Deterministic Optical-Near-Field-Assisted Positioning of Nitrogen-Vacancy Centers

Michael Geiselmann, Renaud Marty, Jan Renger, F. Javier García de Abajo and Romain Quidant

Nanopositioning of single quantum emitters to control their coupling to integrated photonic structures is a crucial step in the fabrication of solid-state quantum optics devices. We use the optical near-field enhancement produced by nanofabricated gold antennas subject to near-infrared illumination to deterministically trap and position single nanodiamonds (NDs) hosting nitrogen-vacancy (NV) centers. The positioning of the NDs at the antenna regions of maximum field intensity is first characterized using both fluorescence and electron microscopy imaging. We further study the interaction between the nanoantenna and the delivered NV center by analyzing its change in fluorescence lifetime, which is driven by the increase in the local density of optical states at the trapping positions. Additionally, the plasmonic enhancement of the near-field intensity allows us to optically control the NV excited lifetime using relatively low NIR illumination intensities, some 20 times lower than in the absence of the antennas.

DOI

Spectral analysis by a video camera in a holographic optical tweezers setup

SŁAWOMIR DROBCZYŃSKI, KAMILA DUŚ-SZACHNIEWICZ, KRZYSZTOF SYMONOWICZ, DARIA GŁOGOCKA

We discuss the basic parameters of the holographic tweezers equipped with a diode laser and cheap video camera. We compare these parameters with the system using a fast camera and high power Nd:YAG laser. The measured parameters are: the power spectra density calculated from tracing the position of the micron polystyrene beads and the trap stiffness. We show that this cheap optical tweezers system is sufficient for experiments in microbiology.

DOI

Optical gradient force of cosh-Gaussian with sine-azimuthal and half-space phase modulation

RUI FU, XIUMIN GAO, HAIBIN SHEN, QING XIN, XINMIAO LU, LINGWEI GUO

The optical gradient force distributions in focal plane of cosh-Gaussian beams with sine-azimuthal variation wavefront and half space phase modulation were investigated. Results show that optical gradient force distributions can be affected considerably by the phase retardation of half-space phase modulation, of a phase parameter that indicates the phase change frequency on an increasing azimuthal angle, and beam parameters in cosh terms of the incident beams. Many gradient force patterns occur, including cross-shape, multiple optical trap arrays, multiple-trap wheel, and many kinds of gradient force lines and curves. Symmetry of the whole gradient force pattern can also be altered remarkably. Above results may find wide applications in optical trapping systems.

DOI

Single-molecule manipulation and detection platform for studying cancer cell chemotaxis

May-Show Chen, Pei-Wen Peng, Bing-Chun Liou, Hsiao-Chen Kuo, Keng-Liang Ou, Tzu-Sen Yang

Chemotaxis of cancer cells is an essential component of tumor dissemination. The chemotactic response is comprised of three separate steps, including chemosensing, polarization and locomotion. We present an innovative approach on chemotaxis assay to address cancer cell chemotaxis. We applied a high-resolution optical tweezers system to manipulate epidermal growth factor (EGF)-coated beads positioned close to the filopodia, to locally stimulate HT29 cells expressing the EGF receptor (EGFR). We demonstrated that membrane protrusion at the leading edge induced by an EGF chemotaxis occurred at about 30∼40 s. In addition, the present observation revealed that the locomotion of HT29 cell depended on whether the HT29 cell sensed the presence of the chemoattractant EGF. We anticipate the proposed approach based on optical tweezers, together with the platform at single-cell level, could be applied to build a quick screening method for detection and treatment evaluation of many types of cancer during chemotaxis.

DOI

Efficient Optical Trapping of CdTe Quantum Dots by Femtosecond Laser Pulses

Wei-Yi Chiang, Tomoki Okuhata, Anwar Usman, Naoto Tamai, and Hiroshi Masuhara

The development in optical trapping and manipulation has been showing rapid progress, most of it is in the small particle sizes in nanometer scales, substituting the conventional continuous-wave lasers with high-repetition-rate ultrashort laser pulse train and nonlinear optical effects. Here, we evaluate two-photon absorption in optical trapping of 2.7 nm-sized CdTe quantum dots (QDs) with high-repetition-rate femtosecond pulse train by probing laser intensity dependence of both Rayleigh scattering image and the two-photon-induced luminescence spectrum of the optically trapped QDs. The Rayleigh scattering imaging indicates that the two-photon absorption (TPA) process enhances trapping ability of the QDs. Similarly, a nonlinear increase of the two-photon-induced luminescence with the incident laser intensity fairly indicates the existence of the TPA process.

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Rotation, oscillation and hydrodynamic synchronization of optically trapped oblate spheroidal microparticles

Alejandro V. Arzola, Petr Jákl, Lukáš Chvátal, and Pavel Zemánek

While the behavior of optically trapped dielectric spherical particles has been extensively studied, the behavior of non-spherical particles remains mainly unexplored. In this work we focus on the dynamics of oblate spheroidal particles trapped in a tightly focused elliptically-polarized vortex beam. In our experiments we used polystyrene spheroids of aspect ratio of major to minor axes equal to 2.55 and of a volume equal to a sphere of diameter 1.7μm. We demonstrate that such particles can be trapped in three dimensions, with the minor axis oriented perpendicular to both the beam polarization (linear) and the beam propagation, can spin in a circularly polarized beam and an optical vortex beam around the axis parallel with the beam propagation. We also observed that these particles can exhibit a periodic motion in the plane transversal to the beam propagation. We measured that the transfer of the orbital angular momentum from the vortex beam to the spheroid gives rise to torques one order of magnitude stronger comparing to the circularly polarized Gaussian beam. We employed a phase-only spatial light modulator to generate several vortex beam traps with one spheroid in each of them. Due to independent setting of beams parameters we controlled spheroids frequency and sense of rotation and observed hydrodynamic phase and frequency locking of rotating spheroids. These optically driven spheroids offer a simple alternative approach to the former techniques based on birefringent, absorbing or chiral microrotors.

