Friday, December 30, 2011

Simulation of the acceleration mechanism by light-propulsion for the powder particles at laser direct material deposition

I.О. Kovaleva, O.B. Kovalev

The results of the numerical analysis of heat- and mass-transfer processes at powder particles' motion in a gas flow and laser beam by light-propulsion force during the laser cladding and direct material deposition are presented. Under consideration were the stainless steel particles, the radiation power range of the CO2 laser were 1000, 3000 and 5000 W. Finally, the particles of 45 μm in diameter reach the maximum velocity of about 80, 220, 280 m/s. It is shown that as particles are heated by the laser up to the temperature approaching the boiling point, the particles' velocity in the light field by the vapor recoil pressure may increase significantly. The radius of the particles slightly varies due to the evaporation; the losses in the clad material mass are negligibly small. Comparisons of numerical results with known experimental data on light-propulsion acceleration of single particles (aluminum, aluminum oxide and graphite) under the influence of pulse laser radiation are also presented. Particle acceleration resulting from the laser evaporation depends on the particle diameter, powder material properties, focusing degree and attenuation laser beam intensity by the direction of its propagation.


Thursday, December 29, 2011

Optical trapping: A review of essential concepts

I.Verdeny, A.Farré, J.Mas, C.López-Quesada, E.Martín-Badosa, M.Montes-Usategui

Optical tweezers are an innovative technique for the non-contact, all-optical manipulation of small material samples, which has extraordinarily expanded and evolved since its inception in the mid-80s of the last century. Nowadays, the potential of optical tweezers has been clearly proven and a wide range of applications both from the physical and biological sciences have solidly emerged, turning the early ideas and techniques into a powerful paradigm for experimentation in the micro- and nanoworld. This review aims at highlighting the fundamental concepts that are essential for a thorough understanding of optical trapping, making emphasis on both its manipulation and measurement capabilities, as well as on the vast array of important biological applications appeared in the last years.


Multimodal biophotonic workstation for live cell analysis

Michael Esseling, Björn Kemper, Maciej Antkowiak, David J. Stevenson, Lionel Chaudet, Mark A. A. Neil, Paul W. French, Gert von Bally, Kishan Dholakia, Cornelia Denz

A reliable description and quantification of the complex physiology and reactions of living cells requires a multimodal analysis with various measurement techniques. We have investigated the integration of different techniques into a biophotonic workstation that can provide biological researchers with these capabilities. The combination of a micromanipulation tool with three different imaging principles is accomplished in a single inverted microscope which makes the results from all the techniques directly comparable. Chinese Hamster Ovary (CHO) cells were manipulated by optical tweezers while the feedback was directly analyzed by fluorescence lifetime imaging, digital holographic microscopy and dynamic phase-contrast microscopy.


Microfabricated continuous cubic phase plate induced Airy beams for optical manipulation with high power efficiency

Rui Cao, Yong Yang, Jingang Wang, Jing Bu, Mingwei Wang, and X.-C. Yuan

We studied and demonstrated optical trapping capabilities of an Airy beam generated with a cubic phase plate incorporated into a conventional optical tweezer system. The power efficiency and damage threshold of the cubic phase plate were found to be much higher when spatial light modulators were employed in beam generation.


Saturday, December 24, 2011

A multi-mode fiber probe for holographic micromanipulation and microscopy

Silvio Bianchi and Roberto Di Leonardo

Holographic tweezers have revolutionized the way we do experiments at the micron scale. The possibility of applying controlled force fields on simultaneously trapped micro-particles has allowed to directly probe interactions and mechanical properties of colloids, macromolecules and living cells. Holographic micromanipulation requires the careful shaping of a laser beam that is then focused by a microscope objective onto a micro-hologram in the sample volume. The same objective is used for imaging. That approach is therefore limited to in vitro samples contained in transparent cells that are easily accessed optically. Here we demonstrate that the complex light propagator of a real multimode fiber can be directly measured. That allows to transmit a micro-hologram through a 1 metre long (60 μm core) optical fiber and produce dynamic arrays of focused spots at the fiber output. We show that those spots can be used for interactive holographic micromanipulation of micron sized beads facing the fiber tip. Scanning a single spot across the output fiber we can employ the same fiber as a probe for scanning fluorescence microscopy. Our findings open the way towards the fabrication of endoscopic probes which could be capable of seeing and manipulating single cells deep into biological tissues.


