Optical trapping by a metal thin-film edge

Dongxiao Li, Yonggang Xi, and Hong Koo Kim

We present a new method of optical trapping based on the intensity gradient that is created by boundary diffraction of light at a metal thin-film edge. The structure consists of an optically thick metal-film step formed on a semi-transparent thin-film-metal-coated glass substrate. While the underlying thin layer of metal serves the purpose of suppressing the thermophoretic effect, the metal film step is found to induce a highly localized intensity distribution of light around the edge via self-interference of an incident wave and its boundary diffraction wave. Two-dimensional (2D) optical trapping of micron-sized dielectric particles is experimentally demonstrated with a 100-nm-thick Au film edge formed on a 10-nm-thick-Cr-coated glass slide. For a 2-µm polystyrene sphere, ∼2-pN trapping force is measured at 30-mW incident power of a 1064-nm laser beam. Not involving surface plasmon fields, this thin-film edge trapping is polarization independent and can be easily incorporated into an on-chip microfluidic configuration.

DOI

Incandescent porous carbon microspheres to light up cells: solution phenomena and cellular uptake

Paul Duffy, Luís M. Magno, Rahul B. Yadav, Selene K. Roberts, Andrew D. Ward, Stanley W. Botchway, Paula E. Colavita and Susan J. Quinn

Carbon based materials are attractive for biological applications because of their excellent biocompatibility profile. Porous carbons with high specific surface area are particularly interesting because it is possible in principle to leverage their properties to deliver high drug payloads. In this work, porous carbon microspheres with high specific surface area were prepared and studied in solution and in cells. Raman optical tweezer trapping of microspheres, excited at 532 nm, results in graphitization and incandescence in solvents that display poor heat conduction. Fluorescence confocal microscopy imaging was used to demonstrate the uptake of fluorescently labelled microspheres by cells and the ability to leverage their optical absorptivity in order to cause carbon graphitization and cell death.

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Optical trapping of porous silicon nanoparticles

Maria G Donato, Marco A Monaca, Giuliana Faggio, Luca De Stefano, Philip H Jones, Pietro G Gucciardi and Onofrio M Maragò

Silicon nanoparticles obtained by ball-milling of a 50% porosity silicon layer have been optically trapped when dispersed in a water–surfactant environment. We measured the optical force constants using linearly and radially polarized trapping beams finding a reshaping of the optical potential and an enhanced axial spring constant for the latter. These measurements open perspectives for the control and handling of silicon nanoparticles as labeling agents in biological analysis and fluorescence imaging techniques.

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Nanoengineering a single-molecule mechanical switch using DNA self-assembly

Ken Halvorsen, Diane Schaak and Wesley P Wong

The ability to manipulate and observe single biological molecules has led to both fundamental scientific discoveries and new methods in nanoscale engineering. A common challenge in many single-molecule experiments is reliably linking molecules to surfaces, and identifying their interactions. We have met this challenge by nanoengineering a novel DNA-based linker that behaves as a force-activated switch, providing a molecular signature that can eliminate errant data arising from non-specific and multiple interactions. By integrating a receptor and ligand into a single piece of DNA using DNA self-assembly, a single tether can be positively identified by force–extension behavior, and receptor–ligand unbinding easily identified by a sudden increase in tether length. Additionally, under proper conditions the exact same pair of molecules can be repeatedly bound and unbound. Our approach is simple, versatile and modular, and can be easily implemented using standard commercial reagents and laboratory equipment. In addition to improving the reliability and accuracy of force measurements, this single-molecule mechanical switch paves the way for high-throughput serial measurements, single-molecule on-rate studies, and investigations of population heterogeneity.

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Three-dimensional positioning of optically trapped nanoparticles

Takayuki Higuchi, Quang Duc Pham, Satoshi Hasegawa, and Yoshio Hayasaki

We firstly demonstrate the three-dimensional (3D) measurement of a nanometer-sized sphere held in optical tweezers in water using an in-line digital holographic microscope with a green light emitting diode. Suppressing the movement with optical tweezers enabled us to detect the three-dimensional position of a polystyrene sphere with a diameter of 200 nm. The positioning resolutions of the microscope were 3.2 nm in the transverse direction and 3.4 nm in the axial direction, from the standard deviation of measurements of the 200 nm sphere fixed on glass. Changes in the Brownian motion in response to a change in the trapping laser power were measured. We also demonstrated that this holographic measurement is an effective method for determining the threshold power of the optical trapping.