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Raman spectroscopy provides a rapid, non-invasive method for quantitation of starch in live, unicellular microalgae

Yuetong Ji, Yuehui He, Yanbin Cui, Tingting Wang, Yun Wang, Yuanguang Li, Wei E. Huang and Jian Xu

Conventional methods for quantitation of starch content in cells generally involve starch extraction steps and are usually labor intensive, thus a rapid and noninvasive method will be valuable. Using the starch-producing unicellular microalga Chlamydomonas reinhardtii as a model, we employed a customized Raman Spectrometer to capture the Raman spectra of individual single-cells under distinct culture conditions and along various growth stages. The results revealed a nearly linear correlation (R2=0.9893) between the signal intensity at 478 cm-1 and the starch content of the cells. We validated the specific correlation by showing that the starch-associated Raman peaks were eliminated in a mutant strain where the AGPase gene was disrupted and consequentially the biosynthesis of starch blocked. Furthermore, the method was validated in an industrial algal strain of Chlorella pyrenoidosa. This is the first demonstration of starch quantitation at individual live cells. Compared to existing cellular-starch quantitation methods, this single-cell Raman spectra based approach is rapid, label-free, noninvasive, culture-independent, low-cost and potentially able to simultaneously track multiple metabolites in individual live cells, therefore should enable many new applications.

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Mechanisms of Cellular Proteostasis: Insights from Single-Molecule Approaches

Carlos J. Bustamante, Christian M. Kaiser, Rodrigo A. Maillard, Daniel H. Goldman, and Christian A.M. Wilson

Cells employ a variety of strategies to maintain proteome homeostasis. Beginning during protein biogenesis, the translation machinery and a number of molecular chaperones promote correct de novo folding of nascent proteins even before synthesis is complete. Another set of molecular chaperones helps to maintain proteins in their functional, native state. Polypeptides that are no longer needed or pose a threat to the cell, such as misfolded proteins and aggregates, are removed in an efficient and timely fashion by ATP-dependent proteases. In this review, we describe how applications of single-molecule manipulation methods, in particular optical tweezers, are shedding new light on the molecular mechanisms of quality control during the life cycles of proteins.

DOI

Modification of the surface plasmon enhanced optical forces on metal nanorod pairs by axial rotation and by dielectric intralayer

Aybike Ural Yalçın, Özgür E. Müstecaplıoğlu, Kaan Güven

We investigate numerically the effect of axial rotation and the presence of a dielectric intralayer on the spectral behavior of the optical force on a gold nanorod pair. The frequency spectrum of the optical force is obtained through the Maxwell stress tensor formulation and the full vectorial solution of electromagnetic waves. The common and the relative forces, which are defined through the optical force acting on each nanorod, are computed for different axial rotations and for different permittivity and thickness of the dielectric intralayer. We found that both the misalignment and the dielectric intralayer can be utilized to tailor the magnitude and direction of the relative optical force, providing a tunable attractive or repulsive response between the nanorods.

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Optical Trapping Effect and Its Calibration Method in Resonance Light Scattering Correlation Spectroscopy of Gold Nanoparticles in Solution

Bocheng Zhang, Tao Lan, Xiangyi Huang, Chaoqing Dong, and Jicun Ren

In this work, we reported an efficient method for eliminating the optical trapping effect on characterization of nanoparticle diffusion parameters by resonance light scattering correlation spectroscopy (RLSCS). The RLSCS represents a new single nanoparticle method and its principle was based on measuring the resonance light scattering fluctuations in a highly focused laser beam due to the Brownian motion of single nanoparticles such as gold nanoparticles (GNPs), which resembled fluorescence correlation spectroscopy (FCS). In RLSCS analysis, the polarizability of nanoparticles are much higher than fluorescent molecules in FCS, and the sizes of them are larger, therefore, the optical trapping force significantly affects the diffusion behaviors of nanoparticles under a highly focused laser beam. In this study, we used the 632.8 nm He—Ne laser as the light source, which was close to the resonance scattering band of GNPs, and chose GNPs (from 20 to 100 nm) as model samples. We theoretically and experimentally investigated the optical trapping effect of GNPs in RLSCS, and observed a good linear relation between the characteristic diffusion times of GNPs and laser intensity in the certain condition (below 100 μW). This result was in line with the theoretical deduction. By the extrapolation strategy, we effectively eliminated the optical trapping effect and accurately obtained the diameter of GNPs, which was in good agreement with that obtained by transmission electron microscopy. The method described here can extend to FCS analysis of fluorescent nanoparticles as well.

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A Rapidly Modulated Multifocal Detection Scheme for Parallel Acquisition of Raman Spectra from a 2-D Focal Array

Lingbo Kong and James Chan

We report the development of a rapidly modulated multifocal detection scheme that enables full Raman spectra (500–2000 cm–1) from a 2-D focal array to be acquired simultaneously. A spatial light modulator splits a laser beam to generate an m × n multifocal array. Raman signals generated within each focus are projected simultaneously into a spectrometer and imaged onto a TE-cooled CCD camera. A shuttering system using different masks is constructed to collect the superimposed Raman spectra of different multifocal patterns. The individual Raman spectrum from each focus is then retrieved from the superimposed spectra with no crosstalk using a postacquisition data processing algorithm. This system is expected to significantly improve the speed of current Raman-based instruments such as laser tweezers Raman spectroscopy and hyperspectral Raman imaging.

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Optofluidic sorting of material chirality by chiral light

Georgiy Tkachenko & Etienne Brasselet

The lack of mirror symmetry, chirality, plays a fundamental role in physics, chemistry and life sciences. The passive separation of entities that only differ by their handedness without need of a chiral material environment remains a challenging task with attractive scientific and industrial benefits. To date, only a few experimental attempts have been reported and remained limited down to the micron scale, most of them relying on hydrodynamical forces associated with the chiral shape of the micro-objects to be sorted. Here we experimentally demonstrate that material chirality can be passively sorted in a fluidic environment by chiral light owing to spin-dependent optical forces without chiral morphology prerequisite. This brings a new twist to the state-of-the-art optofluidic toolbox and the development of a novel kind of passive integrated optofluidic sorters able to deal with molecular scale entities is envisioned.

DOI

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

Yasuharu Takagi, Rachel E. Farrow, Neil Billington, Attila Nagy, Christopher Batters, Yi Yanga, James R. Sellersa, and Justin E. Molloy

Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein’s mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s−1) is similar to the maximum ATPase activity (∼12–14 s−1) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.

