## Friday, January 29, 2016

### The Role of Rac1 in the Growth Cone Dynamics and Force Generation of DRG Neurons

Wasim A. Sayyad, Paolo Fabris, Vincent Torre

We used optical tweezers, video imaging, immunocytochemistry and a variety of inhibitors to analyze the role of Rac1 in the motility and force generation of lamellipodia and filopodia from developing growth cones of isolated Dorsal Root Ganglia neurons. When the activity of Rac1 was inhibited by the drug EHop-016, the period of lamellipodia protrusion/retraction cycles increased and the lamellipodia retrograde flow rate decreased; moreover, the axial force exerted by lamellipodia was reduced dramatically. Inhibition of Arp2/3 by a moderate amount of the drug CK-548 caused a transient retraction of lamellipodia followed by a complete recovery of their usual motility. This recovery was abolished by the concomitant inhibition of Rac1. The filopodia length increased upon inhibition of both Rac1 and Arp2/3, but the speed of filopodia protrusion increased when Rac1 was inhibited and decreased instead when Arp2/3 was inhibited. These results suggest that Rac1 acts as a switch that activates upon inhibition of Arp2/3. Rac1 also controls the filopodia dynamics necessary to explore the environment.

DOI

### Diffractive optical devices produced by light-assisted trapping of nanoparticles

J. F. Muñoz-Martínez, M. Jubera, J. Matarrubia, A. García-Cabañes, F. Agulló-López, and M. Carrascosa

One- and two-dimensional diffractive optical devices have been fabricated by light-assisted trapping and patterning of nanoparticles. The method is based on the dielectrophoretic forces appearing in the vicinity of a photovoltaic crystal, such as Fe:LiNbO3, during or after illumination. By illumination with the appropriate light distribution, the nanoparticles are organized along patterns designed at will. One- and two-dimensional diffractive components have been achieved on X- and Z-cut Fe:LiNbO3 crystals, with their polar axes parallel and perpendicular to the crystal surface, respectively. Diffraction gratings with periods down to around a few micrometers have been produced using metal (Al, Ag) nanoparticles with radii in the range of 70–100 nm. Moreover, several 2D devices, such as Fresnel zone plates, have been also produced showing the potential of the method. The diffractive particle patterns remain stable when light is removed. A method to transfer the diffractive patterns to other nonphotovoltaic substrates, such as silica glass, has been also reported.

DOI

### Structure-Based Derivation of Protein Folding Intermediates and Energies from Optical Tweezers

Aleksander A. Rebane, Lu Ma, Yongli Zhang

Optical tweezers (OTs) measure the force-dependent time-resolved extension of a single macromolecule tethered between two trapped beads. From this measurement, it is possible to determine the folding intermediates, energies, and kinetics of the macromolecule. Previous data analysis generally has used the extension as a reaction coordinate to characterize the observed folding transitions. Despite its convenience, the extension poorly describes folding in the absence of force. Here, we chose the contour length of the unfolded polypeptide as a reaction coordinate and modeled the extensions of protein structures along their predicted folding pathways based on high-resolution structures of the proteins in their native states. We included the extension in our model to calculate the total extensions, energies, and transition rates of the proteins as a function of force. We fit these calculations to the corresponding experimental measurements and obtained the best-fit conformations and energies of proteins in different folding states. We applied our method to analyze single-molecule trajectories of two representative protein complexes responsible for membrane fusion, the HIV-1 glycoprotein 41 and the synaptic SNARE proteins, which involved transitions between two and five states, respectively. Nonlinear fitting of the model to the experimental data revealed the structures of folding intermediates and transition states and their associated energies. Our results demonstrate that the contour length is a useful reaction coordinate to characterize protein folding and that intrinsic extensions of protein structures should be taken into account to properly derive the conformations and energies of protein folding intermediates from single-molecule manipulation experiments.

DOI

### Untangling reaction pathways through modern approaches to high-throughput single-molecule force-spectroscopy experiments

David Dulin, Bojk A Berghuis, Martin Depken, Nynke H Dekker

Single-molecule experiments provide a unique means for real-time observation of the activity of individual biomolecular machines. Through such techniques, insights into the mechanics of for example, polymerases, helicases, and packaging motors have been gleaned. Here we describe the recent advances in single-molecule force spectroscopy instrumentation that have facilitated high-throughput acquisition at high spatiotemporal resolution. The large datasets attained by such methods can capture rare but important events, and contain information regarding stochastic behaviors covering many orders of magnitude in time. We further discuss analysis of such data sets, and with a special focus on the pause states described in the general literature on RNA polymerase pausing we compare and contrast the signatures of different reaction pathways.

DOI

### Graded-Index Fiber Enabled Strain-Controllable Optofluidic Manipulation

Chen-Lin Zhang; Yuan Gong; Qun-Feng Liu; Yu Wu; Yun-Jiang Rao; Gang-Ding Peng

Controllable optofluidic manipulation of a microsphere is achieved by tuning the strain applied onto the graded-index fiber (GIF) for the first time. Instead of fiber-optic tapers, the GIF with a flat endface, which is simple to be fabricated with low cost and high repeatability, is employed for enhancing the performance of optical manipulation. By exerting strain onto the GIF, the manipulation length can be periodically controlled and tuned up to 1314.1 μm. Furthermore, we use the force microbalance on the microsphere as a new way to measure the microfluidic flow rate down to 30 nL/min.

DOI

## Thursday, January 28, 2016

### Ultraviolet broadband light scattering for optically-trapped submicron-sized aerosol particles

Gregory David, Kivanc Esat, Irina Ritsch and Ruth Signorell

We describe a broadband light scattering setup for the characterization of size and refractive index of single submicron-to-micron sized aerosol particles. Individual particles are isolated in air by a quadruple Bessel beam optical trap or a counter-propagating optical tweezer. The use of very broadband radiation in the wavelength range from 320 to 700 nm covering the ultraviolet region allows to size submicron particles. We show that a broad wavelength range is required to determine the particle radius and the refractive index with an uncertainty of several nanometers and ~ 0.01, respectively. The smallest particle radius that can be accurately determined lies around 300 nm. Wavelength-dependent refractive index data over a broad range are obtained, including the ultraviolet region where corresponding data are rare. Four different applications are discussed: 1) the sizing of submicron PSL spheres, 2) the evaporation of binary glycerol water droplets, 3) hydration/dehydration cycling of aqueous potassium carbonate droplets, and 4) photochemical reactions of oleic acid droplets.

DOI

### Low-frequency dielectrophoretic response of a single particle in aqueous suspensions

Jingyu Wang, Ming-Tzo Wei and H. Daniel Ou-Yang

We use optical tweezers-based dielectrophoresis(DEP) force spectroscopy to investigate the roles of the electrical double layer in the AC dielectric response of an individual colloidal particle in an aqueous medium. Specifically, we measure the DEP crossover frequency as a function of particles size, medium viscosity, and temperature. Experimental results were compared to low frequency relaxation mechanisms predicted by Schwarz, demonstrating the dielectrophoretic responses in the frequency range between 10 kHz and 1 MHz were dominated by counterion diffusion within the electric double layer.

