Thursday, June 28, 2012

Optically Directed Mesoscale Assembly and Patterning of Electrically Conductive Organic–Inorganic Hybrid Structures

John T. Bahns, Subramanian K. R. S. Sankaranarayanan, Noel C. Giebink, Hui Xiong, Stephen K. Gray

Directed colloidal synthesis of conductive organic–inorganic hybrid mesoscale structuresis reported. The technique is simple but allows hierarchical assembly and 2D patterning of materials. A focused laser spot is used to direct the colloidal assembly of nanoparticles into electrically conductive organic–inorganic hybrid mesoscale filaments with arbitrary permanent patterns on a glass surface.


Surface transport and stable trapping of particles and cells by an optical waveguide loop

Olav Gaute Hellesø , Pål Løvhaugen , Ananth Z Subramanian , James S. Wilkinson and Balpreet Singh Ahluwalia

Waveguide trapping has emerged as a useful technique for parallel and planar transport of particles and biological cells and can be integrated with lab-on-a-chip applications. However, particles trapped on waveguides are continuously propelled forward along the surface of the waveguide. This limits the practical usability of waveguide trapping technique with other functions (e.g analysis, imaging) that require particles to be held stationary during diagnosis. In this paper, an optical waveguide loop with an intentional gap at the centre is proposed to hold propelled particles and cells. The waveguide acts as a conveyor belt to transport and deliver the particles/cells towards the gap. At the gap, the diverging light fields hold the particles at a fixed position. The proposed waveguide design is numerically studied and experimentally implemented. The optical forces on the particle at the gap are calculated using the finite element method. Experimentally, the method is used to transport and trap micro-particles and red blood cells at the gap with varying separations. The waveguides are only 180 nm thick and thus could be integrated with other functions on the chip, e.g. microfluidics or optical detection, to make an on-chip system for single cell analysis and to study the interaction between cells.


Nanonewton optical force trap employing anti-reflection coated, high-refractive-index titania microspheres

Anita Jannasch, Ahmet F. Demirörs, Peter D. J. van Oostrum, Alfons van Blaaderen & Erik Schäffer

Optical tweezers are exquisite position and force transducers and are widely used for high-resolution measurements in fields as varied as physics, biology and materials science. Typically, small dielectric particles are trapped in a tightly focused laser and are often used as handles for sensitive force measurements. Improvement to the technique has largely focused on improving the instrument and shaping the light beam, and there has been little work exploring the benefit of customizing the trapped object. Here, we describe how anti-reflection coated, high-refractive-index core–shell particles composed of titania enable single-beam optical trapping with an optical force greater than a nanonewton. The increased force range broadens the scope of feasible optical trapping experiments and will pave the way towards more efficient light-powered miniature machines, tools and applications.


Wednesday, June 27, 2012

Raman spectroscopy of optically levitated supercooled water droplet

Hidenori Suzuki, Yoshiki Matsuzaki, Azusa Muraoka, and Maki Tachikawa

By use of an optical trap, we can levitate micrometer-sized drops of purified water and cool them below the melting point free from contact freezing. Raman spectra of the OH stretching band were obtained from those supercooled water droplets at temperatures down to −35 °C. According to the two-state model, an enthalpy change due to hydrogen-bond breaking is derived from temperature dependence of the spectral profile. The isobaric heat capacity calculated from the enthalpy data shows a sharp increase as the temperature is lowered below −20 °C in good agreement with conventional thermodynamic measurements.


Microfluidic sorting with a moving array of optical traps

Raktim Dasgupta, Sunita Ahlawat, and Pradeep Kumar Gupta

Optical sorting was demonstrated by selective trapping of a set of microspheres (having specific size or composition) from a flowing mixture and guiding these in the desired direction by a moving array of optical traps. The approach exploits the fact that whereas the fluid drag force varies linearly with particle size, the optical gradient force has a more complex dependence on the particle size and also on its optical properties. Therefore, the ratio of these two forces is unique for different types of flowing particles. Selective trapping of a particular type of particles can thus be achieved by ensuring that the ratio between fluid drag and optical gradient force on these is below unity whereas for others it exceeds unity. Thereafter, the trapped particles can be sorted using a motion of the trapping sites towards the output. Because in this method the trapping force seen by the selected fraction of particles can be suitably higher than the fluid drag force, the particles can be captured and sorted from a fast fluid flow (about 150  μm/s). Therefore, even when using a dilute particle suspension, where the colloidal trafficking issues are naturally minimized, due to high flow rate a good throughput (about 30  particles/s) can be obtained. Experiments were performed to demonstrate sorting between silica spheres of different sizes (2, 3, and 5 μm) and between 3 μm size silica and polystyrene spheres.


Measurement of interaction forces between red blood cells in aggregates by optical tweezers

A Yu Maklygin, A V Priezzhev, A Karmenian, Sergei Yu Nikitin, I S Obolenskii, Andrei E Lugovtsov and Kisun Li

We have fabricated double-beam optical tweezers and demonstrated the possibility of their use for measuring the interaction forces between red blood cells (erythrocytes). It has been established experimentally that prolonged trapping of red blood cells in a tightly focused laser beam does not cause any visible changes in their shape or size. We have measured the interaction between red blood cells in the aggregate, deformed by optical tweezers.