DOI

Mechanical Operation and Intersubunit Coordination of Ring-Shaped Molecular Motors: Insights from Single-Molecule Studies

Shixin Liu, Gheorghe Chistol, Carlos Bustamante

Ring NTPases represent a large and diverse group of proteins that couple their nucleotide hydrolysis activity to a mechanical task involving force generation and some type of transport process in the cell. Because of their shape, these enzymes often operate as gates that separate distinct cellular compartments to control and regulate the passage of chemical species across them. In this manner, ions and small molecules are moved across membranes, biopolymer substrates are segregated between cells or moved into confined spaces, double-stranded nucleic acids are separated into single strands to provide access to the genetic information, and polypeptides are unfolded and processed for recycling. Here we review the recent advances in the characterization of these motors using single-molecule manipulation and detection approaches. We describe the various mechanisms by which ring motors convert chemical energy to mechanical force or torque and coordinate the activities of individual subunits that constitute the ring. We also examine how single-molecule studies have contributed to a better understanding of the structural elements involved in motor-substrate interaction, mechanochemical coupling, and intersubunit coordination. Finally, we discuss how these molecular motors tailor their operation—often through regulation by other cofactors—to suit their unique biological functions.

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A Single-Cell Study of a Highly Effective Hog1 Inhibitor for in Situ Yeast Cell Manipulation

Charlotte Hamngren Blomqvist, Peter Dinér, Morten Grøtli, Mattias Goksör and Caroline B. Adiels

We present a single cell study of a highly effective Hog1 inhibitor. For this application, we used sequential treatment of a Saccharomyces cerevisiae cell array, with the Hog1 inhibitor and osmotic stress. For this purpose, a four-inlet microfluidic chamber with controlled introduction of two different cell strains within the same experimental setting and a subsequent rapid switching between treatments was designed. Multiple cell strains within the same experiment is a unique feature which is necessary for determining the expected absent cellular response. The nuclear translocation of the cytosolic MAPK, Hog1, was monitored by fluorescence imaging of Hog1-GFP on a single-cell level. An optical tweezers setup was used for controlled cell capture and array formation. Nuclear Hog1-GFP localization was impaired for treated cells, providing evidence of a congenial microfluidic setup, where the control cells within the experiments validated its appropriateness. The chamber enables multiple treatments with incubation times in the order of seconds and the possibility to remove either of the treatments during measurement. This flexibility and the possibility to use internal control cells ensures it a valuable scientific tool for unraveling the HOG pathway, similar signal transduction pathways and other biological mechanisms where temporal resolution and real time imaging is a prerequisite.

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Radiation torque exerted on a uniaxial anisotropic sphere: Effects of various parameters

ZhengJun Li, ZhenSen Wu, Tan Qu, HaiYing Li, Lu Bai, Lei Gong

Based on orthogonality of associated Legendre functions and trigonometric functions, the radiation torque exerted on a uniaxial anisotropic sphere located in a linearly and circularly polarized Gaussian beam with arbitrary propagation direction is obtained. The radiation torque exerted on a uniaxial anisotropic sphere with different permittivity and permeability tensor elements illuminated by a linearly polarized Gaussian beam is calculated. The effects of the position of beam center, incident angle, polarization angle of the Gaussian beam, absorption, anisotropy ratio and size parameter on the three components of the radiation torque are numerically discussed in detail. The incident Gaussian beam can be a linear or a circular polarization. Some comparisons between these characteristics and those on an isotropic sphere are given.

DOI

Multidimensional optical trapping of a mirror

Antonio Perreca, James Lough, David Kelley, and Stefan W. Ballmer

Alignment control in gravitational-wave detectors has consistently proven to be a difficult problem due to the stringent noise contamination requirement for the gravitational wave readout and the radiation-pressure-induced angular instability in Fabry-Perot cavities (Sidles-Sigg instability). We present the analysis of a dual-carrier control scheme that uses radiation pressure to control a suspended mirror, trapping it in the longitudinal degree of freedom and one angular degree of freedom. We show that this scheme can control the Sidles-Sigg angular instability. Its limiting fundamental noise source is the quantum radiation pressure noise, providing an advantage compared to the conventional angular control schemes. In the Appendix we also derive an exact expression for the optical spring constant used in the control scheme.

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Artificially-induced organelles are optimal targets for optical trapping experiments in living cells

C. López-Quesada, A.-S. Fontaine, A. Farré, M. Joseph, J. Selva, G. Egea, M. D. Ludevid, E. Martín-Badosa, and M. Montes-Usategui

Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads.

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Plasma etching of single fine particle trapped in Ar plasma by optical tweezers

T Ito, K Koga, D Yamashita, K Kamataki, N Itagaki, G Uchida and M Shiratani

Physical and chemical interactions between plasmas and nano-featured surfaces are one important issue in the plasma processing. Here we optically trap single fine particle levitated at plasma/sheath boundary with an infrared laser to realize in-situ analysis of such interactions. We have measured time evolution of the diameter of the single fine particle in Ar plasma. The trapped particle was etched at an etching rate of 1 nm/min in Ar plasma. We also obtained a Raman peak at around 2950 cm−1 corresponding to C-H bonds in the single fine particle in Ar plasma. The results open a new possibility to observe directly interactions between plasma and single fine particle.

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Focal Activation of Cells by Plasmon Resonance Assisted Optical Injection of Signaling Molecules

Gabriel V. Orsinger, Joshua D. Williams, and Marek Romanowski

Experimental methods for single cell intracellular delivery are essential for probing cell signaling dynamics within complex cellular networks, such as those making up the tumor microenvironment. Here, we show a quantitative and general method of interrogation of signaling pathways. We applied highly focused near-infrared laser light to optically inject gold-coated liposomes encapsulating bioactive molecules into single cells for focal activation of cell signaling. For this demonstration, we encapsulated either inositol trisphosphate (IP3), an endogenous cell signaling second messenger, or adenophostin A (AdA), a potent analogue of IP, within 100 nm gold-coated liposomes, and injected these gold-coated liposomes and their contents into the cytosol of single ovarian carcinoma cells to initiate calcium (Ca2+) release from intracellular stores. Upon optical injection of IP3 or AdA at doses above the activation threshold, we observed increases in cytosolic Ca2+ concentration within the injected cell initiating the propagation of a Ca2+ wave throughout nearby cells. As confirmed by octanol-induced inhibition, the intercellular Ca2+ wave traveled via gap junctions. Optical injection of gold-coated liposomes represents a quantitative method of focal activation of signaling cascades of broad interest in biomedical research.