Metrology of laser-guided particles in air-filled hollow-core photonic crystal fiber

O. A. Schmidt, M. K. Garbos, T. G. Euser, and P. St. J. Russell

Micrometer-sized particles are trapped in front of an air-filled hollow-core photonic crystal fiber using a novel dual-beam trap. A backward guided mode produces a divergent beam that diffracts out of the core, and simultaneously a focused laser beam launches a forward-propagating mode into the core. By changing the backward/forward power balance, a trapped particle can be selectively launched into the hollow core. Once inside, particles can be optically propelled along several meters of fiber with mobilities as high as 19  cm·s−1  W−1 (precisely measured using in-fiber Doppler velocimetry). The results are in excellent agreement with theory. The system allows determination of fiber loss as well as the mass density and refractive index of single particles.


Friday, December 23, 2011

Large-area optoelastic manipulation of colloidal particles in liquid crystals using photoresponsive molecular surface monolayers

Angel Martinez, Hector C. Mireles, and Ivan I. Smalyukh

Noncontact optical trapping and manipulation of micrometer- and nanometer-sized particles are typically achieved by use of forces and torques exerted by tightly focused high-intensity laser beams. Although they were instrumental for many scientific breakthroughs, these approaches find few technological applications mainly because of the small-area manipulation capabilities, the need for using high laser powers, limited application to anisotropic fluids and low-refractive-index particles, as well as complexity of implementation. To overcome these limitations, recent research efforts have been directed toward extending the scope of noncontact optical control through the use of optically-guided electrokinetic forces, vortex laser beams, plasmonics, and optofluidics. Here we demonstrate manipulation of colloidal particles and self-assembled structures in nematic liquid crystals by means of single-molecule-thick, light-controlled surface monolayers. Using polarized light of intensity from 1,000 to 100,000 times smaller than that in conventional optical tweezers, we rotate, translate, localize, and assemble spherical and complex-shaped particles of various sizes and compositions. By controlling boundary conditions through the monolayer, we manipulate the liquid crystal director field and the landscape of ensuing elastic forces exerted on colloids by the host medium. This permits the centimeter-scale, massively parallel manipulation of particles and complex colloidal structures that can be dynamically controlled by changing illumination or assembled into stationary stable configurations dictated by the “memorized” optoelastic potential landscape due to the last illumination pattern. We characterize the strength of optically guided elastic forces and discuss the potential uses of this noncontact manipulation in fabrication of novel optically- and electrically-tunable composites from liquid crystals and colloids.


Realization of a micrometre-sized stochastic heat engine

Valentin Blickle & Clemens Bechinger

The conversion of energy into mechanical work is essential for almost any industrial process. The original description of classical heat engines by Sadi Carnot in 1824 has largely shaped our understanding of work and heat exchange during macroscopic thermodynamic processes. Equipped with our present-day ability to design and control mechanical devices at micro- and nanometre length scales, we are now in a position to explore the limitations of classical thermodynamics, arising on scales for which thermal fluctuations are important. Here we demonstrate the experimental realization of a microscopic heat engine, comprising a single colloidal particle subject to a time-dependent optical laser trap. The work associated with the system is a fluctuating quantity, and depends strongly on the cycle duration time, τ, which in turn determines the efficiency of our heat engine. Our experiments offer a rare insight into the conversion of thermal to mechanical energy on a microscopic level, and pave the way for the design of future micromechanical machines.


Mutations Altering the Interplay between GkDnaC Helicase and DNA Reveal an Insight into Helicase Unwinding

Yu-Hua Lo, Shih-Wei Liu, Yuh-Ju Sun, Hung-Wen Li,Chwan-Deng Hsiao

Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues inGkDnaC are 2–4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI) to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction.