Monday, June 25, 2012

Time-dependent heterogeneity in viscoelastic properties of worm-like micelle solutions

Koichiro Hori , David P. Penaloza , Atsuomi Shundo and Keiji Tanaka

Surfactant molecules often form micelles with a large aspect ratio, “worm-like” micelles, leading to network structures based on their entanglements. By using optical tweezers, we could detect a heterogeneity in the viscoelastic properties of the worm-like micelle solution, which is observed when the measurement timescale is shorter than the relaxation time − the time at which dissolution of the micelle entanglements occurs.


Metallic-Nanostructure-Enhanced Optical Trapping of Flexible Polymer Chains in Aqueous Solution as Revealed by Confocal Fluorescence Microspectroscopy

Mariko Toshimitsu, Yuriko Matsumura, Tatsuya Shoji, Noboru Kitamura, Mai Takase, Kei Murakoshi, Hiroaki Yamauchi, Syoji Ito, Hiroshi Miyasaka, Atsushi Nobuhiro, Yoshihiko Mizumoto, Hajime Ishihara, and Yasuyuki Tsuboi

Optical trapping of flexible polymer chains to a metallic nano-structured surface was explored by microscopic imaging and confocal fluorescence spectroscopy. A fluorescence-labeled poly(N-isopropylacrylamide) was targeted, being a representative thermo-responsive polymer. Upon resonant plasmonic excitation, it was clearly observed that polymers were assembled into the excitation area to form molecular assemblies. Simultaneously, fluorescence from the area was obviously intensified, indicating an increase in the number of polymer chains at the area. The excitation threshold of light intensity that was required for obvious trapping was 1 kW/cm2, which was much lower by a factor of 104 than that for conventional trapping using a focused laser beam. The morphology of the assemblies was sensitive to excitation intensity. We precisely evaluated temperature rise (T) around the metallic nanostructure upon plasmonic excitation: T ~ 10 K at 1 kW/cm2 excitation. This temperature rise was an origin of a repulsive force that blocked stable trapping. Based on experimental observations and theoretical calculations, we quantitatively evaluated the plasmon-enhanced trapping force and the thermal repulsive force (Soret effect). The overall mechanisms that were involved in such plasmon-enhanced optical trapping are discussed in detail. The smooth catch-and-release trapping (manipulation) of polymer chains was successfully demonstrated.


Ultrasensitive detection of a protein by optical trapping in a photonic-plasmonic microcavity

Miguel A. Santiago-Cordoba, Murat Cetinkaya, Svetlana V. Boriskina, Frank Vollmer, Melik C. Demirel

Microcavity and whispering gallery mode (WGM) biosensors derive their sensitivity from monitoring frequency shifts induced by protein binding at sites of highly confined field intensities, where field strengths can be further amplified by excitation of plasmon resonances in nanoparticle layers. Here, we propose a mechanism based on optical trapping of a protein at the site of plasmonic field enhancements for achieving ultra sensitive detection in only microliter-scale sample volumes, and in real-time. We demonstrate femto-Molar sensitivity corresponding to a few 1000 s of macromolecules. Simulations based on Mie theory agree well with the optical trapping concept at plasmonic ‘hotspots’ locations.


Evanescent wave optical trapping and transport of micro- and nanoparticles on tapered optical fibers

S.E. Skelton, M. Sergides, R. Patel, E. Karczewska, O.M. Maragó, P.H. Jones

We investigate the manipulation of microscopic and nanoscopic particles using the evanescent optical field surrounding an optical fiber that is tapered to a micron-scale diameter, and propose that this scheme could be used to discriminate between, and thereby sort, metallic nanoparticles. First we show experimentally the concept of the transport of micron-sized spheres along a tapered fiber and measure the particle velocity. Having demonstrated the principle we then consider theoretically the application to the optical trapping and guiding of metallic nanoparticles, where the presence of a plasmon resonance is used to enhance optical forces. We show that the dynamics of the nanoparticles trapped by the evanescent field can be controlled by the state of polarization of the fiber mode, and by using more than one wavelength differently detuned from the nanoparticle plasmon resonance. Such a scheme could potentially be used for selectively trapping and transporting nano- or microscopic material from a polydisperse suspension.


Thursday, June 21, 2012

Numerical modelling of optical trapping in hollow photonic crystal cavities

Ulagalandha Perumal Dharanipathy and Romuald Houdré

Photonic crystal (PhC) devices owing to their strong confinement of electromagnetic energy are considered to be excellent candidates for on chip optical trapping of dielectric or biological particles in the nanometer range. In this work, we study and present hollow PhC cavities and characterize them for their trapping stiffness, trapping stability and variation of resonance wavelength due to the presence of various sized single particles in the cavity.


Spinning nanorods – active optical manipulation of semiconductor nanorods using polarised light

C. Robin Head , Elena Kammann , Marco Zanella , Liberato Manna and Pavlos G. Lagoudakis

In this letter we show how a single beam optical trap offers the means for three-dimensional manipulation of semiconductor nanorods in solution. Furthermore rotation of the direction of the electric field provides control over the orientation of the nanorods, which is shown by polarisation analysis of two photon induced fluorescence. Statistics over tens of trapped agglomerates reveal a correlation between the measured degree of polarisation (DLP) and the size of the agglomerate which was determined by the escape frequency and the intensity of the emitted fluorescence. We estimate that we have trapped agglomerates with a volume of close to 10 times the volume of a single nanorod, which exhibited DLPs as high as 52%.


