Wednesday, April 26, 2017

Revealing the subfemtosecond dynamics of orbital angular momentum in nanoplasmonic vortices

G. Spektor, D. Kilbane, A. K. Mahro, B. Frank, S. Ristok, L. Gal, P. Kahl, D. Podbiel, S. Mathias, H. Giessen, F.-J. Meyer zu Heringdorf, M. Orenstein, M. Aeschlimann

The ability of light to carry and deliver orbital angular momentum (OAM) in the form of optical vortices has attracted much interest. The physical properties of light with a helical wavefront can be confined onto two-dimensional surfaces with subwavelength dimensions in the form of plasmonic vortices, opening avenues for thus far unknown light-matter interactions. Because of their extreme rotational velocity, the ultrafast dynamics of such vortices remained unexplored. Here we show the detailed spatiotemporal evolution of nanovortices using time-resolved two-photon photoemission electron microscopy. We observe both long- and short-range plasmonic vortices confined to deep subwavelength dimensions on the scale of 100 nanometers with nanometer spatial resolution and subfemtosecond time-step resolution. Finally, by measuring the angular velocity of the vortex, we directly extract the OAM magnitude of light.


AMPK negatively regulates tensin-dependent integrin activity

Maria Georgiadou, Johanna Lilja, Guillaume Jacquemet, Camilo Guzmán, Maria Rafaeva, Charlotte Alibert, Yan Yan, Pranshu Sahgal, Martina Lerche,  Jean-Baptiste Manneville, Tomi P. Mäkelä, Johanna Ivaska

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin–ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a β1-integrin inhibitor in fibroblasts. Loss of AMPK promotes β1-integrin activity, the formation of centrally located active β1-integrin– and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases β1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind β1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.


Single Molecule Investigation of Kinesin-1 Motility Using Engineered Microtubule Defects

Michael W. Gramlich, Leslie Conway, Winnie H. Liang, Joelle A. Labastide, Stephen J. King, Jing Xu & Jennifer L. Ross

The structure of the microtubule is tightly regulated in cells via a number of microtubule associated proteins and enzymes. Microtubules accumulate structural defects during polymerization, and defect size can further increase under mechanical stresses. Intriguingly, microtubule defects have been shown to be targeted for removal via severing enzymes or self-repair. The cell’s control in defect removal suggests that defects can impact microtubule-based processes, including molecular motor-based intracellular transport. We previously demonstrated that microtubule defects influence cargo transport by multiple kinesin motors. However, mechanistic investigations of the observed effects remained challenging, since defects occur randomly during polymerization and are not directly observable in current motility assays. To overcome this challenge, we used end-to-end annealing to generate defects that are directly observable using standard epi-fluorescence microscopy. We demonstrate that the annealed sites recapitulate the effects of polymerization-derived defects on multiple-motor transport, and thus represent a simple and appropriate model for naturally-occurring defects. We found that single kinesins undergo premature dissociation, but not preferential pausing, at the annealed sites. Our findings provide the first mechanistic insight to how defects impact kinesin-based transport. Preferential dissociation on the single-molecule level has the potential to impair cargo delivery at locations of microtubule defect sites in vivo.


Single-molecule observation of DNA compaction by meiotic protein SYCP3

Johanna L Syrjänen Iddo Heller Andrea Candelli Owen R Davies Erwin J G Peterman Gijs J L Wuite Luca Pellegrini

In a previous paper (Syrjänen et al., 2014), we reported the first structural characterisation of a synaptonemal complex (SC) protein, SYCP3, which led us to propose a model for its role in chromosome compaction during meiosis. As a component of the SC lateral element, SYCP3 has a critical role in defining the specific chromosome architecture required for correct meiotic progression. In the model, the reported compaction of chromosomal DNA caused by SYCP3 would result from its ability to bridge distant sites on a DNA molecule with the DNA-binding domains located at each end of its strut-like structure. Here, we describe a single-molecule assay based on optical tweezers, fluorescence microscopy and microfluidics that, in combination with bulk biochemical data, provides direct visual evidence for our proposed mechanism of SYCP3-mediated chromosome organisation.