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A novel video-based microsphere localization algorithm for low contrast silica particles under white light illumination

O. Ueberschär, C. Wagner, T. Stangner, C. Gutsche, F. Kremer

On the basis of a brief review of four common image recognition algorithms for microspheres made of polystyrene or melamine resin, we present a new microsphere localization method for low-contrast silica beads under white light illumination. We compare both the polystyrene and silica procedures with respect to accuracy and precision by means of an optical tweezers setup providing CMOS video microscopy capability. By that we demonstrate that our new silica algorithm achieves a relative position uncertainty of less than ±1 nm for micron-sized microspheres, significantly exceeding the precision of the other silica approaches studied. Second, we present an advancement of our single microsphere tracking method to scenarios where two polystyrene, melamine resin or silica microspheres are in close-to-contact proximity. While the majority of the analysis algorithms studied generate artefacts due to interference effects under these conditions, we show that our new approach yields accurate and precise results.

DOI

The Role of Tropomyosin Domains in Cooperative Activation of the Actin–Myosin Interaction

Yusuke Oguchi, Junji Ishizuka, Sarah E. Hitchcock-DeGregori, Shin'ichi Ishiwata, Masataka Kawai

To establish α-tropomyosin (Tm)'s structure–function relationships in cooperative regulation of muscle contraction, thin filaments were reconstituted with a variety of Tm mutants (Δ2Tm, Δ3Tm, Δ6Tm, P2sTm, P3sTm, P2P3sTm, P1P5Tm, and wtTm), and force and sliding velocity of the thin filament were studied using an in vitro motility assay. In the case of deletion mutants, Δ indicates which of the quasi-equivalent repeats in Tm was deleted. In the case of period (P) mutants, an Ala cluster was introduced into the indicated period to strengthen the Tm–actin interaction. In P1P5Tm, the N-terminal half of period 5 was substituted with that of period 1 to test the quasi-equivalence of these two Tm periods. The reconstitution included bovine cardiac troponin. Deletion studies revealed that period 3 is important for the positive cooperative effect of Tm on actin filament regulation and that period 2 also contributes to this effect at low ionic strength, but to a lesser degree. Furthermore, Tm with one extra Ala cluster at period 2 (P2s) or period 3 (P3s) did not increase force or velocity, whereas Tm with two extra Ala clusters (P2P3s) increased both force and velocity, demonstrating interaction between these periods. Most mutants did not move in the absence of Ca2+. Notable exceptions were Δ6Tm and P1P5Tm, which moved near at the full velocity, but with reduced force, which indicate impaired relaxation. These results are consistent with the mechanism that the Tm–actin interaction cooperatively affects actin to result in generation of greater force and velocity.

DOI

Controlled alignment of bacterial cells with oscillating optical tweezers

Gideon Carmon and Mario Feingold

We used optical tweezers to rotate bacterial cells relative to the optical axis. We rapidly oscillate the optical tweezers along an axis normal to the laser beam, thereby obtaining a linear trap. When the linear trap is longer than a trapped rod-shaped bacterial cell, the cell is aligned along the trap axis. Decreasing the length of the trap, we found that the cell rotates away from the image plane toward the optical axis. In the limit of a nonoscillating trap, the cell aligns along the optical axis. A defocused-edge detection method was devised to measure the orientation of the rotated cell from the corresponding phase-contrast images. Our technique can be used to image three-dimensional sub-cellular structures from different viewpoints and therefore may become a useful tool in fluorescence microscopy.