Wednesday, June 20, 2012

Optical forces on small particles from partially coherent light

Juan Miguel Auñón and Manuel Nieto-Vesperinas

We put forward a theory on the optical force exerted upon a dipolar particle by a stationary and ergodic partially coherent light field. We show through a rigorous analysis that the ensemble averaged electromagnetic force is given in terms of a partial gradient of the space-variable diagonal elements of the coherence tensor. Further, by following this result we characterize the conservative and nonconservative components of this force. In addition, we establish the propagation law for the optical force in terms of the coherence function of light at a diffraction plane. This permits us to evaluate the effect of the degree of coherence on the force components by using the archetypical configuration of Young’s two-aperture diffraction pattern, so often employed to characterize coherence of waves.


Calculation of optical forces on an ellipsoid using vectorial ray tracing method

Jin-Hua Zhou, Min-Cheng Zhong, Zi-Qiang Wang, and Yin-Mei Li

For a triaxial ellipsoid in an optical trap with spherical aberration, the optical forces, torque and stress are analyzed using vectorial ray tracing. The torque will automatically regulate ellipsoid’s long axis parallel to optic axis. For a trapped ellipsoid with principal axes in the ratio 1:2:3, the high stress distribution appears in x-z plane. And the optical force at x-axis is weaker than at y-axis due to the shape size. While the ellipsoid departs laterally from trap center, the measurable maximum transverse forces will be weakened due to axial equilibrium and affected by inclined orientation. For an appropriate ring beam, the maximum optical forces are strong in three dimensions, thus, this optical trap is appropriate to trap cells for avoiding damage from laser.


Tuesday, June 19, 2012

Dielectrophoresis force spectroscopy for colloidal clusters

Hyunjoo Park, Ming-Tzo Wei, H. Daniel Ou-Yang

Optical trapping-based force spectroscopy was used to measure the frequency dependent dielectrophoresis (DEP) forces and DEP crossover frequencies of colloidal PMMA spheres and clusters. A single sphere or cluster, held by an optical tweezer, was positioned near the center of a pair of gold-film electrodes where ACEO flow was negligible. Use of amplitude modulation and phase-sensitive lock-in detection for accurate measurement of the DEP force yielded new insight into dielectric relaxation mechanisms near the crossover frequencies. On one hand, the size dependence of the DEP force near the crossover frequencies indicates that the dominant polarization mechanism is a volume effect. On the other, the power-law dependence of the crossover frequency on the particle radius with an exponent -2 indicates the dielectric relaxation is more likely due to ionic diffusion across the particle surface, suggesting the dominant polarization mechanism may be a surface polarization effect. Better theories are needed to explain the experiment. Nevertheless, the strong size dependence of the crossover frequencies suggests the use of DEP for size sorting of micron-sized particles.


Optical tweezers technique for the study of red blood cells deformation ability

A. Korobtsov, S. Kotova, N. Losevsky, A. Mayorova, V. Patlan, E. Timchenko, N. Lysov and E. Zarubina

The presented here technique is developed for in vitro estimation of the influence of external conditions on the deformation ability of human red blood cells. The method is based on the use of single-beam laser trap for capturing of erythrocytes fixed to the bottom of the liquid cell by the adhesion force. The approach is suitable for evaluation of the properties of individual cells as well as for real-time acquisition of the data on ensemble of erythrocytes large enough for subsequent statistical analysis. It is shown that both a change in the saline solution concentration and presence of certain stabilizing agents lead to measurable shift of the maximum of the cells distribution by their deformability.


Towards 3D modelling and imaging of infection scenarios at the single cell level using holographic optical tweezers and digital holographic microscopy

Björn Kemper, Álvaro Barroso, Mike Woerdemann, Lena Dewenter, Angelika Vollmer, Robin Schubert, Alexander Mellmann, Gert von Bally, Cornelia Denz

The analysis of dynamic interactions of microorganisms with a host cell is of utmost importance for understanding infection processes. We present a biophotonic holographic workstation that allows optical manipulation of bacteria by holographic optical tweezers and simultaneously monitoring of dynamic processes with quantitative multi-focus phase imaging based on self-interference digital holographic microscopy. Our results show that several bacterial cells, even with non-spherical shape, can be aligned precisely on the surface of living host cells and localized reproducibly in three dimensions. In this way a new label-free multipurpose device for modelling and quantitative analysis of infection scenarios at the single cell level is provided.


Monday, June 18, 2012

Influence of semiflexible structural features of actin cytoskeleton on cell stiffness based on actin microstructural modeling

Kaiqun Wang, Dong Sun

A new actin cytoskeleton microstructural model based on the semiflexible polymer nature of the actin filament is proposed. The relationship between the stretching force and the mechanical properties of cells was examined. Experiments on deforming hematopoietic cells with distinct primitiveness from normal and leukemic sources were conducted via optical tweezer manipulation at single-cell level. The modeling results were demonstrated to be in good agreement with the experimental data. We characterized how the structural properties of the actin cytoskeleton, such as prestress, density of cross-links, and actin concentration, affect the mechanical behavior of cells based on the proposed model. Increasing prestress, actin concentration, and density of cross-links reduced cell deformation, and the cell also exhibited strain stiffening behavior with an increase in the stretching force. Compared with existing models, the proposed model exhibits a distinct feature in probing the influence of semiflexible polymer nature of the actin filament on cell mechanical behavior.