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Improving the trapping capability using radially polarized narrow-width annular beam

Hua-Feng Xu, Wei-Jun Zhang, Jun Quc & Wei Huang

A novel optical-trap method for improving the trapping capability using a radially polarized narrow-width annular beam (NWAB) has been proposed. In this paper, we theoretically study the tight focusing properties of a radially polarized NWAB, formed by subtly blocking the central portion of a radially polarized Bessel–Gaussian beam (the original doughnut beam), through a high-numerical aperture objective. It is shown that a sub-wavelength focal spot () with a quite long depth of focus (about ) can be formed in the vicinity of the focus. Furthermore, the optical trapping forces acting on a metallic Rayleigh particle are calculated for the case where a radially polarized annular beam is applied. Numerical results show that the radially polarized NWAB can largely enhance the transverse trap stiffness and broaden the longitudinal trap range compared with the usage of the original doughnut beam. The influence of the annular factor δ on the focusing properties and the trap stiffness is investigated in detail.

DOI

Interactions between Individual Ultrasound-Stimulated Microbubbles and Fibrin Clots

Christopher Acconcia, Ben Y.C. Leung, Anoop Manjunath, David E. Goertz

The use of ultrasound-stimulated microbubbles (USMBs) to promote thrombolysis is well established, but there remains considerable uncertainty about the mechanisms of this process. Here we examine the microscale interactions between individual USMBs and fibrin clots as a function of bubble size, exposure conditions and clot type. Microbubbles (n = 185) were placed adjacent to clot boundaries (“coarse” or “fine”) using optical tweezers and exposed to 1-MHz ultrasound as a function of pressure (0.1–0.39 MPa). High-speed (10 kfps) imaging was employed, and clots were subsequently assessed with 2-photon microscopy. For fine clots, 46% of bubbles “embedded” within 10 μm of the clot boundary at pressures of 0.1 and 0.2 MPa, whereas at 0.39 MPa, 53% of bubbles penetrated and transited into the clots with an incidence inversely related to their diameter. A substantial fraction of penetrating bubbles induced fibrin network damage and promoted the uptake of nanobeads. In coarse clots, penetration occurred more readily and at lower pressures than in fine clots. The results therefore provide direct evidence of therapeutically relevant effects of USMBs and indicate their dependence on size, exposure conditions and clot properties.

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Stochastic Localization of Microswimmers by Photon Nudging

Andreas P. Bregulla, Haw Yang, and Frank Cichos

Force-free trapping and steering of single photophoretically self-propelled Janus-type particles using a feedback mechanism is experimentally demonstrated. Realtime information on particle position and orientation is used to switch the self-propulsion mechanism of the particle optically. The orientational Brownian motion of the particle thereby provides the reorientation mechanism for the microswimmer. The particle size dependence of the photophoretic propulsion velocity reveals that photon nudging provides an increased position accuracy for decreasing particle radius. The explored steering mechanism is suitable for navigation in complex biological environments and in-depth studies of collective swimming effects.

DOI

Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging

Zachary T. Berndsen, Nicholas Keller, Shelley Grimes, Paul J. Jardine, and Douglas E. Smith

Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.

DOI

Enhanced optical confinement of dye-doped dielectric nanoparticles using a picosecond-pulsed near-infrared laser

A Kittiravechote, W-Y Chiang, A Usman, I Liau and H Masuhara
We demonstrate a novel strategy to increase the capability of confining numerous dye-doped polymeric nanobeads (diameter 100 nm) with laser trapping. Unlike most classical works of optical trapping that address mainly the stiffness of the optical trap, our work concerns an increase in the number of particles confined near the laser focus. We developed an imaging system of light scattering in which a condenser lamp was employed to illuminate the focal plane of the objective lens, and the scattering of the incoherent light was specifically measured to determine the number of confined nanobeads. In contrast to preceding work that used mainly continuous-wave or femtosecond-pulsed lasers, we employed a picosecond-pulsed laser with the half-wavelength of the laser particularly falling within the absorption band of the dopant. Our results show that the number of doped nanobeads held by the laser is significantly greater than that of the bare nanobeads of the same dimension. In striking contrast, the confinement of the nanobeads of the two types was comparable when a continuous-wave laser of the same wavelength and power was employed. The number of confined dye-doped nanobeads increased nonlinearly with the power of the pulsed laser; this dependence was fitted satisfactorily with a second-order polynomial. Supported by theoretical analysis, we attribute the enhanced confinement of doped nanobeads in part to an increased effective refractive index resulting from two-photon resonance between the optical field of the laser and the dopant of the nanobead. We envisage that our findings would evoke applications that benefit from controlled confinement or aggregation of nanomaterials with the employment of near-infrared pulsed lasers.

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Crosstalk elimination in the detection of dual-beam optical tweezers by spatial filtering

Dino Ott, S. Nader S. Reihani and Lene B. Oddershede

In dual-beam optical tweezers, the accuracy of position and force measurements is often compromised by crosstalk between the two detected signals, this crosstalk leading to systematic and significant errors on the measured forces and distances. This is true both for dual-beam optical traps where the splitting of the two traps is done by polarization optics and for dual optical traps constructed by other methods, e.g., holographic tweezers. If the two traps are orthogonally polarized, most often crosstalk is minimized by inserting polarization optics in front of the detector; however, this method is not perfect because of the de-polarization of the trapping beam introduced by the required high numerical aperture optics. Here we present a simple and easy-to-implement method to efficiently eliminate crosstalk. The method is based on spatial filtering by simply inserting a pinhole at the correct position and is highly compatible with standard back focal plane photodiode based detection of position and force. Our spatial filtering method reduces crosstalk up to five times better than polarization filtering alone. The effectiveness is dependent on pinhole size and distance between the traps and is here quantified experimentally and reproduced by theoretical modeling. The method here proposed will improve the accuracy of force-distance measurements, e.g., of single molecules, performed by dual-beam optical traps and hence give much more scientific value for the experimental efforts.