Thursday, December 22, 2011

Low-power nano-optical vortex trapping via plasmonic diabolo nanoantennas

Ju-Hyung Kang, Kipom Kim, Ho-Seok Ee, Yong-Hee Lee, Tae-Young Yoon, Min-Kyo Seo & Hong-Gyu Park

Optical vortex trapping can allow the capture and manipulation of micro- and nanometre-sized objects such as damageable biological particles or particles with a refractive index lower than the surrounding material. However, the quest for nanometric optical vortex trapping that overcomes the diffraction limit remains. Here we demonstrate the first experimental implementation of low-power nano-optical vortex trapping using plasmonic resonance in gold diabolo nanoantennas. The vortex trapping potential was formed with a minimum at 170 nm from the central local maximum, and allowed polystyrene nanoparticles in water to be trapped strongly at the boundary of the nanoantenna. Furthermore, a large radial trapping stiffness, ~0.69 pN nm−1W−1, was measured at the position of the minimum potential, showing good agreement with numerical simulations. This subwavelength-scale nanoantenna system capable of low-power trapping represents a significant step toward versatile, efficient nano-optical manipulations in lab-on-a-chip devices.


Three-dimensional imaging and force characterization of multiple trapped particles in low NA counterpropagating optical traps

T. B. Lindballe, M. V. Kristensen, A. P. Kylling, D. Z. Palima, J. Glückstad, S. R. Keiding, H. Stapelfeldt

An experimental characterization of the three-dimensional (3D) position and force constants, acting on one or multiple trapped polystyrene beads in a weak counterpropagating beams geometry is reported. The 3D position of the trapped particles is tracked by imaging with two synchronized CMOS cameras from two orthogonal views and used to determine the stiffness along all three spatial directions through power spectrum analysis and the equipartition method. For the case of three trapped beads we measure the dependence of the force constants on the counterpropagating beams waist separation. The maximal transverse stiffnesses, is about 0.1 pN/µm per mW at a beam waist separation of 67 µm whereas the longitudinal stiffness is approximately 20 times lower. The experimental findings are in reasonable agreement with a recent physical-geometric optics calculation.


Optical Trapping of a Single Protein

Yuanjie Pang and Reuven Gordon

We experimentally demonstrate the optical trapping of a single bovine serum albumin (BSA) molecule that has a hydrodynamic radius of 3.4 nm, using a double-nanohole in an Au film. The strong optical force in the trap not only stably traps the protein molecule but also unfolds it. The unfolding of the BSA is confirmed by experiments with changing optical power and with changing solution pH. The detection of the trapping event has a signal-to-noise ratio of 33, which shows that the setup is extremely sensitive to detect the presence of a protein, even at the single molecule level.


Tuesday, December 20, 2011

Advances in Experiments and Modeling in Micro- and Nano-Biomechanics: A Mini Review

Mian Long, Masaaki Sato, Chwee Teck Lim, Jianhua Wu, Taiji Adachiand Yasuhiro Inoue

Recent advances in micro- and nano-technologies and high-end computing have enabled the development of new experimental and modeling approaches to study biomechanics at the micro- and nano-scales that were previously not possible. These new cutting-edge approaches are contributing toward our understanding in emerging areas such as mechanobiology and mechanochemistry. Another important potential contribution lies in translational medicine, since biomechanical studies at the cellular and molecular levels have direct relevance in areas such disease diagnosis, nano-medicine and drug delivery. Thus, the developed experimental and modeling approaches are critical in elucidating important mechanistic insights in both basic sciences and clinical treatment. While it is hard to cover all the recent advances in this mini-review, we focus on several important approaches. For experimental techniques, we review the assays involving shear flow, cellular imaging, microbead, microcontact printing, and micropillars at the micro-scale, and micropipette aspiration, optical tweezers, parallel flow chamber, and atomic force microscopy at the nano-scale. In modeling and simulations, we outline the theoretical modeling for actin dynamics in migrating cell and actin-based cell motility in cellular mechanics, as well as the receptor–ligand binding in cell adhesion and the application of free, steered, and flow molecular dynamics simulations in molecular biomechanics. Relevant scientific issues and applications are also discussed.


Progress in coagulation rate measurements of colloidal dispersions

Shenghua Xu and Zhiwei Sun

This article reviews recent advances in coagulation rate measurements of colloidal dispersions, with emphasis on the turbidity method. For turbidity method, measurement of the coagulation rate relies upon the turbidity change resulting from the coagulation process, and the measuring sensitivity significantly depends on particle size and the wavelength used. There exists a “zero sensitivity” blind point for measurement at a specific wavelength, suggesting that such measurements should be performed at a wavelength some distance from the blind point. The major difficulty in determining absolute coagulation rate constant (CRC) by light scattering and turbidity measurements is how to theoretically solve the scattering problem of 2-particle aggregates. The T-matrix method accurately solves this problem, showing its superiority over various earlier theoretical approximations (applicable only to small particles). Results from studies on effects of forward scattering, multiple scattering, etc., provide guidelines for choosing proper particle size and volume fraction for the allowed margin of measurement error. Most of these findings on turbidity methods are also valid or applicable to other scattering methods. Finally, we introduce a new microscopic approach to assess the colloidal stability at individual particle levels, by means of directly observing artificially induced collision with the aid of optical tweezers.