Optical Trapping Forces on Non-Spherical Particles in Fluid Flows

Dewan Hasan Ahmed & Hyung Jin Sung

The optical stability of particles above a waveguide surface depends on the forces induced by fluid drag and the electromagnetic field. The optical trapping forces on non-spherical particles were examined for various flow conditions. A three-dimensional finite element method was employed to calculate the electromagnetic field and the fluid flow. It was found that the stability of non-spherical particles is significantly affected by the fluid velocity and the orientation of the particles. The downward trapping force meant that non-spherical particles are more stable at higher Reynolds numbers. The length of the particle in the transverse direction also had a significant impact on particle stability. The present model was tested against previously reported results.


Dynamical hologram generation for high speed optical trapping of smart droplet microtools

P. M. P. Lanigan, I. Munro, E. J. Grace, D. R. Casey, J. Phillips, D. R. Klug, O. Ces, and M. A. A. Neil

This paper demonstrates spatially selective sampling of the plasma membrane by the implementation of time-multiplexed holographic optical tweezers for Smart Droplet Microtools (SDMs). High speed (>1000fps) dynamical hologram generation was computed on the graphics processing unit of a standard display card and controlled by a user friendly LabView interface. Time multiplexed binary holograms were displayed in real time and mirrored to a ferroelectric Spatial Light Modulator. SDMs were manufactured with both liquid cores (as previously described) and solid cores, which confer significant advantages in terms of stability, polydispersity and ease of use. These were coated with a number of detergents, the most successful based upon lipids doped with transfection reagents. In order to validate these, trapped SDMs were maneuvered up to the plasma membrane of giant vesicles containing Nile Red and human biliary epithelial (BE) colon cancer cells with green fluorescent labeled protein (GFP)-labeled CAAX (a motif belonging to the Ras protein). Bright field and fluorescence images showed that successful trapping and manipulation of multiple SDMs in x, y, z was achieved with success rates of 30-50% and that subsequent membrane-SDM interactions led to the uptake of Nile Red or GFP-CAAX into the SDM.


Friday, June 15, 2012

Keratin 8/18 Regulation of Cell Stiffness-Extracellular Matrix Interplay through Modulation of Rho-Mediated Actin Cytoskeleton Dynamics

François Bordeleau, Marie-Eve Myrand Lapierre,Yunlong Sheng, Normand Marceau
Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells.


Chemotactic adaptation kinetics of individual Escherichia coli cells

Taejin L. Min, Patrick J. Mears, Ido Golding, and Yann R. Chemla

Escherichia coli chemotaxis serves as a paradigm for the way living cells respond and adapt to changes in their environment. The chemotactic response has been characterized at the level of individual flagellar motors and in populations of swimming cells. However, it has not been previously possible to quantify accurately the adaptive response of a single, multiflagellated cell. Here, we use our recently developed optical trapping technique to characterize the swimming behavior of individual bacteria as they respond to sudden changes in the chemical environment. We follow the adaptation kinetics of E. coli to varying magnitudes of step-up and step-down changes in concentration of chemoattractant. We quantify two features of adaptation and how they vary with stimulus strength: abruptness (the degree to which return to prestimulus behavior occurs within a small number of run/tumble events) and overshoot (the degree of excessive response before the return to prestimulus behavior). We also characterize the asymmetry between step-up and step-down responses, observed at the single-cell level. Our findings provide clues to an improved understanding of chemotactic adaptation. 

Patch-Clamp Measurements on a Chip with Full Control over the Oxygen Content

A. Alrifaiy, N. Bitaraf, M. Druzin, O. Lindahl and K. Ramser

We present a novel approach to reproduce hypoxia on a chip by patch-clamp investigations on single nerve cells exposed to anoxic and normoxic environments. The patch-clamp technique was combined with microfluidics to enable fast exchange of buffer-solutions. The micropipette was included within the microfluidic chip to allow investigations with full control over the oxygen content in vicinity of the sample. Nerve cells from Sprague Dawley rats were prepared and inserted into the channels of the microchip. Single nerve cells were optically trapped and manipulated to be patched by patch-clamp micropipette. The experiments were aimed to test proof of principle and to perform patchclamp electrophysiological measurements on the cells under well-defined conditions. The oxygen level within the microfluidic channels was in the range of 0.5 to 1.5%. The laser tweezers showed no remarkable photo-induced effect on the investigated nerve cells and no effects on the electrophysiological measurements were detected. The approach of using closed microfluidic system in patch-clamp experiments showed significant advantages to control the oxygen concentration around the investigated cell. This may be adapted to be used in other biological investigations of single cells demanding optimal control of the surroundings.


The Role of Membrane Stiffness and Actin Turnover on the Force Exerted by DRG Lamellipodia

Ladan Amin, Erika Ercolini, Rajesh Shahapure, Elisa Migliorini, Vincent Torre

We used optical tweezers to analyze the effect of jasplakinolide and cyclodextrin on the force exerted by lamellipodia from developing growth cones (GCs) of isolated dorsal root ganglia (DRG) neurons. We found that 25 nM of jasplakinolide, which is known to inhibit actin filament turnover, reduced both the maximal exerted force and maximal velocity during lamellipodia leading-edge protrusion. By using atomic force microscopy, we verified that cyclodextrin, which is known to remove cholesterol from membranes, decreased the membrane stiffness of DRG neurons. Lamellipodia treated with 2.5 mM of cyclodextrin exerted a larger force, and their leading edge could advance with a higher velocity. Neither jasplakinolide nor cyclodextrin affected force or velocity during lamellipodia retraction. The amplitude and frequency of elementary jumps underlying force generation were reduced by jasplakinolide but not by cyclodextrin. The action of both drugs at the used concentration was fully reversible. These results support the notion that membrane stiffness provides a selective pressure that shapes force generation, and confirm the pivotal role of actin turnover during protrusion.