Formation of Large Hypericin Aggregates in Giant Unilamellar Vesicles—Experiments and Modeling

Jaroslava Joniova, Matúš Rebič, Alena Strejčková, Veronika Huntosova, Jana Staničová, Daniel Jancura, Pavol Miskovsky, Gregor Bánó

The incorporation of hypericin (Hyp) from aqueous solutions into giant unilamellar vesicle (GUV) membranes has been studied experimentally and by means of kinetic Monte Carlo modeling. The time evolution of Hyp fluorescence originating from Hyp monomers dissolved in the GUV membrane has been recorded by confocal microscopy and while trapping individual GUVs in optical tweezers. It was shown that after reaching a maximum, the fluorescence intensity gradually decreased toward longer times. Formation of oversized Hyp clusters has been observed on the GUV surface at prolonged time. A simplified kinetic Monte Carlo model is presented to follow the aggregation/dissociation processes of Hyp molecules in the membrane. The simulation results reproduced the basic experimental observations: the scaling of the characteristic fluorescence decay time with the vesicle diameter and the buildup of large Hyp clusters in the GUV membrane.


Wednesday, April 19, 2017

Photonic and Plasmonic Nanotweezing of Nano- and Microscale Particles

Donato Conteduca, Francesco Dell’Olio, Thomas F. Krauss, and Caterina Ciminelli

The ability to manipulate and sense biological molecules is important in many life science domains, such as single-molecule biophysics, the development of new drugs and cancer detection. Although the manipulation of biological matter at the nanoscale continues to be a challenge, several types of nanotweezers based on different technologies have recently been demonstrated to address this challenge. In particular, photonic and plasmonic nanotweezers are attracting a strong research effort especially because they are efficient and stable, they offer fast response time, and avoid any direct physical contact with the target object to be trapped, thus preventing its disruption or damage. In this paper, we critically review photonic and plasmonic resonant technologies for biomolecule trapping, manipulation, and sensing at the nanoscale, with a special emphasis on hybrid photonic/plasmonic nanodevices allowing a very strong light–matter interaction. The state-of-the-art of competing technologies, e.g., electronic, magnetic, acoustic and carbon nanotube-based nanotweezers, and a description of their applications are also included.


Dynamics of an optically bound structure made of particles of unequal sizes

Vítězslav Karásek, Martin Šiler, Oto Brzobohatý, and Pavel Zemánek

This theoretical study based on the coupled dipoles model focuses on the dynamics of two optically bound dielectric spheres of unequal sizes confined in counter-propagating incoherent Bessel beams. We analyzed the relative motion of the particles with respect to each other and defined conditions where they form a stable optically bound structure (OBS). We also investigated the motion of the center of mass of the OBS and found that its direction depends on the particle separation in the structure. Besides the optical interaction between objects, we also considered a hydrodynamic coupling in order to obtain more precise results for moving an OBS.


Computational inverse design of non-intuitive illumination patterns to maximize optical force or torque

Yoonkyung E. Lee, Owen D. Miller, M. T. Homer Reid, Steven G. Johnson, and Nicholas X. Fang

This paper aims to maximize optical force or torque on arbitrary micro- and nanoscale objects using numerically optimized structured illumination. By developing a numerical framework for computer-automated design of 3d vector-field illumination, we demonstrate a 20-fold enhancement in optical torque per intensity over circularly polarized plane wave on a model plasmonic particle. The nonconvex optimization is efficiently performed by combining a compact cylindrical Bessel basis representation with a fast boundary element method and a standard derivative-free, local optimization algorithm. We analyze the optimization results for 2000 random initial configurations, discuss the tradeoff between robustness and enhancement, and compare the different effects of multipolar plasmon resonances on enhancing force or torque. All results are obtained using open-source computational software available online.


