiation site plays a critical role in the survival and infectivity of human immunodeficiency virus. Two dimerization initiation site kissing sequences, Mal and Lai, have been found in most human immunodeficiency virus 1 variants. Formation and stability of these RNA kissing complexes depend crucially on cationic conditions, particularly Mg2+. Using optical tweezers, we investigated the mechanical unfolding of single RNA molecules with either Mal-type (GUGCAC) or Lai-type (GCGCGC) kissing complexes under various ionic conditions. The force required to disrupt the kissing interaction of the two structures, the rip force, is sensitive to concentrations of KCl and MgCl2; addition of 3 mM MgCl2 to 100 mM KCl changes the rip force of Mal from 21 ± 4 to 46 ± 3 pN. From the rip force distribution, the kinetics of breaking the kissing interaction is calculated as a function of force and cation concentration. The two kissing complexes have distinct unfolding transition states, as shown by different values of ΔX‡, which is the distance from the folded structure to the unfolding transition state. The ΔX‡ of Mal is  0.6 nm smaller than that of Lai, suggesting that fewer kissing base pairs are broken at the transition state of the former, consistent with observations that the Lai-type kissing complex is more stable and requires significantly more force to unfold than the Mal type. More importantly, neither K+ nor Mg2+ significantly changes the position of the transition state along the reaction coordinate. However, increasing concentrations of cations increase the kinetic barrier. We derived a cation-specific parameter, m, to describe how the height of the kinetic barrier depends on the concentration of cations. Our results suggest that Mg2+ greatly slows down the unfolding of the kissing complex but has moderate effects on the formation kinetics of the structure.

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