G-Quadruplex and i-Motif Are Mutually Exclusive in ILPR Double-Stranded DNA

Soma Dhakal, Zhongbo Yu, Ryan Konik, Yunxi Cui, Deepak Koirala, Hanbin Mao

G-quadruplex has demonstrated its biological functions in vivo. Although G-quadruplex in single-stranded DNA (ssDNA) has been well characterized, investigation of this species in double-stranded DNA (dsDNA) lags behind. Here we use chemical footprinting and laser-tweezers-based single-molecule approaches to demonstrate that a dsDNA fragment found in the insulin-linked polymorphic region (ILPR), 5′-(ACA GGGG TGT GGGG)2 TGT, can fold into a G-quadruplex at pH 7.4 with 100 mM K+, and an i-motif at pH 5.5 with 100 mM Li+. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif (pH 5.5, 100 mM K+), a unique determination of change in the free energy of unfolding (ΔGunfold) by laser-tweezers experiments provides compelling evidence that only one species is present in each dsDNA. Under this condition, molecules containing G-quadruplex are more stable than those with i-motif. These two species have mechanical stabilities (rupture force ≥ 17 pN) comparable to the stall force of RNA polymerases, which, from a mechanical perspective alone, could justify a regulatory mechanism for tetraplex structures in the expression of human insulin.


Assembling Neurospheres: Dynamics of Neural Progenitor/Stem Cell Aggregation Probed Using an Optical Tra

Uma Ladiwala, Himanish Basu, Deepak Mathur
Optical trapping (tweezing) has been used in conjunction with fluid flow technology to dissect the mechanics and spatio-temporal dynamics of how neural progenitor/stem cells (NSCs) adhere and aggregate. Hitherto unavailable information has been obtained on the most probable minimum time (~5 s) and most probable minimum distance of approach (4–6 µm) required for irreversible adhesion of proximate cells to occur. Our experiments also allow us to study and quantify the spatial characteristics of filopodial- and membrane-mediated adhesion, and to probe the functional dynamics of NSCs to quantify a lower limit of the adhesive force by which NSCs aggregate (~18 pN). Our findings, which we also validate by computational modeling, have important implications for the neurosphere assay: once aggregated, neurospheres cannot disassemble merely by being subjected to shaking or by thermal effects. Our findings provide quantitative affirmation to the notion that the neurosphere assay may not be a valid measure of clonality and “stemness”. Post-adhesion dynamics were also studied and oscillatory motion in filopodia-mediated adhesion was observed. Furthermore, we have also explored the effect of the removal of calcium ions: both filopodia-mediated as well as membrane-membrane adhesion were inhibited. On the other hand, F-actin disrupted the dynamics of such adhesion events such that filopodia-mediated adhesion was inhibited but not membrane-membrane adhesion.


Tuning Multiple Motor Travel Via Single Motor Velocity

Jing Xu, Zhanyong Shu, Stephen J. King, Steven P. Gross

Microtubule-based molecular motors often work in small groups to transport cargos in cells. A key question in understanding transport (and its regulation in vivo) is to identify the sensitivity of multiple-motor-based motion to various single molecule properties. Whereas both single-motor travel distance and microtubule binding rate have been demonstrated to contribute to cargo travel, the role of single-motor velocity is yet to be explored. Here, we recast a previous theoretical study, and make explicit a potential contribution of velocity to cargo travel. We test this possibility experimentally, and demonstrate a strong negative correlation between single-motor velocity and cargo travel for transport driven by two motors. Our study thus discovers a previously unappreciated role of single-motor velocity in regulating multiple-motor transport.


Thursday, June 14, 2012

The Influence of pH on the Specific Adhesion of P Piliated Escherichia coli

Jeanna E. Klinth, Mickaël Castelain, Bernt Eric Uhlin, Ove Axner

Adhesion to host tissues is an initiating step in a majority of bacterial infections. In the case of Gram-negative bacteria this adhesion is often mediated by a specific interaction between an adhesin, positioned at the distal end of bacterial pili, and its receptor on the surface of the host tissue. Furthermore, the rod of the pilus, and particularly its biomechanical properties, is believed to be crucial for the ability of bacteria to withstand external forces caused by, for example, (in the case of urinary tract infections) urinary rinsing flows by redistributing the force to several pili. In this work, the adhesion properties of P-piliated E. coli and their dependence of pH have been investigated in a broad pH range by both the surface plasmon resonance technique and force measuring optical tweezers. We demonstrate that P piliated bacteria have an adhesion ability throughout the entire physiologically relevant pH range (pH 4.5 – 8). We also show that pH has a higher impact on the binding rate than on the binding stability or the biomechanical properties of pili; the binding rate was found to have a maximum around pH 5 while the binding stability was found to have a broader distribution over pH and be significant over the entire physiologically relevant pH range. Force measurements on a single organelle level show that the biomechanical properties of P pili are not significantly affected by pH.