DOI

A Viral Packaging Motor Varies Its DNA Rotation and Step Size to Preserve Subunit Coordination as the Capsid Fills

Shixin Liu, Gheorghe Chistol, Craig L. Hetherington, Sara Tafoya, K. Aathavan, Joerg Schnitzbauer, Shelley Grimes, Paul J. Jardine, Carlos Bustamante

Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motor’s step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated.

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Indirect pushing based automated micromanipulation of biological cells using optical tweezers

Atul Thakur, Sagar Chowdhury, Petr Švec, Chenlu Wang, Wolfgang Losert, Satyandra K. Gupta

In this paper, we introduce an indirect pushing based technique for automated micromanipulation of biological cells. In indirect pushing, an optically trapped glass bead pushes a freely diffusing intermediate bead that in turn pushes a freely diffusing target cell towards a desired goal. Some cells can undergo significant changes in their behaviors as a result of direct exposure to a laser beam. Indirect pushing eliminates this problem by minimizing the exposure of the cell to the laser beam. We report an automated feedback planning algorithm that combines three motion maneuvers, namely, push, align, and backup for micromanipulation of cells. We have developed a dynamics based simulation model of indirect pushing dynamics and also identified parameters of measurement noise using physical experiments. We present an optimization-based approach for automated tuning of planner parameters to enhance its robustness. Finally, we have tested the developed planner using our optical tweezers physical setup and carried out a detailed analysis of the experimental results. The developed approach can be utilized in biological experiments for studying collective cell migration by accurately arranging the cells in arrays without exposing them to a laser beam.

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Tracing optical force fields within graded-index media

Alireza Akbarzadeh, Mohammad Danesh, Cheng-Wei Qiu and Aaron J Danner

The mechanical interaction between light and graded index media (both isotropic and anisotropic) is presented from the geometrical optics (GO) perspective. Utilizing Hamiltonian equations to determine ray trajectories combined with a description of the Lorentz force exerted on bound currents and charges, we provide a general method that we denote 'force tracing' for determining the direction and magnitude of the bulk and surface force density in arbitrarily anisotropic and inhomogeneous media. This technique provides the optical community with machinery which can give a good estimation of the force field distribution in different complex media, and with significantly faster computation speeds than full-wave methods allow. Comparison of force tracing against analytical solutions shows some unusual limitations of GO, which we also illustrate.

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Fabrication of birefringent nanocylinders for single-molecule force and torque measurement

Ping-Chun Li, Jen-Chien Chang, Arthur La Porta and Edward T Yu

Optically anisotropic subwavelength scale dielectric particles have been shown to enable studies of the mechanical properties of bio-molecules via optical trapping and manipulation. However, techniques emphasized to date for fabrication of such particles generally suffer from limited uniformity and control over particle dimensions, or low throughput and high cost. Here, an approach for rapid, low-cost, fabrication of large quantities of birefringent quartz nanocylinders with dimensions optimized for optical torque wrench experiments is described. For a typical process, 108 or more quartz cylinders with diameters of 500 nm and heights of 800 nm, with uniformity of ±5% in each dimension, can be fabricated over ~10 cm2 areas, for binding to a single bio-molecule, and harvested for use in optical trapping experiments. Use of these structures to measure extensional and torsional dynamics of single DNA molecules is demonstrated with measured forces and torques shown to be in very good agreement with previously reported results.

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Fundamental validation for surface texture imaging using a microsphere as a laser-trapping-based microprobe

Masaki Michihata, Kosuke Takami, Terutake Hayashi, Yasuhiro Takaya

A surface imaging technique using a laser-trapped microsphere is proposed. The goal of this research is to image the surface texture, while simultaneously measuring the position of the engineered surfaces using the laser-trapping-based microprobe. This paper presents an investigation of imaging characteristics for the microsphere technique. Depending on the distance from the surface to the microsphere, the available images could be either real or virtual. Virtual images had a higher contrast than real images. Contrast and magnification varies depending on the positions of the focal point of the objective lens and surface.

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Aurora A orchestrates entosis by regulating a dynamic MCAK–TIP150 interaction

Peng Xia, Jinhua Zhou, Xiaoyu Song, Bing Wu, Xing Liu, Di Li, Shuyuan Zhang, Zhikai Wang, Huijuan Yu, Tarsha Ward, Jiancun Zhang, Yinmei Li, Xiaoning Wang, Yong Chen, Zhen Guo and Xuebiao Yao

Entosis, a cell-in-cell process, has been implicated in the formation of aneuploidy associated with an aberrant cell division control. Microtubule plus-end-tracking protein TIP150 facilitates the loading of MCAK onto the microtubule plus ends and orchestrates microtubule plus-end dynamics during cell division. Here we show that TIP150 cooperates with MCAK to govern entosis via a regulatory circuitry that involves Aurora A-mediated phosphorylation of MCAK. Our biochemical analyses show that MCAK forms an intra-molecular association, which is essential for TIP150 binding. Interestingly, Aurora A-mediated phosphorylation of MCAK modulates its intra-molecular association, which perturbs the MCAK–TIP150 interaction in vitro and inhibits entosis in vivo. To probe if MCAK–TIP150 interaction regulates microtubule plasticity to affect the mechanical properties of cells during entosis, we used an optical trap to measure the mechanical rigidity of live MCF7 cells. We find that the MCAK cooperates with TIP150 to promote microtubule dynamics and modulate the mechanical rigidity of the cells during entosis. Our results show that a dynamic interaction of MCAK–TIP150 orchestrated by Aurora A-mediated phosphorylation governs entosis via regulating microtubule plus-end dynamics and cell rigidity. These data reveal a previously unknown mechanism of Aurora A regulation in the control of microtubule plasticity during cell-in-cell processes.