DOI

Applied Force Provides Insight into Transcriptional Pausing and Its Modulation by Transcription Factor NusA

Jing Zhou, Kook Sun Ha, Arthur La Porta, Robert Landick, Steven M. Block

Transcriptional pausing by RNA polymerase (RNAP) plays an essential role in gene regulation. Pausing is modified by various elongation factors, including prokaryotic NusA, but the mechanisms underlying pausing and NusA function remain unclear. Alternative models for pausing invoke blockade events that precede translocation (on-pathway), enzyme backtracking (off-pathway), or isomerization to a nonbacktracked, elemental pause state (off-pathway). We employed an optical trapping assay to probe the motions of individual RNAP molecules transcribing a DNA template carrying tandem repeats encoding the his pause, subjecting these enzymes to controlled forces. NusA significantly decreased the pause-free elongation rate of RNAP while increasing the probability of entry into short- and long-lifetime pauses, in a manner equivalent to exerting a ∼19 pN force opposing transcription. The effects of force and NusA on pause probabilities and lifetimes support a reaction scheme where nonbacktracked, elemental pauses branch off the elongation pathway from the pretranslocated state of RNAP.

DOI

Probing the mechanobiological properties of human embryonic stem cells in cardiac differentiation by optical tweezers

Youhua Tan, Chi-wing Kong, Shuxun Chen, Shuk Han Cheng, Ronald A. Li, Dong Sun

Human embryonic stem cells (hESC) and hESC-derived cardiomyocytes (hESC-CM) hold great promise for the treatment of cardiovascular diseases. However the mechanobiological properties of hESC and hESC-CM remains elusive. In this paper, we examined the dynamic and static micromechanical properties of hESC and hESC-CM, by manipulating via optical tweezers at the single-cell level. Theoretical approaches were developed to model the dynamic and static mechanical responses of cells during optical stretching. Our experiments showed that the mechanical stiffness of differentiated hESC-CM increased after cardiac differentiation. Such stiffening could associate with increasingly organized myofibrillar assembly that underlines the functional characteristics of hESC-CM. In summary, our findings lay the ground work for using hESC-CMs as models to study mechanical and contractile defects in heart diseases.

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Two-photon fluorescence diagnostics of femtosecond laser tweezers

Arijit Kumar De, Debjit Roy and Debabrata Goswami

We show how two-photon fluorescence signal can be used as an effective detection scheme for trapping particles of any size in comparison to methods using back-scattered light. Development of such a diagnostic scheme allows us a direct observation of trapping a single nanoparticle, which shows new directions to spectroscopy at the single-molecule level in solution.

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Measurement of viscosity of lyotropic liquid crystals by means of rotating laser-trapped microparticles

Qingkun Liu, Theodor Asavei, Taewoo Lee, Halina Rubinsztein-Dunlop, Sailing He, and Ivan I. Smalyukh

We describe a simple microrheology method to measure the viscosity coefficients of lyotropic liquid crystals. This approach is based on the use of a rotating laser-trapped optically anisotropic microsphere. In aligned liquid crystals that have negligible effect on trapping beam’s polarization, the optical torque is transferred from circularly polarized laser trapping beam to the optically anisotropic microparticle and creates the shear flow in the liquid crystalline fluid. The balance of optical and viscous torques yields the local effective viscosity coefficients of the studied lyotropic systems in cholesteric and lamellar phases. This simple yet powerful method is capable of probing viscosity of complex anisotropic fluids for small amounts of sample and even in the presence of defects that obstruct the use of conventional rheology techniques.

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Robust control approach to force estimation in a constant position optical tweezers

Tanuj Aggarwal, Hullas Sehgal, and Murti Salapaka

Feedback enhanced optical tweezers with position regulation capability enable detection and estimation of forces in the pico-Newton regime. In this article we delineate the fundamental limitations and challenges of existing approaches for regulating position and force estimation in an optical tweezer. A modern control systems approach is shown to improve the bandwidth of force estimation by three to four times which is corroborated experimentally.

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Multifocal optical trapping using counter-propagating radially-polarized beams

Yaoju Zhang, Yuxing Dai

A model of optical tweezers which can trap a chain of Rayleigh particles is proposed by using two counter-propagating equal highly focused radial polarized beams. Calculations show that a multifocal distribution along the optical axis is formed and the scattering force is equal to zero in the total focal filed, consequently a chain of metallic Rayleigh particles can be stably trapped. The trap force and the trap stiffness using two counter-propagating Radially-polarized beams are larger than those using two counter-propagating linearly-polarized beams. The trapping stability is calculated and analyzed in detail. The trapping number of particles in a trapping chain can be controlled by adjusting the aperture angle of the objective and the parameters of the filter used in the proposed trap system.