Photonic forces in the near field of statistically homogeneous fluctuating sources

Juan Miguel Auñón and Manuel Nieto-Vesperinas

Electromagnetic sources, e.g., lasers, antennas, diffusers, or thermal sources, produce a wave field that interacts with objects to transfer to them its momentum. We show that the photonic force exerted on a dipolar particle in the near field of a planar statistically homogeneous fluctuating source uniquely depends on and acts along the coordinate perpendicular to its surface. The gradient part of this force is contributed by only the evanescent components of the emitted field, its sign being opposite that of the real part of the particle polarizability. The nonconservative force is due to the propagating components, which are repulsive and constant. In addition, the source coherence length adds a degree of freedom since it largely affects these forces. The excitation of plasmons in the source surface drastically enhances the gradient force while slightly weakening the nonconservative scattering plus curl force. Hence these consequences obtained for random and partially coherent wave fields emitted by the sources addressed here should be relevant for particle manipulation at the subwavelength scale.


Helix-like biopolymers can act as dampers of force for bacteria in flows

Johan Zakrisson, Krister Wiklund, Ove Axner and Magnus Andersson

Biopolymers are vital structures for many living organisms; for a variety of bacteria, adhesion polymers play a crucial role for the initiation of colonization. Some bacteria express, on their surface, attachment organelles (pili) that comprise subunits formed into stiff helix-like structures that possess unique biomechanical properties. These helix-like structures possess a high degree of flexibility that gives the biopolymers a unique extendibility. This has been considered beneficial for piliated bacteria adhering to host surfaces in the presence of a fluid flow. We show in this work that helix-like pili have the ability to act as efficient dampers of force that can, for a limited time, lower the load on the force-mediating adhesin-receptor bond on the tip of an individual pilus. The model presented is applied to bacteria adhering with a single pilus of either of the two most common types expressed by uropathogenicEscherichia coli, P or type 1 pili, subjected to realistic flows. The results indicate that for moderate flows (~25 mm/s) the force experienced by the adhesin-receptor interaction at the tip of the pilus can be reduced by a factor of ~6 and ~4, respectively. The uncoiling ability provides a bacterium with a “go with the flow” possibility that acts as a damping. It is surmised that this can be an important factor for the initial part of the adhesion process, in particular in turbulent flows, and thereby be of use for bacteria in their striving to survive a natural defense such as fluid rinsing actions.


Partial Synchronization of Stochastic Oscillators through Hydrodynamic Coupling

Arran Curran, Michael P. Lee, Miles J. Padgett, Jonathan M. Cooper, and Roberto Di Leonardo

Holographic optical tweezers are used to construct a static bistable optical potential energy landscape where a Brownian particle experiences restoring forces from two nearby optical traps and undergoes thermally activated transitions between the two energy minima. Hydrodynamic coupling between two such systems results in their partial synchronization. This is interpreted as an emergence of higher mobility pathways, along which it is easier to overcome barriers to structural rearrangement.


Monday, June 11, 2012

Hepatitis B surface antigen-antibody interactions studied by optical tweezers

Zhou, Z.L., Tang, B.; Ngan, A.H.W.; Dong, Z.N.; Wu, Y.S.

The protein-protein interactions between hepatitis B surface antigen (HBsAg) and its antibodies (anti-HBs) were studied by measuring the binding force between microspheres coated with such proteins using optical tweezers. The interaction force between the protein-coated microspheres was found to be strongly influenced by the acidity of the surrounding liquid medium, as well as the experimental temperature, and it reaches a maximum value at around pH 7.5 and temperature around 37°C. By measuring the protein distribution on the surfaces of the microspheres and their contact areas using scanning electron microscopy, the specific binding force between an HBsAg and anti-HBs protein pair is estimated to be around 4.8 pN at the optimum pH value and temperature at an applied loading rate of around 1 pN/s.


Cellular viscoelasticity probed by active rheology in optical tweezers

Evgeny V. Lyubin, Maria D. Khokhlova, Maria N. Skryabina, and Andrey A. Fedyanin

A novel approach to probe viscoelastic properties of cells based on double trap optical tweezers is reported. Frequency dependence of the tangent of phase difference in the movement of the opposite erythrocyte edges while one of the edges is forced to oscillate by optical tweezers appeared to be highly dependent on the rigidity of the cellular membrane. Effective viscoelastic parameters characterizing red blood cells with different stiffnesses (normal and glutaraldehyde-fixed) are determined. It is shown that the photo-induced effects caused by laser trapping at the power level used in the experiments are negligible giving the possibility to use the offered technique for dynamic monitoring of soft materials viscoelastic properties.


Tuesday, June 5, 2012

Optical alignment and confinement of an ellipsoidal nanorod in optical tweezers: a theoretical study

Jan Trojek, Lukáš Chvátal, and Pavel Zemánek

Within the Rayleigh approximation, we investigate the behavior of an individual ellipsoidal metal nanorod that is optically confined in three dimensions using a single focused laser beam. We focus on the description of the optical torque and optical force acting upon the nanorod placed into a linearly polarized Gaussian beam (scalar description of the electric field) or a strongly focused beam (vector field description). The study comprises the influence of the trapping laser wavelength, the angular aperture of focusing optics, the orientation of the ellipsoidal nanorod, and the aspect ratio of its principal axes. The results reveal a significantly different behavior of the nanorod if the trapping wavelength is longer or shorter than the wavelength corresponding to the longitudinal plasmon resonance mode. Published experimental observations are compared with our theoretical predictions with satisfactory results.