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Independent Synchronized Control and Visualization of Interactions between Living Cells and Organisms

Vincent Rouger, Guillaume Bordet, Carole Couillault, Serge Monneret, Sébastien Mailfert, Jonathan J. Ewbank, Nathalie Pujol, Didier Marguet

To investigate the early stages of cell-cell interactions occurring between living biological samples, imaging methods with appropriate spatiotemporal resolution are required. Among the techniques currently available, those based on optical trapping are promising. Methods to image trapped objects, however, in general suffer from a lack of three-dimensional resolution, due to technical constraints. Here, we have developed an original setup comprising two independent modules: holographic optical tweezers, which offer a versatile and precise way to move multiple objects simultaneously but independently, and a confocal microscope that provides fast three-dimensional image acquisition. The optical decoupling of these two modules through the same objective gives users the possibility to easily investigate very early steps in biological interactions. We illustrate the potential of this setup with an analysis of infection by the fungus Drechmeria coniospora of different developmental stages of Caenorhabditis elegans. This has allowed us to identify specific areas on the nematode’s surface where fungal spores adhere preferentially. We also quantified this adhesion process for different mutant nematode strains, and thereby derive insights into the host factors that mediate fungal spore adhesion.

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Double nanohole optical tweezers visualize protein p53 suppressing unzipping of single DNA-hairpins

Abhay Kotnala and Reuven Gordon

Here we report on the use of double-nanohole (DNH) optical tweezers as a label-free and free-solution single-molecule probe for protein–DNA interactions. Using this approach, we demonstrate the unzipping of individual 10 base pair DNA-hairpins, and quantify how tumor suppressor p53 protein delays the unzipping. From the Arrhenius behavior, we find the energy barrier to unzipping introduced by p53 to be 2 × 10−20 J, whereas cys135ser mutant p53 does not show suppression of unzipping, which gives clues to its functional inability to suppress tumor growth. This transformative approach to single molecule analysis allows for ultra-sensitive detection and quantification of protein–DNA interactions to revolutionize the fight against genetic diseases.

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Force-induced melting of DNA—evidence for peeling and internal melting from force spectra on short synthetic duplex sequences

Niklas Bosaeus, Afaf H. El-Sagheer, Tom Brown, Björn Åkerman and Bengt Nordén

Overstretching of DNA occurs at about 60–70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive.

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Nascent RNA transcripts facilitate the formation of G-quadruplexes

Prakash Shrestha, Shan Xiao, Soma Dhakal, Zheng Tan and Hanbin Mao

Recent discovery of the RNA/DNA hybrid G-quadruplexes (HQs) and their potential wide-spread occurrence in human genome during transcription have suggested a new and generic transcriptional control mechanism. The G-rich sequence in which HQ may form can coincide with that for DNA G-quadruplexes (GQs), which are well known to modulate transcriptions. Understanding the molecular interaction between HQ and GQ is, therefore, of pivotal importance to dissect the new mechanism for transcriptional regulation. Using a T7 transcription model, herein we found that GQ and HQ form in a natural sequence, (GGGGA)4, downstream of many transcription start sites. Using a newly-developed single-molecular stalled-transcription assay, we revealed that RNA transcripts helped to populate quadruplexes at the expense of duplexes. Among quadruplexes, HQ predominates GQ in population and mechanical stabilities, suggesting HQ may serve as a better mechanical block during transcription. The fact that HQ and GQ folded within tens of milliseconds in the presence of RNA transcripts provided justification for the co-transcriptional folding of these species. The catalytic role of RNA transcripts in the GQ formation was strongly suggested as the GQ folded >7 times slower without transcription. These results shed light on the possible synergistic effect of GQs and HQs on transcriptional controls.

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Construction of a system for single-cell transgene induction in Caenorhabditis elegans using a pulsed infrared laser

Matthew A. Churgin, Liping He, John I. Murray, Christopher Fang-Yen

The spatial and temporal control of transgene expression is an important tool in Caenorhabditis elegans biology. We previously described a method for evoking gene expression in arbitrary cells by using a focused pulsed infrared laser to induce a heat shock response (Churgin et al., 2013) [1]. Here we describe detailed methods for building and testing a system for performing single-cell heat shock. Steps include setting up the laser and associated components, coupling the laser beam to a microscope, and testing heat shock protocols. All steps can be carried out using readily available off-the-shelf components.

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Single molecule techniques in DNA repair: A primer

Craig D. Hughes, Michelle Simons, Cassidy E. Mackenzie, Bennett Van Houten, Neil M. Kad

A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit – searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair.

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Parasite impairment by targeting Plasmodium-infected RBCs using glyceryl-dilaurate nanostructured lipid carriers

Soniya A. Jain, Himanish Basu, Priyanka S. Prabhu, Umangi Soni, Medha D. Joshi, Deepak Mathur, Vandana B. Patravale, Sulabha Pathak, Shobhona Sharma

Antimalarial therapy is a major contributor to declining malaria morbidity and mortality. However, the high toxicity and low bioavailability of current antimalarials and emerging drug resistance necessitates drug-delivery research. We have previously developed glyceryl-dilaurate nanolipid carriers (GDL-NLCs) for antimalarial drug delivery. Here, we show evidence that GDL-NLCs themselves selectively target Plasmodium-infected red blood cells (iRBCs), and cause severe parasite impairment. The glyceryl-dilaurate lipid-moiety was important in the targeting. GDL-NLCs localized to the parasite mitochondrion and uptake led to mitochondrial-membrane polarization and Ca2+ ion accumulation, ROS release, and stage-specific iRBC lysis. GDL-NLC treatment also resulted in externalization of iRBC-membrane phosphatidylserine and enhanced iRBC clearance by macrophages. GDL-NLC uptake disrupted the parasite-induced tubulovesicular network, which is vital for nutrient import by the parasite. Laser optical trap studies revealed that GDL-NLCs also restored iRBC flexibility. Such restoration of iRBC flexibility may help mitigate the vasculature clogging that can lead to cerebral malaria. We demonstrate the suitability of GDL-NLCs for intravenous delivery of antimalarial combinations artemether–clindamycin and artemether–lumefantrine in the murine model. Complete parasite clearance was achieved at 5−20% of the therapeutic dose of these combinations. Thus, this nanostructured lipid formulation can solubilize lipophilic drugs, selectively target and impair the parasite-infected red cell, and therefore constitutes a potent delivery vehicle for antimalarials.