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Inertial Effects of a Small Brownian Particle Cause a Colored Power Spectral Density of Thermal Noise

Anita Jannasch, Mohammed Mahamdeh, and Erik Schäffer

The random thermal force acting on Brownian particles is often approximated in Langevin models by a “white-noise” process. However, fluid entrainment results in a frequency dependence of this thermal force giving it a “color.” While theoretically well understood, direct experimental evidence for this colored nature of the noise term and how it is influenced by a nearby wall is lacking. Here, we directly measured the color of the thermal noise intensity by tracking a particle strongly confined in an ultrastable optical trap. All our measurements are in quantitative agreement with the theoretical predictions. Since Brownian motion is important for microscopic, in particular, biological systems, the colored nature of the noise and its distance dependence to nearby objects need to be accounted for and may even be utilized for advanced sensor applications.

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Chiral Self-Assembled Solid Microspheres: A Novel Multifunctional Microphotonic Device

Gabriella Cipparrone, Alfredo Mazzulla, Alfredo Pane, Raul Josue Hernandez, Roberto Bartolino

Solid chiral microspheres with unique and multifunctional optical properties are produced from cholesteric liquid crystal-water emulsions using photopolymerization processes. These self-organizing microspheres exhibit different internal configurations of helicoidal structures with radial, conical or cylindrical geometries, depending on the physicochemical characteristics of the precursor liquid crystal emulsion.

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Mechanical stochastic tug-of-war models cannot explain bidirectional lipid-droplet transport

Ambarish Kunwar, Suvranta K. Tripathy, Jing Xu, Michelle K. Mattson, Preetha Anand, Roby Sigua, Michael Vershinin,Richard J. McKenney, Clare C. Yu, Alexander Mogilner, and Steven P. Gross

Intracellular transport via the microtubule motors kinesin and dynein plays an important role in maintaining cell structure and function. Often, multiple kinesin or dynein motors move the same cargo. Their collective function depends critically on the single motors’ detachment kinetics under load, which we experimentally measure here. This experimental constraint—combined with other experimentally determined parameters—is then incorporated into theoretical stochastic and mean-field models. Comparison of modeling results and in vitro data shows good agreement for the stochastic, but not mean-field, model. Many cargos in vivo move bidirectionally, frequently reversing course. Because both kinesin and dynein are present on the cargos, one popular hypothesis explaining the frequent reversals is that the opposite-polarity motors engage in unregulated stochastic tugs-of-war. Then, the cargos’ motion can be explained entirely by the outcome of these opposite-motor competitions. Here, we use fully calibrated stochastic and mean-field models to test the tug-of-war hypothesis. Neither model agrees well with our in vivo data, suggesting that, in addition to inevitable tugs-of-war between opposite motors, there is an additional level of regulation not included in the models.

DOI

Colored noise in the fluctuations of an extended DNA molecule detected by optical trapping

Ignacio A. Martínez, Saurabh Raj and Dmitri Petrov

We studied fluctuations of an optically trapped bead connected to a single DNA molecule anchored between the bead and a cover glass or between two optically trapped beads. Power spectral densities of the bead position for different extensions of the molecule were compared with the power spectral density of the position fluctuations of the same bead without the molecule attached. Experiments showed that the fluctuations of the DNA molecule extended up to 80% by a force of 3 pN include the colored noise contribution with spectral dependence 1/f α with α ∼ 0.75.

DOI

Momentum of light scattered from collections of particles

Zhisong Tong and Olga Korotkova

The angular dependence of the momentum flow of a polychromatic plane wave scattered from deterministic and random collections of particles is determined, within the occuracy of the first-order Born approximation, as a function of individual and collective properties of particles. The results are of importance for optimization of optical tweezers.