DOI

### Trapping and patterning of biological objects using photovoltaic tweezers

M. Jubera, I. Elvira, A. García-Cabañes, J. L. Bella and M. Carrascosa

Photovoltaic tweezers are a recently proposed technique for manipulation and patterning of micro- and nano-objects. It is based in the dielectrophoretic forces associated to the electric fields induced by illumination of certain ferroelectrics due to the bulk photovoltaic effect. The technique has been applied to the patterning of dielectric and metal micro- and nano-particles. In this work, we report the use of photovoltaic tweezers to pattern biological objects on LiNbO3:Fe. Specifically, spores and pollen grains and their nanometric fragments have been trapped and patterned. 1D and 2D arrangements have been achieved by deposition in air or from a hexane suspension. The quality of patterns obtained with nanometric fragments is even better than previous results using photovoltaic tweezers with inorganic micro- and nano-particles. In fact, 1D patterns with a period of 2 μm, almost half of the minimum reported period achieved with photovoltaic tweezers, have been obtained with pollen fragments.

DOI

### Metal-Enhanced Fluorescence of Silver Island Associated with Silver Nanoparticle

Jiunn-Woei Liaw, Hsin-Yu Wu, Chu-Chuan Huang and Mao-Kuen Kuo

The coupling plasmon of a hybrid nanostructure, silver island (SI) associated with silver nanoparticle (SNP), on metal-enhanced fluorescence (MEF) was studied theoretically. We used the multiple multipole method to analyze the plasmon-mediated enhancement factor on the fluorescence of a molecule immobilized on SNP and located in the gap zone between SI and SNP; herein, the SI was modeled as an oblate spheroid. Numerical results show that the enhancement factor of the hybrid nanostructure is higher than that of a SNP or a SI alone due to the coupled gap mode. This finding is in agreement with the previous experimental results. In addition, the plasmon band of the structure is broadband and tunable, which can be red-shifted and broadened by flattening or enlarging SI. Based on this property, the hybrid nanostructure can be tailored to obtain the optimal enhancement factor on a specific molecule according to its excitation spectrum. Moreover, we found that there is an induced optical force allowing SNP be attracted by SI. Consequently, the gap is reduced gradually to perform a stronger MEF effect.

DOI

### Plasmon–Exciton Interactions Probed Using Spatial Coentrapment of Nanoparticles by Topological Singularities

Paul J. Ackerman, Haridas Mundoor, Ivan I. Smalyukh, and Jao van de Lagemaat

We study plasmon–exciton interaction by using topological singularities to spatially confine, selectively deliver, cotrap and optically probe colloidal semiconductor and plasmonic nanoparticles. The interaction is monitored in a single quantum system in the bulk of a liquid crystal medium where nanoparticles are manipulated and nanoconfined far from dielectric interfaces using laser tweezers and topological configurations containing singularities. When quantum dot-in-a-rod particles are spatially colocated with a plasmonic gold nanoburst particle in a topological singularity core, its fluorescence increases because blinking is significantly suppressed and the radiative decay rate increases by nearly an order of magnitude owing to the Purcell effect. We argue that the blinking suppression is the result of the radiative rate change that mitigates Auger recombination and quantum dot ionization, consequently reducing nonradiative recombination. Our work demonstrates that topological singularities are an effective platform for studying and controlling plasmon–exciton interactions.

DOI

## Wednesday, January 27, 2016

### Theoretical Study on Surface Mode in Photonic Crystal Fishbone Nanocavity

Tsan-Wen Lu; Po-Tsung Lee

We propose and theoretically investigate a novel 1-D photonic crystal fishbone (FB) that can sustain surface waves. By designing a nanocavity in an FB, the confined surface mode with a high quality factor (~ 105) and extremely concentrated field near the FB surface (small mode volume, ~ 2.3 × 10-2 (λ/2)3) cause strong interactions between light and the surrounding medium for optical sensing and manipulation. In simulation, as an optical sensor, the proposed design achieved a high index sensitivity of 650 nm/RIU and minimum detectable index variation of 2 × 10-5. As optical tweezers, a simulated injected optical threshold power of only 80 μW is needed for stably trapping a polystyrene sphere (PS) 100 nm in diameter. In addition, a method of selectively trapping a PS of specific size is theoretically proposed via our design. We believe that our proposed FB nanocavity with a surface mode would provide enhanced features for on-chip optical sensors and tweezers.

DOI

### High-Throughput Assessment of Cellular Mechanical Properties

Eric M. Darling and Dino Di Carlo

Traditionally, cell analysis has focused on using molecular biomarkers for basic research, cell preparation, and clinical diagnostics; however, new microtechnologies are enabling evaluation of the mechanical properties of cells at throughputs that make them amenable to widespread use. We review the current understanding of how the mechanical characteristics of cells relate to underlying molecular and architectural changes, describe how these changes evolve with cell-state and disease processes, and propose promising biomedical applications that will be facilitated by the increased throughput of mechanical testing: from diagnosing cancer and monitoring immune states to preparing cells for regenerative medicine. We provide background about techniques that laid the groundwork for the quantitative understanding of cell mechanics and discuss current efforts to develop robust techniques for rapid analysis that aim to implement mechanophenotyping as a routine tool in biomedicine. Looking forward, we describe additional milestones that will facilitate broad adoption, as well as new directions not only in mechanically assessing cells but also in perturbing them to passively engineer cell state.

DOI

### Exploiting Optical Asymmetry for Controlled Guiding of Particles with Light

Ognjen Ilic, Ido Kaminer, Yoav Lahini, Hrvoje Buljan, and Marin Soljačić

Conventional methods of manipulating particles with light, such as optical tweezers and optical tractor beams, rely on beam-shaping to realize complex electromagnetic field profiles and are thus sensitive to scattering. Here, we show that, by introducing tailored optical asymmetry in the particle, we can realize a novel guiding method that is controllable by the frequency of light, without regard to the direction or the shape of the light beam. With detailed stochastic simulations, we demonstrate guiding of a two-faced nanoparticle where the optically induced thermophoretic drift serves as the propulsion mechanism. Exploiting the difference in resonant absorption spectra of the two materials, we create a bidirectional local thermal gradient that is externally switchable. This is advantageous because the frequency of a light beam, unlike its shape or coherence, is preserved even in strongly scattering environments. Since this approach is insensitive to scattering and applicable to many particles at once, as well as particles that cannot be optically resolved, it may enable useful applications in biology, microfluidics, in vivo tasks, and colloidal science.

DOI

### Raman Spectroscopy of Isotopic Water Diffusion in Ultraviscous, Glassy, and Gel States in Aerosol by Use of Optical Tweezers

James F. Davies and Kevin R. Wilson

The formation of ultraviscous, glassy, and amorphous gel states in aqueous aerosol following the loss of water results in nonequilibrium dynamics due to the extended time scales for diffusive mixing. Existing techniques for measuring water diffusion by isotopic exchange are limited by contact of samples with the substrate, and methods applied to infer diffusion coefficients from mass transport in levitated droplets requires analysis by complex coupled differential equations to derive diffusion coefficients. We present a new technique that combines contactless levitation with aerosol optical tweezers with isotopic exchange (D2O/H2O) to measure the water diffusion coefficient over a broad range (Dw ≈ 10–12–10–17 m2·s–1) in viscous organic liquids (citric acid, sucrose, and shikimic acid) and inorganic gels (magnesium sulfate, MgSO4). For the organic liquids in binary and ternary mixtures, Dw depends on relative humidity and follows a simple compositional Vignes relationship. In MgSO4 droplets, water diffusivity decreases sharply with water activity and is consistent with predictions from percolation theory. These measurements show that, by combining micrometer-sized particle levitation (a contactless measurement with rapid mixing times) with an established probe of water diffusion, Dw can be simply and directly quantified for amorphous and glassy states that are inaccessible to existing methods.