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Optical stacking of microparticles in a pyramidal structure created with a symmetric cubic phase

Pedro A. Quinto-Su and Rocío Jáuregui
We show a simple way to generate three dimensional optical potentials consisting of tightly localized high intensity spots arranged in a structure with a pyramidal geometry. The three dimensional patterns are created by focusing a Gaussian beam with a symmetric cubic phase abs((ax)3) + abs((ay)3) imprinted by a spatial light modulator. We show that it is possible to trap and stack around a hundred dielectric microspheres (silica mean diameter 2.47 μm) in pyramidal structures (characteristic dimensions H, W ∼ 15 – 20μm) held together by optical binding with moderate laser power (P < 20 mW). Axial stability is mainly provided by balancing the light scattering force with the axial gradient and gravity. The microparticle structures are sufficiently stable to be easily displaced by moving the microscope stage.

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Confocal Raman Microscopy for Investigating Synthesis and Characterization of Individual Optically Trapped Vinyl-Polymerized Surfactant Particles

Jonathan J. Schaefer, Alexis C. Crawford, Marc D. Porter, and Joel M. Harris

Small polymeric particles are increasingly employed as adsorbent materials, as molecular carriers, as delivery vehicles, and in preconcentration applications. The rational development of these materials requires in situ methods of analysis to characterize their synthesis, structure, and applications. Optical-trapping confocal Raman microscopy is a spectroscopic method capable of acquiring information at several stages of the development of such dispersed particulate materials. In the present study, an example material is developed and tested using confocal Raman microscopy for characterization at each stage of the process. Specifically, the method is used to investigate the synthesis, structure, and applications of individual polymeric surfactant particles produced by the vinyl polymerization of sodium 11-acrylamidoundecanoate (SAAU). The kinetics of polymerization can be monitored over time by measuring the loss of the acrylamide C=C functional groups using confocal Raman microscopy of particles optically trapped by the excitation laser, where, within the limits of detecting the vinyl functional group, the complete polymerization of the SAAU monomer was achieved. The polymerized SAAU particles are spherical, and they exhibit uniform access to water throughout their structure, as tested by the penetration of heavy water (D2O) and collection of spatially resolved Raman spectra from the interior of the particle. These porous particles contain hydrophobic domains that can be used to accumulate molecules for adsorption or carrier applications. This property was tested by using confocal Raman microscopy to measure the accumulation equilibria and kinetics of a model compound, dioxybenzone. The partitioning of this compound into the polymer surfactant could be determined on a quantitative basis using relative scattering cross sections of the SAAU monomer and the adsorbate. The study points out the utility of optical-trapping confocal Raman microscopy for investigating the synthesis, structure, and potential carrier applications of polymeric particle materials.

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Radiation forces on a Rayleigh particle by a highly focused elliptically polarized beam

Jianhua Shu, Yongxin Liu, Ziyang Chen & Jixiong Pu

The radiation force of highly focused elliptically polarized beams on a Rayleigh particle is theoretically studied. The numerical results show that elliptically polarized beams can be used to trap particles. The influence of the beam widths, phase retardations of the incident beam, and numerical apertures of an objective lens on the radiation force distribution has been studied. Studies in transverse scattering forces reveal that torques can be produced by elliptically polarized beams carrying spin angular momentum, and that the torque, in the focal plane, produced by elliptically polarized beams can be regarded as the superposition of those by right-hand circularly and left-hand circularly polarized beams with different ratios between them.

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Enhancing DNA binding rate using optical trapping of high-density gold nanodisks

En-Hung Lin, Ming-Yang Pan, Ming-Chang Lee and Pei-Kuen Wei

We present the dynamic study of optical trapping of fluorescent molecules using high-density gold nanodisk arrays. The gold nanodisks were fabricated by electron beam lithography with a diameter of 500 nm and a period of 1 μm. Dark-field illumination showed ∼15 times enhancement of fluorescence near edges of nanodisks. Such enhanced near-field generated an optical trapping force of ∼10 fN under 3.58 × 103 W/m2 illumination intensity as calculated from the Brownian motions of 590 nm polystyrene beads. Kinetic observation of thiolated DNA modified with Cy5 dye showed different binding rates of DNA under different illumination intensity. The binding rate increased from 2.14 × 103 s−1 (I = 0.7 × 103 W/m2) to 1.15 × 105 s−1 (I = 3.58 × 103 W/m2). Both enhanced fluorescence and binding rate indicate that gold nanodisks efficiently improve both detection limit and interaction time for microarrays.

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Extraordinary momentum and spin in evanescent waves

Konstantin Y. Bliokh, Aleksandr Y. Bekshaev & Franco Nori

Momentum and spin represent fundamental dynamic properties of quantum particles and fields. In particular, propagating optical waves (photons) carry momentum and longitudinal spin determined by the wave vector and circular polarization, respectively. Here we show that exactly the opposite can be the case for evanescent optical waves. A single evanescent wave possesses a spin component, which is independent of the polarization and is orthogonal to the wave vector. Furthermore, such a wave carries a momentum component, which is determined by the circular polarization and is also orthogonal to the wave vector. We show that these extraordinary properties reveal a fundamental Belinfante’s spin momentum, known in field theory and unobservable in propagating fields. We demonstrate that the transverse momentum and spin push and twist a probe Mie particle in an evanescent field. This allows the observation of ‘impossible’ properties of light and of a fundamental field-theory quantity, which was previously considered as ‘virtual’.

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Interconnection of polarization properties and coherence of optical fields

Claudia Yu. Zenkova

Theoretical and experimental approaches to diagnosing internal spin and orbital optical flows and the corresponding optical forces caused by these flows are offered. These approaches are based on the investigation of the motion of the particles tested in the formed optical field. The dependence of the above-mentioned forces upon the size and optical properties of the particles is demonstrated. The possibility of using kinematic values defining the motion dynamics of particles of the Rayleigh light scattering mechanism to make a quantitative assessment of the degree of coherence of mutually orthogonal waves that are linearly polarized in the incidence plane is demonstrated. The feasibility of using the above mentioned approach, its shortcomings, and its advantages over the interfering method for estimating the degree of coherence are analyzed.

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Moving average process underlying the holographic–optical–tweezers experiments

Jakub Ślęzak, Sławomir Drobczyński, Karina Weron, and Jan Masajada

We study the statistical properties of recordings that contain time-dependent positions of a bead trapped in optical tweezers. Analysis of such a time series indicates that the commonly accepted model, i.e., the autoregressive process of first-order, is not sufficient to fit the data. We show the presence of a first-order moving average part in the dynamical model of the system. We explain the origin of this part as an influence of the high-frequency CCD camera on the measurements. We show that this influence evidently depends on the applied exposure time. The proposed autoregressive moving average model appears to reflect perfectly all statistical features of the high-frequency recording data.