DOI

Optical Trapping of Beads and Jurkat Cells Using Micromachined Fresnel Zone Plate Integrated with Microfluidic Chip

Ju-Nan Kuo and Han-Zhong Hu

This paper presents a method for trapping beads and cells using a single-beam optical tweezer and a Fresnel zone plate integrated with a microfluidic chip. The experimental results show that a laser power of 2.4 mW is sufficient to trap 3-µm-diameter polystyrene beads, while a laser power of 1.5 mW is sufficient to trap individual Jurkat cells. The Fresnel zone plate developed in this study has many advantages, including a small size, a straightforward fabrication process, and a simple integration with microfluidic chips. Consequently, it provides an ideal solution for the trapping of a wide range of biological cells for analysis purposes.

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Expanding the Optical Trapping Range of Lipid Vesicles to the Nanoscale

Poul M. Bendix and Lene B. Oddershede

Small unilamellar lipid vesicles with diameters down to 50 nm enclosing high refractive index sucrose cores can be optically trapped individually in three dimensions using a focused laser beam. Combined optical trapping and confocal microscopy allows for simultaneous quantitative measurements of the forces exerted on individual vesicles and of their size and shape. The position of individual vesicles in three dimensions is measured with nanometer spatial and 10 μs temporal resolution.

DOI

Holographic aberration correction: optimising the stiffness of an optical trap deep in the sample

Maria Dienerowitz, Graham Gibson, Richard Bowman, and Miles Padgett

We investigate the effects of 1st order spherical aberration and defocus upon the stiffness of an optical trap tens of μm into the sample. We control both these aberrations with a spatial light modulator. The key to maintain optimum trap stiffness over a range of depths is a specific non-trivial combination of defocus and axial objective position. This optimisation increases the trap stiffness by up to a factor of 3 and allows trapping of 1μm polystyrene beads up to 50μm deep in the sample.

DOI

Shear-flow-enhanced barrier crossing

Diego Kienle, Jochen Bammert, and Walter Zimmermann

We consider a single Brownian particle confined in a double well potential (DWP) and investigate its response to a linear shear flow by means of the probability density and current determined via numerical solution of the Fokker-Planck equation. Besides a shear-dependent distortion of the probability distribution, we find that the associated current crossing the potential barrier exhibits a convex dependency on the shear rate when the DWP's minima are far apart. With decreasing distance this functional dependency changes from a convex to concave characteristics accompanied with an increase of the probability current crossing the DWP's barrier. Through the difference map of the particle density distribution it is possible to extract the shear-flow-induced contribution to the particle density driving the barrier-crossing current. This may open the possibility to design specific flow profiles to optimize flow-induced activated transport of nanoparticles.

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Optical manipulation of microparticles using whispering-gallery modes in a silicon nitride microdisk resonator

Hong Cai and Andrew W. Poon

We demonstrate optical manipulation of 1 μm sized polystyrene microparticles on silicon nitride microdisk resonator devices using whispering-gallery modes in an integrated optofluidic chip. We demonstrate multiple trapping tracks and extended trapping ranges within single wavelengths through exciting high-order modes. We observe various sets of trapping tracks and ranges through exciting various resonance modes. We switch particle traveling tracks by tuning the laser wavelength to various wavelengths. We also observe microparticles assembling along the trapping tracks.

DOI

The elementary events underlying force generation in neuronal lamellipodia

Ladan Amin, Erika Ercolini, Rajesh Shahapure, Giacomo Bisson & Vincent Torre

We have used optical tweezers to identify the elementary events underlying force generation in neuronal lamellipodia. When an optically trapped bead seals on the lamellipodium membrane, Brownian fluctuations decrease revealing the underlying elementary events. The distribution of bead velocities has long tails with frequent large positive and negative values associated to forward and backward jumps occurring in 0.1–0.2 ms with varying amplitudes up to 20 nm. Jump frequency and amplitude are reduced when actin turnover is slowed down by the addition of 25 nM Jasplakinolide. When myosin II is inhibited by the addition of 20 μM Blebbistatin, jump frequency is reduced but to a lesser extent than by Jasplainolide. These jumps constitute the elementary events underlying force generation.