Optical trapping for analytical biotechnology

Praveen C Ashok, Kishan Dholakia

We describe the exciting advances of using optical trapping in the field of analytical biotechnology. This technique has opened up opportunities to manipulate biological particles at the single cell or even at subcellular levels which has allowed an insight into the physical and chemical mechanisms of many biological processes. The ability of this technique to manipulate microparticles and measure pico-Newton forces has found several applications such as understanding the dynamics of biological macromolecules, cell–cell interactions and the micro-rheology of both cells and fluids. Furthermore we may probe and analyse the biological world when combining trapping with analytical techniques such as Raman spectroscopy and imaging.


Wednesday, December 14, 2011

Quasiperiodic Distribution of Rigor Cross-Bridges Along a Reconstituted Thin Filament in a Skeletal Myofibril

Madoka Suzuki, Shin'ichi Ishiwata

Electron microscopy has shown that cross-bridges (CBs) are formed at the target zone that is periodically distributed on the thin filament in striated muscle. Here, by manipulating a single bead-tailed actin filament with optical tweezers, we measured the unbinding events of rigor CBs one by one on the surface of the A-band in rabbit skeletal myofibrils. We found that the spacings between adjacent CBs were not always the same, and instead were 36, 72, or 108 nm. Tropomyosin and troponin did not affect the CB spacing except for a relative increase in the appearance of longer spacing in the presence of Ca2+. In addition, in an in vitro assay where myosin molecules were randomly distributed, were obtained the same spacing, i.e., a multiple of 36 nm. These results indicate that the one-dimensional distribution of CBs matches with the 36-nm half pitch of a long helical structure of actin filaments. A stereospecific model composed of three actin protomers per target zone was shown to explain the experimental results. Additionally, the unbinding force (i.e., the binding affinity) of CBs for the reconstituted thin filaments was found to be larger and smaller relative to that for actin filaments with and without Ca2+, respectively.


Cooperative Responses of Multiple Kinesins to Variable and Constant Loads

D. Kenneth Jamison, Jonathan W. Driver and Michael R. Diehl

Microtubule-dependent transport is most often driven by collections of kinesins and dyneins that function in either a concerted fa-shion or antagonistically. Several lines of evi-dence suggest that cargo transport may not be influenced appreciably by the combined action of multiple kinesins. Yet, as in previous optical trapping experiments, the forces imposed on cargos will vary spatially and temporally in cells depending on a number of local environ-mental factors, and the influence of these con-ditions has been largely overlooked. Here, we characterize the dynamics of structurally-defined complexes containing multiple kinesins under controlled loads of an optical force clamp. While demonstrating that there are ge-neric kinetic barriers that restrict the ability of multiple kinesins to cooperate productively, the spatial and temporal properties of applied loads is found to play an important role in the collective dynamics of multiple-motor systems. We propose these dependencies have implica-tions for intracellular transport processes, es-pecially for bidirectional transport.


Optimisation of a low cost SLM for diffraction efficiency and ghost order suppression

R. Bowman, V. D’Ambrosio, E. Rubino, O. Jedrkiewicz, P. Di Trapani and M. J. Padgett

Spatial Light Modulators (SLMs) are a powerful tool in many optics laboratories, but due to the technology required for their fabrication, they are usually very expensive. Recently some inexpensive devices have been produced, however their phase shift range is less than 2π, leading to a loss of diffraction efficiency for the SLM. We show how to improve the first order diffraction efficiency of such an SLM by adjusting the blazing function, and obtain a 1.5 times increase in first order diffracted power. Even a perfect SLM with 2π phase throw can produce undesired effects in some situations; for example in holographic optical tweezers it is common to find unwanted “ghost spots” near to the array of first-order spots. Modulating the amplitude, by spatially modulating the blazing function, allows us to suppress the ghost spots. This increases the contrast between desired and unwanted spots by more than an order of magnitude.