## Tuesday, January 26, 2016

### Optical Trapping Dynamics of a Single Polystyrene Sphere: Continuous Wave versus Femtosecond Lasers

Tsung-Han Liu, Wei-Yi Chiang, Anwar Usman, and Hiroshi Masuhara

Understanding of optical trapping dynamics of a single particle in the trapping site is important to develop its optical manipulation for molecular assembly and chemical application. For micrometer-sized Mie particles, similar trapping efficiency of the conventional continuous wave (cw) laser or high-repetition-rate femtosecond (fs) laser pulse train has been established [Dholakia et al., Opt. Express 2010, 18, 7554–7568], in contrast to higher efficiency of the laser pulses to trap dielectric Rayleigh particles. To further explore and clarify the switching phenomena of optical trapping efficiency with cw laser and fs laser pulse and to elucidate its nature, we study the immobilization dynamics of a single polystyrene sphere with 500 nm in diameter (which is comparable to focal beam size) in shallow potential well. By observing trapping events and immobilization time of the particle with a size in Lorenz–Mie regime, distinct from well-known Rayleigh particle and ray optics approximations, we found that immobilization time is only linearly related to the incident laser power ≤40 mW, and at higher laser powers cw laser is more efficient than fs laser pulses to immobilize the particle. This finding means that the dynamics of the particle in this size region is still affected by the strong transient force fields induced by high-repetition-rate ultrashort pulse train as usually observed for Rayleigh particles. This may provide an understanding that the dynamics of the target particle in the trapping site is size- and laser mode-dependent.

### Investigation of the polarization-dependent optical force in optical tweezers by using generalized Lorenz-Mie theory

Jai-Min Choi, Heeso Noh

In vectorial diffraction theory, tight focusing of a linearly-polarized laser beam produces an anisotropic field distribution around the focal plane. We present a numerical investigation of the electromagnetic field distribution of a focused beam in terms of the input beam’s polarization state and the associated effects on the trap stiffness asymmetry of optical tweezers. We also explore the symmetry change of a polarization-dependent optical force due to the electromagnetic field redistribution in the presence of dielectric spheres of selected diameters ranging from the Rayleigh scattering regime to the Mie scattering regime.

DOI

### Wave propagation and Lorentz force density in gain chiral structures

Guiping Li, Maoyan Wang, Hailong Li, Mengxia Yu, Yuliang Dong, and Jun Xu

The electromagnetic coupling and mechanical interaction between a plane wave and dispersive gain chiral structures are investigated using the Auxiliary Differential Equation Finite Difference Time Domain (ADE-FDTD) method. Utilizing the constitutive relations containing frequency-dependent Lorentzian models and a Condon model, the wave equations and time-averaged Lorentz force density for the magneto-electric coupling chiral media are presented. Numerical results show that the cross-polarized transmission coefficient is larger than the co-polarized transmission coefficient for a gain chiral slab with certain thickness. The gradient force engendered by bound currents of the cross-polarized waves in chiral media is larger than the scattering force to pull the slab towards the incident source. The complicated optical pulling or pushing force density among slabs, which is illuminated by a normally incident plane wave, containing chiral materials with different medium parameters is achieved.

DOI

### Grading plasmonic nanoparticles with light

Alexander A. Zharov, Jr., Alexander A. Zharov, Ilya V. Shadrivov, and Nina A. Zharova

We introduce an approach for fine grading of plasmonic ellipsoidal nanoparticles by two interfering light beams. We consider electrically neutral subwavelength metal nanoparticles whose response is described within the dipole approximation. For the ellipsoidal nanoparticles, we find that their polarizability tensor is strongly dispersive due to the existence of two orthogonal plasmon modes. These modes can be resonantly excited by light and the optical force experienced by particles depends on the ratio of ellipsoid semiaxes. This dependence allows us to spatially separate ellipsoidal particles with different aspect ratio. The eigenfrequencies of plasmons depend on the depolarization factor as well as on the permittivity of the environment and therefore our results can potentially be employed in a wide frequency range including near infrared, visible, and ultraviolet.

DOI

### Dual-color dynamic tracking of GM-CSF receptors/JAK2 kinases signaling activation using temporal focusing multiphoton fluorescence excitation and astigmatic imaging

Fan-Ching Chien, Chi-Hsiang Lien, and Yang-Hong Dai

The dual-color dynamic particle tracking approach that uses temporal focusing multiphoton fluorescence excitation and two-channel astigmatic imaging is utilized to track molecular trajectories in three dimensions to explore molecular interactions. Images of two fluorophores were obtained to extract their positions by optical sectioning excitation using a fast temporal focusing multiphoton excitation microscope (TFMPEM) and by the simultaneous collection of data in two channels. The presented pair of cylindrical lenses, which was used to adjust the astigmatism effect with the minimum shifting of the imaging plane, was more feasible and flexible than single cylindrical lens for aligning two separate detection channels in astigmatic imaging. The lateral and axial positioning resolutions were observed to be approximately 9-13 nm and 23-30 nm respectively, for the two fluorescence channels. The dynamic movement and binding behavior of clusters of GM-CSF receptors and JAK2 kinases in HeLa cells in the presence of GM-CSF ligands were observed. Therefore, the proposed dual-color tracking strategy is useful for the dynamic study of molecular interactions in living specimens with a fast frame rate, less photobleaching, better penetration depth, and minimum optical trapping force.

DOI

## Monday, January 25, 2016

### Cooperative optical trapping in asymmetric plasmon nanocavity arrays

Ling Guo and Zhijun Sun

We propose a scheme using cooperative interaction of antiphase resonance modes to enhance optical trapping in plasmonic nanostructures. This is implemented with a subwavelength array of asymmetric binary nanogrooves (e.g. different depths) in metal. When damping and inter-coupling of antiphase fields in the nanogrooves are mediated satisfying a critical condition, light can be cooperatively trapped in the nanogrooves, demonstrating perfect absorption at nearly the intrinsic resonance frequency of the deeper nanogrooves. A harmonic oscillator model is developed to interpret the cooperative interaction processes. The phenomenon has been also implemented in asymmetric ternary nanogroove arrays. In terms of compositions and intra-coupling mechanisms, the asymmetric binary/ternary plasmonic nanostructure arrays are crystalline molecular-metamaterials, analogous to electronic crystals composed of covalence-bond molecules.

DOI

### Precise, contactless measurements of the surface tension of picolitre aerosol droplets

Bryan R. Bzdek, Rory M. Power, Stephen H. Simpson, Jonathan P. Reid and C. Patrick Royall
The surface composition and surface tension of aqueous droplets can influence key aerosol characteristics and processes including the critical supersaturation required for activation to form cloud droplets in the atmosphere. Despite its fundamental importance, surface tension measurements on droplets represent a considerable challenge owing to their small volumes. In this work, we utilize holographic optical tweezers to study the damped surface oscillations of a suspended droplet (<10 μm radius) following the controlled coalescence of a pair of droplets and report the first contactless measurements of the surface tension and viscosity of droplets containing only 1–4 pL of material. An advantage of performing the measurement in aerosol is that supersaturated solute states (common in atmospheric aerosol) may be accessed. For pairs of droplets starting at their equilibrium surface composition, surface tensions and viscosities are consistent with bulk equilibrium values, indicating that droplet surfaces respond to changes in surface area on microsecond timescales and suggesting that equilibrium values can be assumed for growing atmospheric droplets. Furthermore, droplet surfaces are shown to be rapidly modified by trace species thereby altering their surface tension. This equilibration of droplet surface tension to the local environmental conditions is illustrated for unknown contaminants in laboratory air and also for droplets exposed to gas passing through a water–ethanol solution. This approach enables precise measurements of surface tension and viscosity over long time periods, properties that currently are poorly constrained.