Characterization of single airborne particle extinction using the tunable optical trap-cavity ringdown spectroscopy (OT-CRDS) in the UV

Zhiyong Gong, Yong-Le Pan, and Chuji Wang

We integrated a rigid optical trap into a tunable pulsed cavity ringdown spectroscopy (OT-CRDS) system to characterize the extinction of single airborne particles in the UV spectral region (306-315 nm). Single solid particles from a multi-walled carbon nanotube (MWCNT), Bermuda grass smut spore, carbon microsphere, and blackened polyethylene microsphere were trapped in air based on the photophoretic force. The improved OT-CRDS system was highly sensitive and able to resolve extinctions of single particles from different materials and sizes at a given wavelength. Further, we successfully manipulated the number of particles, e.g., 1, 2 or more particles, in the trap and measured their distinguishable extinctions using the OT-CRDS. We also show that the particle size and extinction have a good linear correlation from the measurements of 24 single MWCNT particles. Material- and wavelength-dependent extinctions of the four types of airborne particles were also characterized. Results reveal that single airborne particles regardless of their differences in material and size, due to their heterogeneous morphology, have individual-particle dependent extinctions and that dependence can be resolved and characterized using the OT-CRDS technique.


Thermometry of levitated nanoparticles in a hybrid electro-optical trap

E B Aranas, P Z G Fonseca, P F Barker and T S Monteiro

There have been recent rapid developments in stable trapping of levitated nanoparticles in high vacuum. Cooling of nanoparticles, from phonon occupancies of 107 down to $\simeq \,100\mbox{--}1000$ phonons, have already been achieved by several groups. Prospects for quantum ground-state cooling seem extremely promising. Cavity-cooling without added stabilisation by feedback cooling remains challenging, but trapping at high vacuum in a cavity is now possible through the addition of a Paul trap. However, the Paul trap has been found to qualitatively modify the cavity output spectrum, with the latter acquiring an atypical 'split-sideband' structure, of different form from the displacement spectrum, and which depends on N, the optical well at which the particle localises. In the present work we investigate the N-dependence of the dynamics, in particular with respect to thermometry: we show that in strong cooling regions $N\gtrsim 100$, the temperature may still be reliably inferred from the cavity output spectra. We also explain the N-dependence of the mechanical frequencies and optomechanical coupling showing that these may be accurately estimated. We present a simple 'fast-cavity' model for the cavity output and test all our findings against full numerical solutions of the nonlinear stochastic equations of motion for the system.


Tuesday, April 18, 2017

Laser-assisted biofabrication in tissue engineering and regenerative medicine

Sangmo Koo, Samantha M. Santoni, Bruce Z. Gao, Costas P. Grigoropoulos and Zhen Ma

Controlling the spatial arrangement of biomaterials and living cells provides the foundation for fabricating complex biological systems. Such level of spatial resolution (less than 10 µm) is difficult to be obtained through conventional cell processing techniques, which lack the precision, reproducibility, automation, and speed required for the rapid fabrication of engineered tissue constructs. Recently, laser-assisted biofabrication techniques are being intensively developed with the use of computer-aided processes for patterning and assembling both living and nonliving materials with prescribed 2D or 3D organization. In this review, we discuss laser-assisted fabrication methods, including laser tweezers, multi-photon polymerization, laser-induced forward transfer (LIFT), matrix assisted pulsed laser evaporation (MAPLE), and laser ablation as well as their applications in biological science and biomedical engineering. These advanced technologies enable the precise manipulation of in vitro cellular microenvironments and the ability to engineer functional tissue constructs with high complexity and heterogeneity, which serve in regenerative medicine, pharmacology, and basic cell biology studies.


Optical Manipulation of Dielectric Nanoparticles with Au Micro-racetrack Resonator by Constructive Interference of Surface Plasmon Waves