On-Chip Cellomics Assay Enabling Algebraic and Geometric Understanding of Epigenetic Information in Cellular Networks of Living Systems. 1. Temporal Aspects of Epigenetic Information in Bacteria

Kenji Yasuda

A series of studies aimed at developing methods and systems of analyzing epigenetic information in cells and in cell networks, as well as that of genetic information, was examined to expand our understanding of how living systems are determined. Because cells are minimum units reflecting epigenetic information, which is considered to map the history of a parallel-processing recurrent network of biochemical reactions, their behaviors cannot be explained by considering only conventional DNA information-processing events. The role of epigenetic information on cells, which complements their genetic information, was inferred by comparing predictions from genetic information with cell behaviour observed under conditions chosen to reveal adaptation processes, population effects and community effects. A system of analyzing epigenetic information was developed starting from the twin complementary viewpoints of cell regulation as an “algebraic” system (emphasis on temporal aspects) and as a “geometric” system (emphasis on spatial aspects). Exploiting the combination of latest microfabrication technology and measurement technologies, which we call on-chip cellomics assay, we can control and re-construct the environments and interaction of cells from “algebraic” and “geometric” viewpoints. In this review, temporal viewpoint of epigenetic information, a part of the series of single-cell-based “algebraic” and “geometric” studies of celluler systems in our research groups, are summerized and reported. The knowlege acquired from this study may lead to the use of cells that fully control practical applications like cell-based drug screening and the regeneration of organs.


Microfluidic sorting of arbitrary cells with dynamic optical tweezers

Benjamin Landenberger

Optical gradient forces generated by fast steerable optical tweezers are highly effective for sorting small populations of cells in a lab-on-a-chip environment. The presented system can sort a broad range of different biological specimen by an automated optimisation of the tweezers path and velocity profile. The optimal grab positions for subsequent trap and cell displacements are estimated from the intensity of the bright field image, which is derived theoretically and proven experimentally. We exhibit rapid displacements of 2 µm small mitochondria, yeast cells, rod-shaped bacteria and 30 µm large protoplasts. Reliable sorting of yeast cells in a micro-fluidic chamber by both morphological criteria and by fluorescence emission is demonstrated.


Optical Tweezers Studies on Notch: Single-Molecule Interaction Strength Is Independent of Ligand Endocytosis

Bhupinder Shergill, Laurence Meloty-Kapella, Abdiwahab A. Musse, Gerry Weinmaster, Elliot Botvinick

Notch signaling controls diverse cellular processes critical to development and disease.
 Cell surface ligands bind Notch on neighboring cells but require endocytosis to activate signaling. The role ligand endocytosis plays in Notch activation has not been established. Here we integrate optical tweezers with cell biological and biochemical methods to test the prevailing model that ligand endocytosis facilitates recycling to enhance ligand interactions with Notch necessary to trigger signaling. Specifically, single-molecule measurements indicate that interference of ligand endocytosis and/or recycling does not alter the force required to rupture bonds formed between cells expressing the Notch ligand Delta-like1 (Dll1) and laser-trapped Notch1 beads. Together, our analyses eliminate roles for ligand endocytosis and recycling in Dll1-Notch1 interactions and indicate that recycling indirectly affects signaling by regulating the accumulation of cell surface ligand. Importantly, our study demonstrates the utility of optical tweezers to test a role for ligand endocytosis in generating cell-mediated mechanical force. 

Notch Ligand Endocytosis Generates Mechanical Pulling Force Dependent on Dynamin, Epsins, and Actin

Laurence Meloty-Kapella, Bhupinder Shergill, Jane Kuon, Elliot Botvinick, Gerry Weinmaster

Notch signaling induced by cell surface ligands is critical to development and maintenance of many eukaryotic organisms. Notch and its ligands are integral membrane proteins that facilitate direct cell-cell interactions to activate Notch proteolysis and release the intracellular domain that directs Notch-specific cellular responses. Genetic studies suggest that Notch ligands require endocytosis, ubiquitylation, and epsin endocytic adaptors to activate signaling, but the exact role of ligand endocytosis remains unresolved. Here we characterize a molecularly distinct mode of clathrin-mediated endocytosis requiring ligand ubiquitylation, epsins, and actin for ligand cells to activate signaling in Notch cells. Using a cell-bead optical tweezers system, we obtained evidence for cell-mediated mechanical force dependent on this distinct mode of ligand endocytosis. We propose that the mechanical pulling force produced by endocytosis of Notch-bound ligand drives conformational changes in Notch that permit activating proteolysis.


Monday, June 4, 2012

Single-cell adhesion probed in-situ using optical tweezers: A case study with Saccharomyces cerevisiae

Mickaël Castelain, Paul G. Rouxhet, Frédéric Pignon, Albert Magnin, and Jean-Michel Piau

A facile method of using optical trapping to measure cell adhesion forces is presented and applied to the adhesion of Saccharomyces cerevisiae on glass, in contact with solutions of different compositions. Trapping yeast cells with optical tweezers (OT) is not perturbed by cell wall deformation or cell deviation from a spherical shape. The trapping force calibration requires correction not only for the hydrodynamic effect of the neighboring wall but also for spherical aberrations affecting the focal volume and the trap stiffness. Yeast cells trapped for up to 5 h were still able to undergo budding but showed an increase of doubling time. The proportion of adhering cells showed the expected variation according to the solution composition. The detachment force varied in the same way. This observation and the fact that the detachment stress was exerted parallel to the substrate surface point to the role of interactions involving solvated macromolecules. Both the proportion of adhering cells and the removal force showed a distribution which, in our experimental conditions, must be attributed to a heterogeneity of surface properties at the cell level or at the subcellular scale. As compared with magnetic tweezers, atomic force microscopy, and more conventional ways of studying cell adhesion (shear-flow cells), OT present several advantages that are emphasized in this paper.