DOI

### Mode Excitation in Finite Dust Clusters Using an Optical Trap

Block, D.; Wieben, F.; Schablinski, J.
With the ability to trap a single particle in a dusty plasma, it becomes possible to alter the structural properties of the particle assembly or to manipulate its dynamical behavior. Here, we report on a first experiment to excite the eigenmodes of a finite dusty plasma crystal by means of a controlled oscillation of a single particle being trapped in a modified optical tweezer. The eigenmode structure and the excitation process are analyzed.

DOI

### Optical force exerted on a Rayleigh particle by a vector arbitrary-order Bessel beam

Ruiping Yang, Renxian Li

An analytical description of optical force on a Rayleigh particle by a vector Bessel beam is investigated. Linearly, radially, azimuthally, and circularly polarized Bessel beams are considered. The radial, azimuthal, and axial forces by a vector Bessel beam are numerically simulated. The effect of polarization, order of beams, and half-cone angle to the optical force are mainly discussed. For Bessel beams of larger half-cone angle, the non-paraxiality of beams plays an important role in optical forces. Numerical calculations show that optical forces, especially azimuthal forces, are very sensitive to the polarization of beams.

DOI

### Systematic analysis of optical gradient force in photonic crystal nanobeam cavities

Shoubao Han and Yaocheng Shi
In this work, we provide systematic analysis of the optical force for a particle trapped in the waveguide/cavity/waveguide system. The theoretical analysis shows that the optical trapping force is proportional to QT1/2/V for the particle at a fixed position. We provide numerical proof for the proposed principles and systematically optimize the design recipe to investigate optical tapping force of photonic crystal (PhC) nanobeam cavities. The numerical results are in accordance with theoretical analysis and show that the maximum optical trapping force for a particle occurs at the transmission of cavity around 0.25 and the maxima are almost the same for different PhC cavities.

DOI

## Thursday, January 21, 2016

### Optical trapping and manipulation of Mie particles with Airy beam

Ziyu Zhao, Weiping Zang and Jianguo Tian

In this paper we calculate the radiation forces and moving trajectories of Mie particles induced by 1D Airy beams using the plane wave spectrum method and arbitrary beam theory. Numerical results show that both the transverse and the longitudinal radiation forces are deeply dependent on the relative refractive index, radii and positions of the scattering particles illuminated by the Airy beam. Due to the radiation forces, Mie particles with different radii and initial positions can be dragged into the nearest main intensity lobes, and move along parabolic trajectories in the direction of the Poynting vector. At the ends of these trajectories, in the presence of Brownian force, the trapped scattering particles show irregular Brownian movement near their equilibrium positions. This characteristic property of Airy beams enables optical sorting to be used more easily in the colloidal and biological sciences.

DOI

### Connecting Metallic Nanoparticles by Optical Printing

Julián Gargiulo, Santiago Cerrota, Emiliano Cortés, Ianina L. Violi, and Fernando D. Stefani

Optical printing is a simple and flexible method to bring colloidal nanoparticles from suspension to specific locations of a substrate. However, its application has been limited to the fabrication of arrays of isolated nanoparticles because, until now, it was never possible to bring nanoparticles closer together than approximately 300 nm. Here, we propose this limitation is due to thermophoretic repulsive forces generated by plasmonic heating of the NPs. We show how to overcome this obstacle and demonstrate the optical printing of connected nanoparticles with well-defined orientation. These experiments constitute a key step toward the fabrication by optical printing of functional nanostructures and microcircuits based on colloidal nanoparticles.

DOI

### A new approach to follow a single extracellular vesicle–cell interaction using optical tweezers

Ilaria Prada, Ladan Amin, Roberto Furlan, Giuseppe Legname, Claudia Verderio, and Dan Cojoc

Extracellular vesicles (EVs) are spherical membrane structures released by most cells. These highly conserved mediators of intercellular communication carry proteins, lipids, and nucleic acids, and transfer these cellular components between cells by different mechanisms, such as endocytosis, macropinocytosis, or fusion. However, the temporal and spatial dynamics of vesicle–cell interactions still remain largely unexplored. Here we used optical tweezers to drive single EVs produced by microglial cells onto the surface of astrocytes or microglia in primary culture. By visualizing single EV–cell contacts, we observed that microglial vesicles displayed different motilities on the surface of astrocytes compared with microglia. After contact, EVs positioned on astrocytes displayed some minor oscillatory motion around the point of adhesion, while vesicles dragged to microglia displayed quite regular directional movement on the plasma membrane. Both the adhesion and motion of vesicles on glial cells were strongly reduced by cloaking phosphatidylserine (PS) residues, which are externalized on the vesicle membrane and act as determinants for vesicle recognition by target cells. These data identify optical manipulation as a powerful tool to monitor in vitro vesicle–cell dynamics with high temporal and spatial resolution and to determine in a quantitative manner the contribution of surface receptors/extracellular protein ligands to the contact.

DOI

### Hybrid PDMS/glass microfluidics for high resolution imaging and application to sub-wavelength particle trapping

Mario Tonin, Nicolas Descharmes and Romuald Houdré

We demonstrate the fabrication of a hybrid PDMS/glass microfluidic layer that can be placed on top of non-transparent samples and allows high-resolution optical microscopy through it. The layer mimics a glass coverslip to limit optical aberrations and can be applied on the sample without the use of permanent bonding. The bonding strength can withstand to hold up to 7 bars of injected pressure in the channel without leaking or breaking. We show that this process is compatible with multilayer soft lithography for the implementation of flexible valves. The benefits of this application is illustrated by optically trapping sub-wavelength particles and manipulate them around photonic nano-structures. Among others, we achieve close to diffraction limited imaging through the microfluidic assembly, full control on the flow with no dynamical deformations of the membrane and a 20-fold improvement on the stiffness of the trap at equivalent trapping power.

DOI

### Optical Pushing: A Tool for Parallelized Biomolecule Manipulation

Gerrit Sitters, Niels Laurens, Emilie J. de Rijk, Holger Kress, Erwin J.G. Peterman, Gijs J.L. Wuite

The ability to measure and manipulate single molecules has greatly advanced the field of biophysics. Yet, the addition of more single-molecule tools that enable one to measure in a parallel fashion is important to diversify the questions that can be addressed. Here we present optical pushing (OP), a single-molecule technique that is used to exert forces on many individual biomolecules tethered to microspheres using a single collimated laser beam. Forces ranging from a few femtoNewtons to several picoNewtons can be applied with a submillisecond response time. To determine forces exerted on the tethered particles by the laser, we analyzed their measured Brownian motion using, to our knowledge, a newly derived analytical model and numerical simulations. In the model, Brownian rotation of the microspheres is taken into account, which proved to be a critical component to correctly determine the applied forces. We used our OP technique to map the energy landscape of the protein-induced looping dynamics of DNA. OP can be used to apply loading rates in the range of 10−4–106 pN/s to many molecules at the same time, which makes it a tool suitable for dynamic force spectroscopy.

DOI

## Wednesday, January 20, 2016

### Optical tweezing by photomigration

Zouheir Sekkat

Photomigration in azo polymers is an area of research that witnessed intensive studies owing to its potential in optical manipulation, e.g., optical tweezing, the physical mechanism of which remains unsolved since its discovery about two decades ago. In this paper, a detailed theoretical study that reproduces the phenomena associated with photomigration is presented, including the physical models and the associated master equations. Polarization effects are discussed and analytical solutions are given to describe the steady-state and the dynamics of photomigration. Such a theory leads to new theoretical experiments relating material properties to light action. A photoisomerization force which is described by a spring-type model is introduced. This force is derived from a harmonic light potential that moves the azo polymer. This force is parenting to optical tweezers, but it is quite different in the sense that it requires photoisomerization to occur. The azo polymer’s motion is governed by four competing forces: the photoisomerization force, and the restoring optical gradient and elastic forces, as well as the random forces due to spontaneous diffusion.