DOI

Single Gradientless Light Beam Drags Particles as Tractor Beams

Andrey Novitsky, Cheng-Wei Qiu, and Haifeng Wang

Usually a light beam pushes a particle when the photons act upon it. We investigate the optical forces by nonparaxial gradientless beams and find that the forces can drag suitable particles all the way towards the light source. The major criterion of realizing the backward dragging force is the strong nonparaxiality of the light beam, which contributes to the pulling force owing to momentum conservation. The nonparaxiality of the Bessel beam can be manipulated to possess a dragging force along both the radial longitudinal directions, i.e., a “tractor beam” with stable trajectories is achieved.

DOI

Expanding the optical trapping range of lipid vesicles to the nano-scale

Poul Martin Bendix and Lene B Oddershede

Small unilamellar lipid vesicles with diameters down to 50 nm enclosing high refractive index sucrose cores can be optically trapped individually in three dimensions using a focused laser beam. Combined optical trapping and confocal microscopy allows for simultaneous quantitative measurements of the forces exerted on individual vesicles and of their size and shape. The position of individual vesicles in three dimensions is measured with nanometer spatial and ~10 μs temporal resolution.

DOI

Noise reduction by signal combination in Fourier space applied to drift correction in optical tweezers

Alireza Mashaghi, Peter J. Vach, and Sander J. Tans

A general method is proposed to reduce noise by combining signals. Different measurements of the same physical quantity often exhibit different noise levels in different frequency ranges. Hence, a single high-fidelity signal can be constructed by combining the low-noise parts of the signals in Fourier space. We demonstrate this method by reducing noise in the measured bead-to-bead distance in an optical tweezers setup.

DOI

Negative Nonconservative Forces: Optical “Tractor Beams” for Arbitrary Objects

S. Sukhov and A. Dogariu

Based on the conservation of linear momentum on scattering from arbitrary objects, we demonstrate the generation of nonconservative optical forces that act in a direction opposite to the propagation of the incident beam. The concept can be applied to tailor the force fields produced on nonabsorbing bodies regardless of their sizes and shapes.

DOI

Modeling of optical traps for aerosols

Daniel R. Burnham and David McGloin

Experimental observations suggest that there are differences between the behavior of particles optically trapped in air and trapped in a liquid phase. We have modified the Mie–Debye spherical aberration theory to numerically simulate an aerosol optical trap in an attempt to explain and predict the differences. The model incorporates Mie scattering and a trapping beam focused through media of stratified refractive index. We show that geometrical optics cannot correctly describe the aerosol optical trap and that spherical aberration must be included. We qualitatively explain the observed phenomena before discussing the limits of the experimental techniques and methods to improve it. We conclude that the system does not behave as a true “optical tweezers,” varying between levitation and single beam gradient force trapping, depending on particle and beam parameters.

DOI

Controlling the transverse momentum distribution of a light field via azimuth division of a hologram in holographic optical tweezers

Sheng-Yang Tseng and Long Hsu
This study proposes a method for creating a light field with controlled distribution of transverse momentum (TM) by displaying a hologram only in an azimuth region that centers at θ0 and has a range of Δθ of a spatial light modulator in holographic optical tweezers. This study utilized ray optics to analyze the TM of the resultant field, revealing that the direction of the TM is determined by the center angle of the azimuth region and that the magnitude of the TM is proportional to sin⁡(Δθ/2), without regarding the intensity. The relationship was verified experimentally. In addition, this study demonstrated moving particles along a designed path and depleting particles by the fields.

DOI

Single Nuclei Raman spectroscopy for Drug Evaluation

Hsin-Hung Lin , Yen-Chang Li , Chih-Hao Chang , Chun Liu , Alice L Yu , and Chung-Hsuan Chen

Detection of cellular changes at single-cell level has a great potential for biomedical and biopharmaceutical applications. Raman spectroscopy is an important tool for single-cell molecular imaging analysis. Raman spectroscopy can provide time-resolved information of the selected biomolecular distributions inside a single cell without the need of chemical labeling. In this study, we monitored the cellular responses to antineoplastic drug at a single cell basis with Raman spectroscopy. We demonstrated that single nuclei Raman spectroscopy has the ability to detect and identify nuclear changes related to cytotoxicity at lower concentrations and in shorter time span than conventional cell based assays. Thus, this strategy of using Raman spectroscopy of single, isolated nuclei may be very valuable for rapid and sensitive detection of cellular changes in response to chemotherapeutic agents.