Mingrui Yuan, Lin Cheng, Pengfei Cao, Xu Li, Xiaodong He, Xiaoping Zhang

We design a gold micro-racetrack resonator (Au-MRR) which can tightly trap and drive the dielectric nanoparticle to rotate around the circuit of racetrack with an adjustable velocity. Since the surface plasmon waves can be excited and obey the resonance condition of the Au-MRR, the optics force can be strengthened observably due to the resonance. The optical forces applied on dielectric nanoparticle are discussed by utilizing the Maxwell’s stress tensor integration with a numerical finite element method. The depth of longitudinal trapping potential well in the Au-MRR is four times as large as that of a straight waveguide. At the same level of input power, the velocity of particle with radius of 50 nm driven by optical forces on Au-MRR is 200 times larger than that on a straight waveguide. Further, we explore the motion behavior of single nanoparticle lies on different position of Au-MRR, which can provide the details to trap and manipulate multiple nanoparticles and predict their trace of movement. This optimum geometry of Au-MRR allows further enhancement of the optical forces which is expected to realize all-optical on-chip manipulation of nanoparticles, biomolecules, and many other nanomanipulation applications.


Omnidirectional Transport in Fully Reconfigurable Two Dimensional Optical Ratchets

Alejandro V. Arzola, Mario Villasante-Barahona, Karen Volke-Sepúlveda, Petr Jákl, and Pavel Zemánek

A fully reconfigurable two-dimensional (2D) rocking ratchet system created with holographic optical micromanipulation is presented. We can generate optical potentials with the geometry of any Bravais lattice in 2D and introduce a spatial asymmetry with arbitrary orientation. Nontrivial directed transport of Brownian particles along different directions is demonstrated numerically and experimentally, including on axis, perpendicular, and oblique with respect to an unbiased ac driving. The most important aspect to define the current direction is shown to be the asymmetry and not the driving orientation, and yet we show a system in which the asymmetry orientation of each potential well does not coincide with the transport direction, suggesting an additional symmetry breaking as a result of a coupling with the lattice configuration. Our experimental device, due to its versatility, opens up a new range of possibilities in the study of nonequilibrium dynamics at the microscopic level.


Monday, April 17, 2017

Effect of lipopolysaccharide O-side chains on the adhesiveness of Yersinia pseudotuberculosis to J774 macrophages as revealed by optical tweezers

A. A. Byvalov, V. L. KononenkoI. V. Konyshev

A method has been developed for the quantitative estimation of the binding force of a model microsphere with a eukaryocyte based on the optical trap in order to study the molecular mechanism of adhesion between an individual bacterium and a host cell. The substantial role of LPS O-side chains in the adhesiveness of Yersinia pseudotuberculosis 1b to J774 macrophages has been revealed with the use of a set of microspheres functionalized with lipopolysaccharide (LPS) preparations and antibodies with different specificities. The results indicate the significance of the O-antigen as a pathogenicity factor of Y. pseudotuberculosis in colonization of a macroorganism. The developed methodical approaches can be applied to the study of molecular mechanisms of the pathogenesis of pseudotuberculosis and other infectious diseases to improve antiepidemic service.


Laser-mediated rupture of chlamydial inclusions triggers pathogen egress and host cell necrosis

Markus C. Kerr, Guillermo A. Gomez, Charles Ferguson, Maria C. Tanzer, James M. Murphy, Alpha S. Yap, Robert G. Parton, Wilhelmina M. Huston & Rohan D Teasdale

Remarkably little is known about how intracellular pathogens exit the host cell in order to infect new hosts. Pathogenic chlamydiae egress by first rupturing their replicative niche (the inclusion) before rapidly lysing the host cell. Here we apply a laser ablation strategy to specifically disrupt the chlamydial inclusion, thereby uncoupling inclusion rupture from the subsequent cell lysis and allowing us to dissect the molecular events involved in each step. Pharmacological inhibition of host cell calpains inhibits inclusion rupture, but not subsequent cell lysis. Further, we demonstrate that inclusion rupture triggers a rapid necrotic cell death pathway independent of BAK, BAX, RIP1 and caspases. Both processes work sequentially to efficiently liberate the pathogen from the host cytoplasm, promoting secondary infection. These results reconcile the pathogen's known capacity to promote host cell survival and induce cell death.