DOI

### Investigations of Molecular Mechanisms of Actin–Myosin Interactions in Cardiac Muscle

L. V. Nikitina , G. V. Kopylova, D. V. Shchepkin, S. R. Nabiev, S. Y. Bershitsky

The functional characteristics of cardiac muscle depend on the composition of protein isoforms in the cardiomyocyte contractile machinery. In the ventricular myocardium of mammals, several isoforms of contractile and regulatory proteins are expressed–two isoforms of myosin (V1 and V3) and three isoforms of tropomyosin chains (α, β, and κ). Expression of protein isoforms depends on the animal species, its age and hormonal status, and this can change with pathologies of the myocardium. Mutations in these proteins can lead to cardiomyopathies. The functional significance of the protein isoform composition has been studied mainly on intact hearts or on isolated preparations of myocardium, which could not provide a clear comprehension of the role of each particular isoform. Present-day experimental techniques such as an optical trap and in vitro motility assay make it possible to investigate the phenomena of interactions of contractile and regulatory proteins on the molecular level, thus avoiding effects associated with properties of a whole muscle or muscle tissue. These methods enable free combining of the isoforms to test the molecular mechanisms of their participation in the actin–myosin interaction. Using the optical trap and the in vitro motility assay, we have studied functional characteristics of the cardiac myosin isoforms, molecular mechanisms of the calcium-dependent regulation of actin–myosin interaction, and the role of myosin and tropomyosin isoforms in the cooperativity mechanisms in myocardium. The knowledge of molecular mechanisms underlying myocardial contractility and its regulation is necessary for comprehension of cardiac muscle functioning, its disorders in pathologies, and for development of approaches for their correction.

DOI

### Fabrication of ring assemblies of nematic colloids and their electric response

Yuta Tamura and Yasuyuki Kimura

Colloidal particles with a limited number of interactive sites are called colloidal molecules, and their assemblies have been intensively studied to reveal complex micro-structures. In this study, we examine colloidal particles in nematic liquid crystals, so-called nematic colloids, as colloidal molecules and fabricated some non-close-packed assemblies. Micrometer-sized particles with homeotropic surface anchoring of liquid crystal in a homeotropic cell interact with each other through dipolar-type anisotropic interactions arising from the elastic deformation of the nematic field around the particles. Using optical tweezers, we have built two-dimensional colloidal assemblies with low packing densities, including polygon-rings, chains of polygon-rings, and lattices composed of octagon-rings in a hierarchical way from smaller structure units. Because the nematic field is sensitive to the electric field, the response of the polygon-rings to an alternative electric field has been studied. They exhibited homogeneous reversible shrink as large as 15%–22% to their original sizes under several volts.

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### Mechanical Folding and Unfolding of Protein Barnase at the Single-Molecule Level

Anna Alemany, Blanca Rey-Serra, Silvia Frutos, Ciro Cecconi, Felix Ritort

The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale.

DOI

### Atomic force microscopy combined with optical tweezers (AFM/OT)

F Pierini, K Zembrzycki, P Nakielski, S Pawłowska and T A Kowalewski

The role of mechanical properties is essential to understand molecular, biological materials, and nanostructures dynamics and interaction processes. Atomic force microscopy (AFM) is the most commonly used method of direct force evaluation, but due to its technical limitations this single probe technique is unable to detect forces with femtonewton resolution. In this paper we present the development of a combined atomic force microscopy and optical tweezers (AFM/OT) instrument. The focused laser beam, on which optical tweezers are based, provides us with the ability to manipulate small dielectric objects and to use it as a high spatial and temporal resolution displacement and force sensor in the same AFM scanning zone. We demonstrate the possibility to develop a combined instrument with high potential in nanomechanics, molecules manipulation and biological studies. AFM/OT equipment is described and characterized by studying the ability to trap dielectric objects and quantifying the detectable and applicable forces. Finally, optical tweezers calibration methods and instrument applications are given.

DOI

## Monday, January 18, 2016

### Clad photon sieve for generating localized hollow beams

Yiguang Cheng, Junmin Tong, Jiangping Zhu, Junbo Liu, Song Hu, Yu He

A novel photon sieve structure called clad photon sieve is proposed to generate localized hollow beams and its design principle and focusing properties are studied. The clad photon sieve is composed of the internal zone and external zone with pinholes being positioned on the dark zones. Pinholes in the internal zone and in the external zone give destructive interference to the focus, leading to localized hollow beams being generated on the focal plane. Focusing properties of clad photon sieve with different focal lengths, zone numbers and modulation factors are also studied by theoretical calculations, numerical simulations and experiments, showing that the central dark spot size can be controlled by the focal length and rings number, and the intensity of the central dark spot varies with different modulation factors related with the internal zone and the external zone. This photon sieve can be useful for trapping and manipulating of particles and cooling of atoms.

### Active microrheology with optical tweezers: a versatile tool to investigate anisotropies in intermediate filament networks

T Neckernuss, L K Mertens, I Martin, T Paust, M Beil and O Marti

Mechanical properties of cells are determined by the cytoskeleton and especially by intermediate filaments (IFs). To measure the contribution of IFs to the mechanics of the cytoskeleton, we determine the shear moduli of in vitro assembled IF networks consisting of keratin 8/18 and MgCl2, serving as a crosslinker. In this study we want to present a new method, a combination of active and passive microrheology, to characterize these networks. We also show the applicability of the new method and discuss new findings on the organization and force transmission in keratin networks gained by the new method. We trap and move embedded polystyrene particles with an optical tweezers setup in an oscillatory manner. The amplitude response of the trapped particle is measured and evaluated with a lock-in approach in order to suppress random motions. With this technique we determine the degree of isotropy of the assembled network and sense preferred directions due to inhomogeneities of the network. Furthermore, we show that we can deliberately create anisotropic networks by adjusting the assembly process and chamber geometry. To determine whether there are local network anisotropies in a globally isotropic network, we altered the evaluation method and included the motion of embedded particles in the vicinity of the trapped one. The correlations of the observed motions enable us to map local network anisotropies. Finally, we compare mechanical properties determined from passive with ones from active microrheology. We find the networks measured with the active technique to be approximately 20% more compliant than the ones from passive measurements.

DOI

### Cell paintballing using optically targeted coacervate microdroplets

James P. K. Armstrong, Sam N. Olof, Monika D. Jakimowicz, Anthony P. Hollander, Stephen Mann, Sean A. Davis, Mervyn J. Miles, Avinash J. Patil and Adam W. Perriman

We present a new approach for the directed delivery of biomolecular payloads to individual cells with high spatial precision. This was accomplished via active sequestration of proteins, oligonucleotides or molecular dyes into coacervate microdroplets, which were then delivered to specific regions of stem cell membranes using a dynamic holographic assembler, resulting in spontaneous coacervate microdroplet–membrane fusion. The facile preparation, high sequestration efficiency and inherent membrane affinity of the microdroplets make this novel “cell paintballing” technology a highly advantageous option for spatially-directed cell functionalization, with potential applications in single cell stimulation, transfection and differentiation.