Monday, December 12, 2011

A photon-driven micromotor can direct nerve fibre growth

Tao Wu, Timo A. Nieminen, Samarendra Mohanty, Jill Miotke, Ronald L. Meyer, Halina Rubinsztein-Dunlop & Michael W. Berns

Axonal path-finding is important in the development of the nervous system, nerve repair and nerve regeneration. The behaviour of the growth cone at the tip of the growing axon determines the direction of axonal growth and migration. We have developed an optical-based system to control the direction of growth of individual axons (nerve fibres) using laser-driven spinning birefringent spheres. One or two optical traps position birefringent beads adjacent to growth cones of cultured goldfish retinal ganglion cell axons. Circularly polarized light with angular momentum causes the trapped bead to spin. This creates a localized microfluidic flow generating an estimated 0.17 pN shear force against the growth cone that turns in response to the shear. The direction of axonal growth can be precisely manipulated by changing the rotation direction and position of this optically driven micromotor. A physical model estimating the shear force density on the axon is described.


Non-specific interactions of CdTe/Cds Quantum Dots with human blood mononuclear cells

Rafael B. Lira, Mariana B. Cavalcanti, Maria A.B.L. Seabra, Diego C.N. Silva, Ademir J. Amaral, Beate S. Santos, Adriana Fontes

In order to study biological events, researchers commonly use methods based on fluorescence. These techniques generally use fluorescent probes, commonly small organic molecules or fluorescent proteins. However, these probes still present some drawbacks, limiting the detection. Semiconductor nanocrystals – Quantum Dots (QDs) – have emerged as an alternative tool to conventional fluorescent dyes in biological detection due to its topping properties – wide absorption cross section, brightness and high photostability. Some questions have emerged about the use of QDs for biological applications. Here, we use optical tools to study non-specific interactions between aqueous synthesized QDs and peripheral blood mononuclear cells. By fluorescence microscopy we observed that bare QDs can label cell membrane in live cells and also label intracellular compartments in artificially permeabilized cells, indicating that non-specific labeling of sub-structures inside the cells must be considered when investigating an internal target by specific conjugation. Since fluorescence microscopy and flow cytometry are complementary techniques (fluorescence microscopy provides a morphological image of a few samples and flow cytometry is a powerful technique to quantify biological events in a large number of cells), in this work we also used flow cytometry to investigate non-specific labeling. Moreover, by using optical tweezers, we observed that, after QDs incubation, zeta potentials in live cells changed to a less negative value, which may indicate that oxidative adverse effects were caused by QDs to the cells.


The formation of actin waves during regeneration after axonal lesion is enhanced by BDNF

Francesco Difato, Hanako Tsushima, Mattia Pesce, Fabio Benfenati, Axel Blau & Evelina Chieregatti

During development, axons of neurons in the mammalian central nervous system lose their ability to regenerate. To study the regeneration process, axons of mouse hippocampal neurons were partially damaged by an UVA laser dissector system. The possibility to deliver very low average power to the sample reduced the collateral thermal damage and allowed studying axonal regeneration of mouse neurons during early days in vitro. Force spectroscopy measurements were performed during and after axon ablation with a bead attached to the axonal membrane and held in an optical trap. With this approach, we quantified the adhesion of the axon to the substrate and the viscoelastic properties of the membrane during regeneration. The reorganization and regeneration of the axon was documented by long-term live imaging. Here we demonstrate that BDNF regulates neuronal adhesion and favors the formation of actin waves during regeneration after axonal lesion.


Thursday, December 8, 2011

Single cell Raman spectroscopy for cell sorting and imaging

Mengqiu Li, Jian Xu, Maria Romero-Gonzalez, Steve A Banwart, Wei E Huang
Single cell Raman spectroscopy (SCRS) is a non-invasive and label-free technology, allowing in vivo and multiple parameter analysis of individual living cells. A single cell Raman spectrum usually contains more than 1000 Raman bands which provide rich and intrinsic information of the cell (e.g. nucleic acids, protein, carbohydrates and lipids), reflecting cellular genotypes, phenotypes and physiological states. A Raman spectrum serves as a molecular ‘fingerprint’ of a single cell, making it possible to differentiate various cells including bacterial, protistan and animal cells without prior knowledge of the cells. However, a key drawback of SCRS is the fact that spontaneous Raman signals are naturally weak; this review discusses recent research progress in significantly enhancing and improving the signal of spontaneous Raman spectroscopy, including resonance Raman spectroscopy (RRS), coherent anti-Stokes Raman spectroscopy (CARS), stimulated Raman spectroscopy (SRS) and surface enhanced Raman scattering (SERS). This review focuses on the biotechnological development and the associated applications of SCRS, including Raman activated cell sorting (RACS) and Raman imaging and mapping.