DOI

Computational study of the optical trapping of ellipsoidal particles

Stephen H. Simpson and Simon Hanna

Ellipsoidal dielectric particles may be trapped in a linearly polarized Gaussian beam such that they are harmonically bound with respect to each of their rotational and translational degrees of freedom. The ellipsoid belongs to the highest symmetry class for which this is possible. Typically, the longest axis of the ellipsoid aligns itself with the incident beam axis and the second longest with the polarization direction. We investigate this special property by evaluating the trap stiffness matrix for dielectric ellipsoids with aspect ratios (largest:smallest dimension) in the range 1–10, using the discrete dipole approximation. The results are interpreted using a simple phenomenological model and conclusions are drawn concerning optimization of the trap stiffness for specific applications.

DOI

Optical tweezers setup with optical height detection and active height regulation under white light illumination

Carolin Wagner, Tim Stangner, Christof Gutsche, Olaf Ueberschär and Friedrich Kremer

An optical tweezers setup with optical detection in three dimensions and active height regulation has been developed. The presented novel method to determine the relative height of a microparticle from its microscopic image is based on the analysis of the integrated light intensity of the main maximum of the diffraction pattern. After the determination of a master curve as reference, the height can be detected with an accuracy of up to 2 nm. The method is applicable under microscopic white light illumination and is simple to implement. As an example of measurements where active height regulation is indispensable, force–distance curves are discussed. Furthermore, the colloid height is calculated geometrically. In the range where the geometrical estimation provides reliable results, the values are found to be in quantitative agreement with the suggested algorithm.

DOI

Attachment of Anti-GFP Antibodies to Microspheres for Optical Trapping Experiments

James A. Spudich, Sarah E. Rice, Ronald S. Rock, Thomas J. Purcell and Hans M. Warrick

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes a method for attaching anti-GFP (green fluorescent protein) antibodies to microspheres. GFP-motor fusion proteins can then be adsorbed to the microspheres for use in single-molecule motility studies and optical trapping experiments.

DOI

The Optical Trapping Dumbbell Assay for Nonprocessive Motors or Motors That Turn around Filaments

James A. Spudich, Sarah E. Rice, Ronald S. Rock, Thomas J. Purcell and Hans M. Warrick

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes the preparation of biotin–actin filaments and coverslips coated with polystyrene beads. These are then used in optical trapping dumbbell assays to study interactions between motors and filaments.

DOI

Optical Traps to Study Properties of Molecular Motors

James A. Spudich, Sarah E. Rice, Ronald S. Rock, Thomas J. Purcell and Hans M. Warrick

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This article describes the use of optical traps to study processive and nonprocessive molecular motor proteins, focusing on the design of the instrument and the assays to characterize motility.

DOI

Detachment and reorientation of cells using near-infrared laser microbeam

Ling Gu, Samarendra K. Mohanty, Ninad Ingle

Reorientation of adhering cell(s) with respect to other cell(s) has not been yet possible, thus limiting study of controlled interaction between cells. Here, we report cell detachment upon irradiation with a focused near-infrared laser beam, and reorientation of adherent cells. The detached cell was transported along the axial direction by scattering force and trapped at a higher plane inside the media using the same laser beam by a gravito-optical trap. The trapped cell could then be repositioned by movement of the sample stage and reoriented by rotation of the astigmatic trapping beam. The height at which the cell was stably held was found to depend on the laser beam power. Viability of the detached and manipulated cell was found not to be compromised as confirmed by propidium iodide fluorescence exclusion assay. The reoriented cell was allowed to reattach to the substrate at a controlled distance and orientation with respect to other cells. Further, the cell was found to retain its shape even after multiple detachments and manipulation using the laser beam. This technique opens up new avenues for noncontact modification of cellular orientations that will enable study of intercellular interactions and design of engineered tissue.