DOI

### Scattering and Extinction Torques: How Plasmon Resonances Affect the Orientation Behavior of a Nanorod in Linearly Polarized Light

Xiaohao Xu, Chang Cheng, Yao Zhang, Hongxiang Lei, and Baojun Li

Linearly polarized light can exert an orienting torque on plasmonic nanorods. The torque direction has generally been considered to change when the light wavelength passes through a plasmon longitudinal resonance. Here, we use the Maxwell stress tensor to evaluate this torque in general terms. According to distinct light–matter interaction processes, the total torque is decomposed into scattering and extinction torques. The scattering torque tends to orient plasmonic nanorods parallel to the light polarization, independent of the choice of light wavelength. The direction of the extinction torque is not only closely tied to the excitation of plasmon resonance but also depends on the specific plasmon mode around which the light wavelength is tuned. Our findings show that the conventional wisdom that simply associates the total torque with the plasmon longitudinal resonances needs to be replaced with an understanding based on the different torque components and the details of spectral distribution.

DOI

## Saturday, January 16, 2016

### Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer

Van K Duesterberg, Irena T Fischer-Hwang, Christian F Perez, Daniel W Hogan, Steven M Block

The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The 'Force-FRET' approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity.

### Radiation Forces on a Dielectric Sphere Produced by Finite Olver-Gaussian Beams

Salima Hennani, Lahcen Ez-zariy, Abdelmajid Belafhal
In this work, we use the analytical expression of the propagation of Finite Olver-Gaussian beams (FOGBs) through a paraxial ABCD optical system to study the action of radiation forces produced by highly focused FOGBs on a Rayleigh dielectric sphere. Our numerical results show that the FOGBs can be employed to trap and manipulate particles with the refractive index larger than that of the ambient. The radiation force distribution has been studied under different beam widths. The trapping stability under different conditions is also analyzed.

DOI

### Plasmon-induced strong interaction between chiral molecules and orbital angular momentum of light

Tong Wu, Rongyao Wang & Xiangdong Zhang

Whether or not chiral interaction exists between the optical orbital angular momentum (OAM) and a chiral molecule remains unanswered. So far, such an interaction has not been observed experimentally. Here we present a T-matrix method to study the interaction between optical OAM and the chiral molecule in a cluster of nanoparticles. We find that strong interaction between the chiral molecule and OAM can be induced by the excitation of plasmon resonances. An experimental scheme to observe such an interaction has been proposed. Furthermore, we have found that the signal of the OAM dichroism can be either positive or negative, depending on the spatial positions of nanocomposites in the cross-sections of OAM beams. The cancellation between positive and negative signals in the spatial average can explain why the interaction has not been observed in former experiments.

DOI

### Two particle tracking and detection in a single Gaussian beam optical trap

P. Praveen, Yogesha, Shruthi S. Iyengar, Sarbari Bhattacharya, and Sharath Ananthamurthy

We have studied in detail the situation wherein two microbeads are trapped axially in a single-beam Gaussian intensity profile optical trap. We find that the corner frequency extracted from a power spectral density analysis of intensity fluctuations recorded on a quadrant photodetector (QPD) is dependent on the detection scheme. Using forward- and backscattering detection schemes with single and two laser wavelengths along with computer simulations, we conclude that fluctuations detected in backscattering bear true position information of the bead encountered first in the beam propagation direction. Forward scattering, on the other hand, carries position information of both beads with substantial contribution from the bead encountered first along the beam propagation direction. Mie scattering analysis further reveals that the interference term from the scattering of the two beads contributes significantly to the signal, precluding the ability to resolve the positions of the individual beads in forward scattering. In QPD-based detection schemes, detection through backscattering, thereby, is imperative to track the true displacements of axially trapped microbeads for possible studies on light-mediated interbead interactions.

DOI

## Friday, January 15, 2016

### Munc18-1-regulated stage-wise SNARE assembly underlying synaptic exocytosis

Lu Ma, Aleksander A Rebane, Guangcan Yang, Zhiqun Xi, Yuhao Kang, Ying Gao, Yongli Zhang
Synaptic soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins couple their stage-wise folding/assembly to rapid exocytosis of neurotransmitters in a Munc18-1-dependent manner. The functions of the different assembly stages in exocytosis and the role of Munc18-1 in SNARE assembly are not well understood. Using optical tweezers, we observed four distinct stages of assembly in SNARE N-terminal, middle, C-terminal, and linker domains (or NTD, MD, CTD and LD, respectively). We found that SNARE layer mutations differentially affect SNARE assembly. Comparison of their effects on SNARE assembly and on exocytosis reveals that NTD and CTD are responsible for vesicle docking and fusion, respectively, whereas MD regulates SNARE assembly and fusion. Munc18-1 initiates SNARE assembly and structures t-SNARE C-terminus independent of syntaxin N-terminal regulatory domain (NRD) and stabilizes the half-zippered SNARE complex dependent upon the NRD. Our observations demonstrate distinct functions of SNARE domains whose assembly is intimately chaperoned by Munc18-1.

DOI

### All-dielectric reciprocal bianisotropic nanoparticles

The study of high-index dielectric nanoparticles currently attracts a lot of attention. They do not suffer from absorption but promise to provide control of the properties of light comparable to plasmonic nanoparticles. To further advance the field, it is important to identify versatile dielectric nanoparticles with unconventional properties. Here, we show that breaking the symmetry of an all-dielectric nanoparticle leads to a geometrically tunable magnetoelectric coupling, i.e., an omega-type bianisotropy. The suggested nanoparticle exhibits different backscatterings and, as an interesting consequence, different optical scattering forces for opposite illumination directions. An array of such nanoparticles provides different reflection phases when illuminated from opposite directions. With a proper geometrical tuning, this bianisotropic nanoparticle is capable of providing a 2π phase change in the reflection spectrum while possessing a rather large and constant amplitude. This allows the creation of reflectarrays with near-perfect transmission out of the resonance band due to the absence of a usually employed metallic screen.

DOI

### Surface plasmon polariton assisted optical pulling force

Mihail I. Petrov, Sergey V. Sukhov, Andrey A. Bogdanov, Alexander S. Shalin and Aristide Dogariu

We demonstrate both analytically and numerically the existence of optical pulling forces acting on particles located near plasmonic interfaces. Two main factors contribute to the appearance of this negative recoil force. The interference between the incident and reflected waves induces a rotating dipole with an asymmetric scattering pattern, while the directional excitation of surface plasmon polaritons (SPPs) enhances the linear momentum of scattered light. The strongly asymmetric SPP excitation is determined by spin–orbit coupling of the rotating dipole and surface plasmon polariton. As a result of the total momentum conservation, the force acting on the particle points in a direction opposite to the incident wave propagation. We derive analytical expressions for the force acting on dipolar particles placed in the proximity of plasmonic surfaces. Analytical expressions for this pulling force are derived within the dipole approximation and are in excellent agreement with results of electromagnetic numerical calculations. The forces acting on larger particles are analyzed numerically, beyond the dipole approximation.

DOI

## Wednesday, January 13, 2016

### New prospects of using tested particles for investigating optical fields and optical flows

O. V. ANGELSKY, C. YU. ZENKOVA, I. V. SOLTYS

We have shown that internal energy flows (spin and orbital) can be diagnosed by test particles of different sizes and properties using the methods of singular optics. The motion velocity of these particles both in the energy and polarization inhomogeneous optical fields can be employed as a diagnostic parameter of the degree of coherence of mutually orthogonal linearly polarized in the incidence plane superposing waves. The feasibilities of using the above mentioned approach, its shortcomings and its advantages over the interfering method for estimating the degree of coherence is analyzed.