Tuesday, December 6, 2011

Phase-Transition-like Properties of Double-Beam Optical Tweezers

A. B. Stilgoe, N. R. Heckenberg, T. A. Nieminen, and H. Rubinsztein-Dunlop

We report on double-beam optical tweezers that undergo previously unknown phase-transition-like behavior resulting in the formation of more optical traps than the number of beams used to create them. We classify the optical force fields which produce multiple traps for a double-beam system including the critical behavior. This effect is demonstrated experimentally in orthogonally polarized (noninterfering) dual-beam optical tweezers for a silica particle of 2.32  μm diameter. Phase transitions of multiple beam trapping systems have implications for hopping rates between traps and detection of forces between biomolecules using dual-beam optical tweezers. It is an example of a novel dynamic system with multiple states where force fields undergo a series of sign inversions as a function of parameters such as size and beam separation.


Extending vaterite microviscometry to ex vivo blood vessels by serial calibration

Samir G. Shreim, Earl Steward, and Elliot L. Botvinick

The endothelial glycocalyx layer is a ~2 µm thick glycosaminoglycan rich pericellular matrix expressed on the luminal surface of vascular endothelial cells, which has implications in vessel mechanics and mechanotransduction. Despite its role in vascular physiology, no direct measurement has of yet been made of vessel glycocalyx material properties. Vaterite microviscometry is a laser tweezers based microrheological method, which has been previously utilized to measure the viscosity of linear and complex fluids under flow. This form of microrheology has until now relied on complete recollection of the forward scattered light. Here we present a novel method to extend vaterite microviscometry to relatively thick samples. We validate our method and its assumptions and measure the apparent viscosity as a function of distance from the vascular endothelium. We observe a differential response in conditions designed to preserve the EGL in comparison to those designed to collapse it.


Monday, December 5, 2011

Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament

Kimihide Hayakawa, Hitoshi Tatsumi, and Masahiro Sokabe

Intracellular and extracellular mechanical forces affect the structure and dynamics of the actin cytoskeleton. However, the underlying molecular and biophysical mechanisms, including how mechanical forces are sensed, are largely unknown. Actin-depolymerizing factor/cofilin proteins are actin-modulating proteins that are ubiquitously distributed in eukaryotes, and they are the most likely candidate as proteins to drive stress fiber disassembly in response to changes in tension in the fiber. In this study, we propose a novel hypothesis that tension in an actin filament prevents the filament from being severed by cofilin. To test this, we placed single actin filaments under tension using optical tweezers. When a fiber was tensed, it was severed after the application of cofilin with a significantly larger delay in comparison with control filaments suspended in solution. The binding rate of cofilin to an actin bundle decreased when the bundle was tensed. These results suggest that tension in an actin filament reduces the cofilin binding, resulting in a decrease in its effective severing activity.


Bimolecular integrin–ligand interactions quantified using peptide-functionalized dextran-coated microparticles