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Nano-Optical Conveyor Belt, Part II: Demonstration of Handoff Between Near-Field Optical Traps

Yuxin Zheng, Jason Ryan, Paul Hansen, Yao-Te Cheng, Tsung-Ju Lu, and Lambertus Hesselink

Optical tweezers have been widely used to manipulate biological and colloidal material, but the diffraction limit of far-field optics makes focused beams unsuitable for manipulating nanoscale objects with dimensions much smaller than the wavelength of light. While plasmonic structures have recently been successful in trapping nanoscale objects with high positioning accuracy, using such structures for manipulation over longer range has remained a significant challenge. In this work, we introduce a conveyor belt design based on a novel plasmonic structure, the resonant C-shaped engraving (CSE). We show how long-range manipulation is made possible by means of handoff between neighboring CSEs, and we present a simple technique for controlling handoff by rotating the polarization of laser illumination. We experimentally demonstrate handoff between a pair of CSEs for polystyrene spheres 200, 390, and 500 nm in diameter. We then extend this technique and demonstrate controlled particle transport down a 4.5 μm long “nano-optical conveyor belt.”

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Nano-Optical Conveyor Belt, Part I: Theory

Paul Hansen, Yuxin Zheng, Jason Ryan, and Lambertus Hesselink

We propose a method for peristaltic transport of nanoparticles using the optical force field over a nanostructured surface. Nanostructures may be designed to produce strong near-field hot spots when illuminated. The hot spots function as optical traps, separately addressable by their resonant wavelengths and polarizations. By activating closely packed traps sequentially, nanoparticles may be handed off between adjacent traps in a peristaltic fashion. A linear repeating structure of three separately addressable traps forms a “nano-optical conveyor belt”; a unit cell with four separately addressable traps permits controlled peristaltic transport in the plane. Using specifically designed activation sequences allows particle sorting.

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Bacterial twitching motility is coordinated by a two-dimensional tug-of-war with directional memory

Rahul Marathe, Claudia Meel, Nora C. Schmidt, Lena Dewenter, Rainer Kurre, Lilo Greune, M. Alexander Schmidt, Melanie J.I. Müller, Reinhard Lipowsky, Berenike Maier & Stefan Klumpp

Type IV pili are ubiquitous bacterial motors that power surface motility. In peritrichously piliated species, it is unclear how multiple pili are coordinated to generate movement with directional persistence. Here we use a combined theoretical and experimental approach to test the hypothesis that multiple pili of Neisseria gonorrhoeae are coordinated through a tug-of-war. Based on force-dependent unbinding rates and pilus retraction speeds measured at the level of single pili, we build a tug-of-war model. Whereas the one-dimensional model robustly predicts persistent movement, the two-dimensional model requires a mechanism of directional memory provided by re-elongation of fully retracted pili and pilus bundling. Experimentally, we confirm memory in the form of bursts of pilus retractions. Bursts are seen even with bundling suppressed, indicating re-elongation from stable core complexes as the key mechanism of directional memory. Directional memory increases the surface range explored by motile bacteria and likely facilitates surface colonization.

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Sub-diffraction positioning of a two-photon excited and optically trapped quantum dot

Liselotte Jauffred, Anders Kyrsting, Eva C. Arnspang, S. Nader S. Reihani and Lene B. Oddershede

Colloidal quantum dots are luminescent long-lived probes that can be two-photon excited and manipulated by a single laser beam. Therefore, quantum dots can be used for simultaneous single molecule visualization and force manipulation using an infra-red laser. Here, we show that even a single optically trapped quantum dot, performing restricted Brownian motion within the focal volume, can be two-photon excited by the trapping laser beam and its luminescence can be detected by a camera. After two-photon excitation for a time long enough, the emitted light from the quantum dot is shown to blueshift. A quantum dot is much smaller than a diffraction limited laser focus and by mapping out the intensity of the focal volume and overlaying this with the positions visited by a quantum dot, a quantum dot is shown often to explore regions of the focal volume where the intensity is too low to render two-photon absorption likely. This is in accordance with the observation that a trapped quantum dot is only fluorescing 5–10 percent of the time. The results are important for realizing nano-scale quantum dot control and visualization and for correct interpretation of experiments using two-photon excited quantum dots as markers.

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Ultrafast Redistribution of E. coli SSB along Long Single-Stranded DNA via Intersegment Transfer

Kyung Suk Lee, Amanda B. Marciel, Alexander G. Kozlov, Charles M. Schroeder, Timothy M. Lohman, Taekjip Ha

Single-stranded DNA binding proteins (SSBs) selectively bind single-stranded DNA (ssDNA) and facilitate recruitment of additional proteins and enzymes to their sites of action on DNA. SSB can also locally diffuse on ssDNA, which allows it to quickly reposition itself while remaining bound to ssDNA. In this work, we used a hybrid instrument that combines single-molecule fluorescence and force spectroscopy to directly visualize the movement of Escherichia coli SSB on long polymeric ssDNA. Long ssDNA was synthesized without secondary structure that can hinder quantitative analysis of SSB movement. The apparent diffusion coefficient of E. coli SSB thus determined ranged from 70,000 to 170,000 nt2/s, which is at least 600 times higher than that determined from SSB diffusion on short ssDNA oligomers, and is within the range of values reported for protein diffusion on double-stranded DNA. Our work suggests that SSB can also migrate via a long-range intersegment transfer on long ssDNA. The force dependence of SSB movement on ssDNA further supports this interpretation.

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Manipulation of ZnO nanowires using a tapered fiber probe

Chang Cheng, Xiaohao Xu, Hongxiang Lei and Baojun Li

Manipulating ZnO nanowires with high precision and flexibility is of great importance for fabrication of semiconductor-based optoelectronic devices. Here, we report the manipulation of ZnO nanowires using a tapered fiber probe. With a light of 980 nm launched into the probe, ZnO nanowires were trapped and manipulated by the probe through optical forces. Migration and alignment were also realized. Experiments were interpreted by numerical simulations.

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