DOI

### Epitope mapping of monoclonal antibody HPT-101: a study combining dynamic force spectroscopy, ELISA and molecular dynamics simulations

Tim Stangner, Stefano Angioletti-Uberti, Daniel Knappe, David Singer, Carolin Wagner, Ralf Hoffmann and Friedrich Kremer

By combining enzyme-linked immunosorbent assay (ELISA) and optical tweezers-assisted dynamic force spectroscopy (DFS), we identify for the first time the binding epitope of the phosphorylation-specific monoclonal antibody (mAb) HPT-101 to the Alzheimer's disease relevant peptide tau[pThr231/pSer235] on the level of single amino acids. In particular, seven tau isoforms are synthesized by replacing binding relevant amino acids by a neutral alanine (alanine scanning). From the binding between mAb HPT-101 and the alanine-scan derivatives, we extract specific binding parameters such as bond lifetime ${\tau }_{0}$, binding length ${x}_{\mathrm{ts}}$, free energy of activation ${\rm{\Delta }}G$ (DFS) and affinity constant ${K}_{{\rm{a}}}$ (ELISA, DFS). Based on these quantities, we propose criteria to identify essential, secondary and non-essential amino acids, being representative of the antibody binding epitope. The obtained results are found to be in full accord for both experimental techniques. In order to elucidate the microscopic origin of the change in binding parameters, we perform molecular dynamics (MD) simulations of the free epitope in solution for both its parent and modified form. By taking the end-to-end distance ${d}_{{\rm{E}}-{\rm{E}}}$ and the distance between the α-carbons ${d}_{{\rm{C}}-{\rm{C}}}$ of the phosphorylated residues as gauging parameters, we measure how the structure of the epitope depends on the type of substitution. In particular, whereas ${d}_{{\rm{C}}-{\rm{C}}}$ is sometimes conserved between the parent and modified form, ${d}_{{\rm{E}}-{\rm{E}}}$ strongly changes depending on the type of substitution, correlating well with the experimental data. These results are highly significant, offering a detailed microscopic picture of molecular recognition.

DOI

### Graphene based resonance structure to enhance the optical pressure between two planar surfaces

To enhance the optical pressure on a thin dielectric sample, a resonance structure using graphene layers coated over a metal film on a high index prism sputtered with MgF2 was theoretically analyzed. The number of graphene layers and the thicknesses of metal and MgF2 films were optimized to achieve the highest optical pressure on the sample. Effects of three different types of metals on the optical pressure were investigated numerically. In addition, simulations were carried out for samples with various thicknesses. Our numerical results show that the optical pressure increased by more than five orders of magnitude compared to the conventional metal-film-base resonance structure. The highest optical pressure was obtained for 10 layers of graphene deposited on 29-nm thick Au film and 650 nm thickness of MgF2 at 633nm wavelength, The proposed graphene based resonance structure can open new possibilities for optical tweezers, nanomechnical devices and surface plasmon based sensing and imaging techniques.

DOI

### In vivo tissue has non-linear rheological behavior distinct from 3D biomimetic hydrogels, as determined by AMOTIV microscopy

Benjamin H. Blehm, Alexus Devine, Jack R. Staunton, Kandice Tanner

Variation in matrix elasticity has been shown to determine cell fate in both differentiation and development of malignant phenotype. The tissue microenvironment provides complex biochemical and biophysical signals in part due to the architectural heterogeneities found in extracellular matrices (ECMs). Three dimensional cell cultures can partially mimic in vivo tissue architecture, but to truly understand the role of viscoelasticity on cell fate, we must first determine in vivo tissue mechanical properties to improve in vitro models. We employed Active Microrheology by Optical Trapping InVivo (AMOTIV), using in situ calibration to measure in vivo zebrafish tissue mechanics. Previously used trap calibration methods overestimate complex moduli by ∼2–20 fold compared to AMOTIV. Applying differential microscale stresses and strains showed that hyaluronic acid (HA) gels display semi-flexible polymer behavior, while laminin-rich ECM hydrogels display flexible polymer behavior. In contrast, zebrafish tissues displayed different moduli at different stresses, with higher power law exponents at lower stresses, indicating that living tissue has greater stress dependence than the 3D hydrogels examined. To our knowledge, this work is the first vertebrate tissue rheological characterization performed in vivo. Our fundamental observations are important for the development and refinement of in vitro platforms.

DOI

## Tuesday, January 12, 2016

### After stress comes relax(ation)

Lucio Isa

Viscoelastic materials take a finite time to relax and dissipate stress and this time scale is directly connected to the microstructure of the material itself. In their paper, Gomez-Solano and Bechinger (2015 New J. Phys. 17 103032) perform 'miniaturized' mechanical tests on a range of viscoelastic materials by dragging a micron-sized bead across them using optical tweezers. Upon switching off all the external forces, they watch the bead recoil to its original position and by tracking its motion they pinpoint the relaxation time of the material. These experiments open up a new range of possibilities to characterize stress relaxation at the microscale just by watching it.

DOI

### Light Based Techniques for Improving Health Care: Studies at RRCAT

P. K. Gupta, H. S. Patel, S. Ahlawat

The invention of Lasers in 1960, the phenomenal advances in photonics as well as the information processing capability of the computers has given a major boost to the R&D activity on the use of light for high resolution biomedical imaging, sensitive, non-invasive diagnosis and precision therapy. The effort has resulted in remarkable progress and it is widely believed that light based techniques hold great potential to offer simpler, portable systems which can help provide diagnostics and therapy in a low resource setting. At Raja Ramanna Centre for Advanced Technology (RRCAT) extensive studies have been carried out on fluorescence spectroscopy of native tissue. This work led to two important outcomes. First, a better understanding of tissue fluorescence and insights on the possible use of fluorescence spectroscopy for screening of cancer and second development of diagnostic systems that can serve as standalone tool for non-invasive screening of the cancer of oral cavity. The optical coherence tomography setups and their functional extensions (polarization sensitive, Doppler) have also been developed and used for high resolution (~10 µm) biomedical imaging applications, in particular for non-invasive monitoring of the healing of wounds. Chlorophyll based photo-sensitisers and their derivatives have been synthesized in house and used for photodynamic therapy of tumors in animal models and for antimicrobial applications. Various variants of optical tweezers (holographic, Raman etc.) have also been developed and utilised for different applications notably Raman spectroscopy of optically trapped red blood cells. An overview of these activities carried out at RRCAT is presented in this article.

DOI

### Laser propulsion of nanobullets by adiabatic compression of surface plasmon polaritons

Viola Folli, Giancarlo Ruocco & Claudio Conti

Laser propulsion and guide of nanosized objects is fundamental for a wide number of applications. These applications are often limited by the fact that the optical forces acting on nanoparticles are almost negligible even in the favorable case of metallic particles and hence large laser powers are needed to accelerate and guide nanosize devices in practical applications. Furthermore, metallic nanoparticles exhibit strong absorption bands and scattering and this makes more difficult controlling nanopropulsion. Thus, finding some mechanism enhancing the optomechanical interaction at the nanoscale controlled by laser is specifically challenging and pivotal. Here, we demonstrate a novel physical effect where the well-known adiabatic localization of the enhanced plasmonic surface field on the apex of metallic nanocones produces a significant optical pressure employable as a propulsive mechanism. The proposed method gives the possibility to develop new photonics devices to accelerate metallic nanobullets over long distances for a variety of applications.

DOI

## Wednesday, January 6, 2016

### Raman spectroscopy of single nanoparticles in a double-nanohole optical tweezer system

Steven Jones, Ahmed A Al Balushi and Reuven Gordon

A double nanohole in a metal film was used to trap nanoparticles (20 nm diameter) and simultaneously record their Raman spectrum using the trapping laser as the excitation source. This allowed for the identification of characteristic Stokes lines for titania and polystyrene nanoparticles, showing the capability for material identification of nanoparticles once trapped. Increased Raman signal was observed for the trapping of multiple nanoparticles. This system combines the benefits of nanoparticle isolation and manipulation with unique identification.