DOI

CTGF/CCN2 has a chemoattractive function but a weak adhesive property to embryonic carcinoma cells

Diego P. Aguiar, Bruno Pontes, Fabio A. Mendes, Leonardo R. Andrade, Nathan B. Viana, José G. Abreu

Connective tissue growth factor (CTGF/CCN2) is a protein of the CCN family that modulates cell–ECM interactions in a variety of cell types. In this study, we investigated the chemotactic and adhesive properties of CCN2 protein in embryonic teratocarcinoma P19 cells. Initially, P19 cells were attracted to CCN2-coated agarose beads. In Boyden chamber experiments, CCN2-containing medium induced a threefold greater migration of P19 cells. CCN2 adhesion properties were studied by using optical tweezers. The specific adhesion times of P19 cells to polystyrene beads coated with laminin, fibronectin, CCN2and bovine serum albumin were 1.8 ± 0.5s, 2.7 ± 0.4s, 10 ± 2s and 13 ± 2s, respectively, revealing an unexpectedly low adhesive capacity of CCN2 protein for P19 cells. In conclusion, our findings support the chemoattractive role of CCN2 for P19 cells, but not its adhesive role when compared to laminin or fibronectin.

DOI

Probing ribosomal protein–RNA interactions with an external force

Pierre Mangeol, Thierry Bizebard, Claude Chiaruttini, Marc Dreyfus, Mathias Springer, and  Ulrich Bockelmann

Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites—i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations—i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by “one-side” clamping.

DOI

The Complex Folding Network of Single Calmodulin Molecules

Johannes Stigler, Fabian Ziegler, Anja Gieseke, J. Christof M. Gebhardt, Matthias Rief

Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on- and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.

DOI

Picoliter rheology of gaseous media using a rotating optically trapped birefringent microparticle

Yoshihiko Arita , Andrew W. McKinley , Michael Mazilu , Halina Rubinsztein-Dunlop , and Kishan Dholakia

An optically trapped birefringent microparticle is rotated by a circularly polarized beam in a confined gaseous medium. By recording the terminal rotation velocity and the change in polarization of the incident trapping beam, we determine the viscosity by probing a picolitervolume of air, carbon dioxide and argon in the vicinity of the microparticle. We also characterize the optical force acting on a trapped particle in air using the generalized Lorenz-Mie theory taking into account the aberrations present. This opens up a new potential application of optical tweezers for the accurate measurement of gas viscosity in confined geometries.

DOI

A microfluidic diffusion chamber for reversible environmental changes around flaccid lipid vesicles

Saša Vrhovec, Mojca Mally, Blaž Kavčič and Jure Derganc

The reversible environmental changes around flaccid lipid vesicles represent a considerable experimental challenge, particularly because of remarkable softness of flaccid membranes, which can warp irreversibly under the slightest hydrodynamic flow. As a result, we have developed a microfluidic device for the controlled analysis of individual flaccid, giant lipid vesicles in a changing chemical environment. The setup combines the advantages of a flow-free microfluidic diffusion chamber and optical tweezers, which are used to load the sample vesicles into the chamber. After a vesicle is loaded into the diffusion chamber, its chemical environment is controllably and reversibly changed solely by means of diffusion. The chamber is designed as a 250 micrometres-long and 100 micrometres-wide dead-end microchannel, which extends from a T-junction of the main microchannels. Measurements of the flow-velocity profile in the chamber show that the flow rate decreases exponentially and scales linearly with the flow rate in the main channel. The characteristic length of the exponential decrease is 15 (1 ± 0.13) micrometres, meaning that a large part of the diffusion chamber is effectively flow-free. The diffusion properties are assessed by monitoring the diffusion of a dye into the chamber. It was found that a simple 1D diffusion model fits well to the experimental data. The time needed for the exchange of solutes in the chamber is of the order of minutes, depending on the solute's molecular weight. Here, we demonstrate how the diffusion chamber can be used for reversible environmental changes around flaccid, giant lipid vesicles and membrane tethers (nanotubes).

DOI