Hydrodynamically induced rhythmic motion of optically driven colloidal particles on a ring

Yuriko Sassa, Shuhei Shibata, Yasutaka Iwashita, and Yasuyuki Kimura
We experimentally study the motion of optically driven colloidal particles on a circular path by varying their number N. Although an identical driving force is applied to each particle, their equally spaced configuration is hydrodynamically unstable, and a doublet configuration is spontaneously formed. In small-N systems, the angular difference between neighboring particles exhibits oscillatory or nonoscillatory behavior. The number of oscillatory modes that appear depends on the maximum number of doublets that the system can contain. Frequent switching between different modes was observed with increasing N. The characteristic frequencies of the oscillatory modes are discussed theoretically by linear stability analysis of the equations that govern the motion of hydrodynamically coupled particles. The evaluated frequencies of the slowest modes exhibit reasonably good agreement with those of the mainly observed modes in experiments. The relationship between the characteristic frequencies and specific configurations is confirmed experimentally by setting a specific initial configuration for the particles. An increase in N also enhances the mean angular velocity of the particles owing to the reduced effective viscosity in large-N systems.


Friday, June 1, 2012

Electric and magnetic optical response of dielectric nanospheres: Optical forces and scattering anisotropy

R. Gómez-Medina, B. García-Cámara, I. Suárez-Lacalle, L.S. Froufe-Pérez, F. González, F. Moreno, M. Nieto-Vesperinas, J.J. Sáenz

Nanospheres made of non-magnetic materials are shown to present non-conventional scattering properties similar to those previously reported for somewhat hypothetical magnetodielectric particles. We find a wide window in the near-infrared, where light scattering by lossless submicrometer semiconductor nanospheres is fully described by their induced electric and magnetic dipoles. The interference between electric and magnetic dipolar fields is shown to lead to anisotropic angular distributions of scattered intensity, including zero backward and almost zero forward scattered intensities at specific wavelengths. Interesting new consequences for the corresponding optical forces are derived from the interplay, both in and out of resonance, between the electric- and magnetic-induced dipoles.


Plasmonic Trapping with a Gold Nanopillar

Dr. Kai Wang, Dr. Kenneth B. Crozier

An improved ability to manipulate nanoscale objects could spur the field of nanotechnology. Optical tweezers offer the compelling advantage that manipulation is performed in a non-invasive manner. However, traditional optical tweezers based on laser beams focused with microscope lenses face limitations due to the diffraction limit, which states that conventional lenses can focus light to spots no smaller than roughly half the wavelength. This has motivated recent work on optical trapping based on the sub-wavelength field distributions of surface plasmon nanostructures. This approach offers the benefits of higher precision and resolution, and the possibility of large-scale parallelization. Herein, we discuss the fundamentals of optical manipulation using surface plasmon resonance structures. We describe two important issues in plasmonic trapping: optical design and thermal management strategies. Finally, we describe a surface plasmon nanostructure, consisting of a gold nanopillar that takes these issues into consideration. It is shown to enable the trapping and rotation (manual and passive) of nanoparticles. Methods by which this concept can be extended are discussed.


Simultaneous microscale optical manipulation, fabrication and immobilisation in aqueous media

Farah Dawood , Sijia Qin , Linjie Li , Emily Y. Lin and John T. Fourkas

Efficient multiphoton radical generation chemistry has been developed for use in aqueous media. Through a combination of multiphoton absorption polymerisation (MAP) and optical tweezers, this chemistry has been applied to the fabrication, manipulation, and assembly of 3D polymeric and biomolecular structures. Combining MAP and optical tweezers allows for the direct assembly of 3D structures from microscale objects as well as for the realisation of structures, such as tape-like and rope-like microthreads, that can be used for unconventional microfabrication techniques including microbraiding and microweaving. These capabilities significantly expand the toolbox of methods available for the creation of functional microstructures in aqueous media.


Back-scattered detection yields viable signals in many conditions

Frederick B. Shipley and Ashley R. Carter

Precision position-sensing is required for many microscopy techniques. One promising method, back-scattered detection (BSD), is incredibly sensitive, allowing for position measurements at the level of tens of picometers in three dimensions. In BSD the position of a micron-sized bead is measured by back-scattering a focused laser beam off the bead and imaging the resulting interference pattern onto a detector. Since the detection system geometry is confined to one side of the objective, the technique is compatible with platforms that have restricted optical access (e.g. magnetic tweezers, atomic force microscopy, and microfluidics). However, general adoption of BSD may be limited according to a recent theory [Volpe et al., J. Appl. Phys. 102, 084701, 2007] that predicts diminished signals under certain conditions. We directly measured the BSD response while varying the experimental conditions, including bead radius, numerical aperture, and relative index. Contrary to the proposed theory, we find that all experimental conditions tested produced a viable signal for atomic-scale measurements.