DOI

### Optically trapping Rayleigh particles by using focused partially coherent multi-rotating elliptical Gaussian beams

Xi Peng, Chidao Chen, Bo Chen, Yulian Peng, Meiling Zhou, Xiangbo Yang, and Dongmei Deng

By investigating the cross-spectral density of partially coherent multi-rotating elliptical Gaussian beams (REGBs) that propagate through a focusing optical system, we obtain the radiation force on a Rayleigh particle. The radiation force distribution is studied under different beam indexes, coherence widths, and elliptical ratios of the partially coherent multi REGBs. The transverse and the longitudinal trapping ranges can increase at the focal plane by increasing the beam index or decreasing the coherence width. The range of the trapped particle radii increases as the elliptical ratio increases. Furthermore, we analyze the trapping stability.

DOI

### Investigation into local cell mechanics by atomic force microscopy mapping and optical tweezer vertical indentation

G Coceano, M S Yousafzai, W Ma, F Ndoye, L Venturelli, I Hussain, S Bonin, J Niemela, G Scoles, D Cojoc

Investigating the mechanical properties of cells could reveal a potential source of label-free markers of cancer progression, based on measurable viscoelastic parameters. The Young's modulus has proved to be the most thoroughly studied so far, however, even for the same cell type, the elastic modulus reported in different studies spans a wide range of values, mainly due to the application of different experimental conditions. This complicates the reliable use of elasticity for the mechanical phenotyping of cells. Here we combine two complementary techniques, atomic force microscopy (AFM) and optical tweezer microscopy (OTM), providing a comprehensive mechanical comparison of three human breast cell lines: normal myoepithelial (HBL-100), luminal breast cancer (MCF-7) and basal breast cancer (MDA-MB-231) cells. The elastic modulus was measured locally by AFM and OTM on single cells, using similar indentation approaches but different measurement parameters. Peak force tapping AFM was employed at nanonewton forces and high loading rates to draw a viscoelastic map of each cell and the results indicated that the region on top of the nucleus provided the most meaningful results. OTM was employed at those locations at piconewton forces and low loading rates, to measure the elastic modulus in a real elastic regime and rule out the contribution of viscous forces typical of AFM. When measured by either AFM or OTM, the cell lines' elasticity trend was similar for the aggressive MDA-MB-231 cells, which were found to be significantly softer than the other two cell types in both measurements. However, when comparing HBL-100 and MCF-7 cells, we found significant differences only when using OTM.

DOI

### L1 retrotransposition requires rapid ORF1p oligomerization, a novel coiled coil-dependent property conserved despite extensive remodeling

M. Nabuan Naufer, Kathryn E. Callahan, Pamela R. Cook, Cesar E. Perez-Gonzalez, Mark C. Williams and Anthony V. Furano

Detailed mechanistic understanding of L1 retrotransposition is sparse, particularly with respect to ORF1p, a coiled coil-mediated homotrimeric nucleic acid chaperone that can form tightly packed oligomers on nucleic acids. Although the coiled coil motif is highly conserved, it is uniquely susceptible to evolutionary change. Here we studied three ORF1 proteins: a modern human one (111p), its resuscitated primate ancestor (555p) and a mosaic modern protein (151p) wherein 9 of the 30 coiled coil substitutions retain their ancestral state. While 111p and 555p equally supported retrotransposition, 151p was inactive. Nonetheless, they were fully active in bulk assays of nucleic acid interactions including chaperone activity. However, single molecule assays showed that 151p trimers form stably bound oligomers on ssDNA at <1/10th the rate of the active proteins, revealing that oligomerization rate is a novel critical parameter of ORF1p activity in retrotransposition conserved for at least the last 25 Myr of primate evolution.

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### Optical trap-cavity ringdown spectroscopy as a single-aerosol-particle-scope

Chuji Wang, Zhiyong Gong, Yong-Le Pan and Gorden Videen

We report a single-aerosol-particle-scope using an optical trapping-cavity ringdown spectroscopy technique. The scope can not only view physical parameters such as size, motion, and restoring force constant of a single aerosol particle trapped in air but also display time-, particle-, or wavelength-resolved chemical properties such as single aerosol particle extinction. We demonstrate the scope by trapping and walking single carbon-nanotube particles of ∼50 μm in size and viewing those properties via changes of ringdown time. This single-aerosol-particle-scope offers a powerful tool to study both physical and chemical properties as well as their evolving dynamics.

DOI

## Tuesday, January 5, 2016

### Cell adhesion manipulation through single cell assembly for characterization of initial cell-to-cell interaction

Xue Gou, Ran Wang, Stephen S. Y. Lam, Jundi Hou, Anskar Y. H. Leung and Dong Sun

Cell-to-cell interactions are complex processes that involve physical interactions, chemical binding, and biological signaling pathways. Identification of the functions of special signaling pathway in cell-to-cell interaction from the very first contact will help characterize the mechanism underlying the interaction and advance new drug discovery. This paper reported a case study of characterizing initial interaction between leukemia cancer cells and bone marrow stromal cells, through the use of an optical tweezers-based cell manipulation tool. Optical traps were used to assemble leukemia cells at different positions of the stromal cell layer and enable their interactions by applying a small trapping force to maintain the cell contact for a few minutes. Specific drug was used to inhibit the binding of molecules during receptor-ligand-mediated adhesion. Our results showed that the amount of adhesion molecule could affect cell adhesion during the first few minutes contact. We also found that leukemia cancer cells could migrate on the stromal cell layer, which was dependent on the adhesion state and activation triggered by specific chemokine. The reported approaches provided a new opportunity to investigate cell-to-cell interaction through single cell adhesion manipulation.

DOI

### Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant, Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

Shiwei Wang, Jing Yu, Milomir Suvira, Peter Setlow, Yong-qing Li

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T1) and reached a maximum after the completion of CaDPA release (Trelease) and spore cortex lysis (Tlysis).

DOI

### Plasmonic lateral forces on chiral spheres

Antoine Canaguier-Durand and Cyriaque Genet

We show that the optical force exerted on a finite size chiral sphere by a surface plasmon mode has a component along a direction perpendicular to the plasmon linear momentum. We reveal how this chiral lateral force, pointing in opposite directions for opposite enantiomers, stems from an angular-to-linear crossed momentum transfer involving the plasmon transverse spin angular momentum density and mediated by the chirality of the sphere. Our multipolar approach allows us discussing the inclusion of the recoil term in the force on a small sphere taken in the dipolar limit and observing sign inversions of the lateral chiral force when the size of the sphere increases.

DOI

### Two-Dimensional Growth Rate Control of L-Phenylalanine Crystal by Laser Trapping in Unsaturated Aqueous Solution

Ken-ichi Yuyama, Jino George, K George Thomas, Teruki Sugiyama, and Hiroshi Masuhara

The growth rate control of single L-phenylalanine plate-like anhydrous crystal is successfully demonstrated by laser trapping at an air/solution interface of the unsaturated aqueous solution. Focusing a continuous-wave near-infrared laser beam into the solution surface generates one L-phenylalanine crystal at the focal spot. Subsequently, the formed crystal grows two-dimensionally at a constant rate under unsaturated condition while being trapped by the laser. When the laser power is decreased after the crystallization, the growth rate is slowed down accordingly. Thus, the two-dimensional growth rate is controllable by tuning the power of the trapping laser after the crystallization. As the critical phenomenon underlying the growth rate control, we propose the formation of a dense domain of the liquid-like clusters induced prior to the crystallization.

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