Jessie E. P. Sun, Justin Vranic, Russell J. Composto, Craig Streu, Paul C. Billings, Joel S. Bennett, John W. Weisel and Rustem I. Litvinov
Integrins play a key role in cell–cell and cell–matrix interactions. Artificial surfaces grafted with integrin ligands, mimicking natural interfaces, have been used to study integrin-mediated cell adhesion. Here we report the use of a new chemical engineering technology in combination with single-molecule nanomechanical measurements to quantify peptide binding to integrins. We prepared latex beads with covalently-attached dextran. The beads were then functionalized with the bioactive peptides, cyclic RGDFK (cRGD) and the fibrinogen γC-dodecapeptide (H12), corresponding to the active sites for fibrinogen binding to the platelet integrin αIIbβ3. Using optical tweezers-based force spectroscopy to measure non-specific protein–protein interactions, we found the dextran-coated beads nonreactive towards fibrinogen, thus providing an inert platform for biospecific modifications. Using periodate oxidation followed by reductive amination, we functionalized the bead-attached dextran with either cRGD or H12 and used the peptide-grafted beads to measure single-molecule interactions with the purified αIIbβ3. Bimolecular force spectroscopy revealed that the peptide-functionalized beads were highly and specifically reactive with the immobilized αIIbβ3. Further, the cRGD- and H12-functionalized beads displayed a remarkable interaction profile with a bimodal force distribution up to 90 pN. The cRGD–αIIbβ3 interactions had greater binding strength than that of H12–αIIbβ3, indicating that they are more stable and resistant mechanically, consistent with the platelet reactivity of RGD-containing ligands. Thus, the results reported here describe the mechanistic characteristics of αIIbβ3–ligand interactions, confirming the utility of peptide-functionalized latex beads for the quantitative analysis of molecular recognition.


Enhanced Optical Trapping and Arrangement of Nano-Objects in a Plasmonic Nanocavity

Chang Chen, Mathieu L. Juan, Yi Li, Guido Maes, Gustaaf Borghs, Pol Van Dorpe, and Romain Quidant

Gentle manipulation of micrometer-sized dielectric objects with optical forces has found many applications in both life and physical sciences. To further extend optical trapping toward the true nanometer scale, we present an original approach combining self-induced back action (SIBA) trapping with the latest advances in nanoscale plasmon engineering. The designed resonant trap, formed by a rectangular plasmonic nanopore, is successfully tested on 22 nm polystyrene beads, showing both single- and double-bead trapping events. The mechanism responsible for the higher stability of the double-bead trapping is discussed, in light of the statistical analysis of the experimental data and numerical calculations. Furthermore, we propose a figure of merit that we use to quantify the achieved trapping efficiency and compare it to prior optical nanotweezers. Our approach may open new routes toward ultra-accurate immobilization and arrangement of nanoscale objects, such as biomolecules.


Friday, December 2, 2011

Dark-field optical tweezers for nanometrology of metallic nanoparticles

Kellie Pearce, Fan Wang, and Peter J. Reece

Applications of metallic nanoparticles are based on their strongly size-dependent optical properties. We present a method for combining optical tweezers with dark field microscopy that allows measurement of localised surface plasmon resonance (LSPR) spectra on single isolated nanoparticles without compromising the strength of the optical trap. Using this spectroscopic information in combination with measurements of trap stiffness and hydrodynamic drag, allows us to determine the dimensions of the trapped nanoparticles. A relationship is found between the measured diameters of the particles and the peak wavelengths of their spectra. Using this method we may also resolve complex spectra of particle aggregation and interactions within the tweezers.


Probing ribosomal protein-RNA interactions with an external force

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

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


Thursday, December 1, 2011

Raman spectroscopy and microscopy based on mechanical force detection

I. Rajapaksa and H. Kumar Wickramasinghe

The Raman effect is typically observed by irradiating a sample with an intense light source and detecting the minute amount of frequency shifted scattered light. We demonstrate that Raman molecular vibrational resonances can be detected directly through an entirely different mechanism—namely, a force measurement. We create a force interaction through optical parametric down conversion between stimulated, Raman excited, molecules on a surface and a cantilevered nanometer scale probe tip brought very close to it. Spectroscopy and microscopy on clusters of molecules have been performed. Single molecules within such clusters are clearly resolved in the Raman micrographs. The technique can be readily extended to perform pump probe experiments for measuring inter- and intramolecular couplings and conformational changes at the single molecule level.


Plasmonic Nanoparticle Chain in a Light Field: A Resonant Optical Sail

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

Optical trapping and driving of small objects has become a topic of increasing interest in multidisciplinary sciences. We propose to use a chain made of metallic nanoparticles as a resonant light sail, attached by one end point to a transparent object and propelling it by the use of electromagnetic radiation. Driving forces exerted on the chain are theoretically studied as a function of radiation’s wavelength and chain’s alignments with respect to the direction of radiation. Interestingly, there is a window in the frequency spectrum in which null–torque equilibrium configuration, with minimum geometric cross section, corresponds to a maximum in the driving force.