Real-time monitoring and visualization of the multi-dimensional motion of an anisotropic nanoparticle

Gi-Hyun Go, Seungjin Heo, Jong-Hoi Cho, Yang-Seok Yoo, MinKwan Kim, Chung-Hyun Park & Yong-Hoon Cho

As interest in anisotropic particles has increased in various research fields, methods of tracking such particles have become increasingly desirable. Here, we present a new and intuitive method to monitor the Brownian motion of a nanowire, which can construct and visualize multi-dimensional motion of a nanowire confined in an optical trap, using a dual particle tracking system. We measured the isolated angular fluctuations and translational motion of the nanowire in the optical trap, and determined its physical properties, such as stiffness and torque constants, depending on laser power and polarization direction. This has wide implications in nanoscience and nanotechnology with levitated anisotropic nanoparticles.


Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbβ3

Olga Kononova, Rustem I. Litvinov, Dmitry S. Blokhin, Vladimir V. Klochkov, John W. Weisel, Joel S. Bennett, and Valeri Barsegov

Binding of soluble fibrinogen to the activated conformation of the integrin αIIbβ3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. However, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbβ3 and make substantial contributions to αIIbβ3 binding when fibrinogen is immobilized and when it is converted to fibrin. Here, we employed optical trap-based nanomechanical measurements and computational molecular modeling to determine the kinetics, energetics, and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbβ3. Docking analysis revealed that NMR-determined solution structures of cRGDFK and γC-12 bind to both the open and closed αIIbβ3 conformers at the interface between the αIIb β-propeller domain and the β3 βI domain. The nanomechanical measurements revealed that cRGDFK binds to αIIbβ3 at least as tightly as γC-12. A subsequent analysis of molecular force profiles and the number of peptide−αIIbβ3 binding contacts revealed that both peptides form stable bimolecular complexes with αIIbβ3 that dissociate in the 60–120 pN range. The Gibbs free energy profiles of the αIIbβ3–peptide complexes revealed that the overall stability of the αIIbβ3-cRGDFK complex was comparable with that of the αIIbβ3−γC-12 complex. Thus, these results provide a mechanistic explanation for previous observations that RGD- and AGDV-containing peptides are both potent inhibitors of the αIIbβ3–fibrinogen interactions and are consistent with the observation that RGD motifs, in addition to AGDV, support interaction of αIIbβ3 with immobilized fibrinogen and fibrin.


Two-laser optical tweezers with a blinking beam

Weronika Lamperska, Jan Masajada, Sławomir Drobczyński, Paweł Gusin

We report on a two-laser holographic optical tweezers setup and present its two major advantages over single-laser one. First, the trap stiffness of a weak trapping beam can be measured with a considerable accuracy. Second, a novel method of examining local viscosity of fluid is proposed. Both measurements are performed based on forcing the oscillations of a microscopic polystyrene bead placed between two optical traps. The two beams are generated by separate laser sources and therefore their trapping power can vary. Moreover, a stronger trap ‘blinks’, modulated by an electronic shutter. The blinking frequency can be precisely adjusted to the experimental conditions, which results in high accuracy of the measurements.


Onset of particle trapping and release via acoustic bubbles

Yun Chen, Zecong Fang, Brett Merritt, Dillon Strack, Jie Xu and Sungyon Lee

Trapping and sorting of micro-sized objects is one important application of lab on a chip devices, with the use of acoustic bubbles emerging as an effective, non-contact method. Acoustically actuated bubbles are known to exert a secondary radiation force (FSR) on micro-particles and stabilize them on the bubble surface, when this radiation force exceeds the external hydrodynamic forces that act to keep the particles in motion. While the theoretical expression of FSR has been derived by Nyborg decades ago, no direct experimental validation of this force has been performed, and the relationship between FSR and the bubble's ability to trap particles in a given lab on a chip device remains largely empirical. In order to quantify the connection between the bubble oscillation and the resultant FSR, we experimentally measure the amplitude of bubble oscillations that give rise to FSR and observe the trapping and release of a single microsphere in the presence of the mean flow at the corresponding acoustic parameters using an acoustofluidic device. By combining well-developed theories that connect bubble oscillations to the acoustic actuation, we derive the expression for the critical input voltage that leads to particle release into the flow, in good agreement with the experiments.