Tuesday, December 25, 2012

Quantitative optical trapping on single organelles in cell extract

Pradeep Barak, Ashim Rai, Priyanka Rai & Roop Mallik

We have developed an optical trapping method to precisely measure the force generated by motor proteins on single organelles of unknown size in cell extract. This approach, termed VMatch, permits the functional interrogation of native motor complexes. We apply VMatch to measure the force, number and activity of kinesin-1 on motile lipid droplets isolated from the liver of normally fed and food-deprived rats.

Friday, December 21, 2012

PilT2 enhances the speed of gonococcal type IV pilus retraction and of twitching motility

Rainer Kurre, Andrea Höne, Martin Clausen, Claudia Meel, Berenike Maier

Type IV pilus (T4P) dynamics is important for various bacterial functions including host cell interaction, surface motility, and horizontal gene transfer. T4P retract rapidly by depolymerization, generating large mechanical force. The gene that encodes the pilus retraction ATPase PilT has multiple paralogues, whose number varies between different bacterial species, but their role in regulating physical parameters of T4P dynamics remains unclear. Here, we address this question in the human pathogen Neisseria gonorrhoeae, which possesses two pilT paralogues, namelypilT2 and pilU. We show that the speed of twitching motility is strongly reduced in a pilT2 deletion mutant, while directional persistence time and sensitivity of speed to oxygen are unaffected. Using laser tweezers, we found that the speed of single T4P retraction was reduced by a factor of ≈ 2 in a pilT2 deletion strain, whereas pilU deletion showed a minor effect. The maximum force and the probability for switching from retraction to elongation under application of high force were not significantly affected. We conclude that the physical parameters of T4P are fine-tuned through PilT2.

An optically actuated surface scanning probe

D. B. Phillips, G. M. Gibson, R. Bowman, M. J. Padgett, S. Hanna, D. M. Carberry, M. J. Miles, and S. H. Simpson

We demonstrate the use of an extended, optically trapped probe that is capable of imaging surface topography with nanometre precision, whilst applying ultra-low, femto-Newton sized forces. This degree of precision and sensitivity is acquired through three distinct strategies. First, the probe itself is shaped in such a way as to soften the trap along the sensing axis and stiffen it in transverse directions. Next, these characteristics are enhanced by selectively position clamping independent motions of the probe. Finally, force clamping is used to refine the surface contact response. Detailed analyses are presented for each of these mechanisms. To test our sensor, we scan it laterally over a calibration sample consisting of a series of graduated steps, and demonstrate a height resolution of ∼ 11 nm. Using equipartition theory, we estimate that an average force of only ∼ 140 fN is exerted on the sample during the scan, making this technique ideal for the investigation of delicate biological samples.


Mapping the local dielectric response at the nanoscale by means of plasmonic force spectroscopy

Francesco De Angelis, Remo Proietti Zaccaria, and Enzo Di Fabrizio
At the present, the local optical properties of nanostructured materials are difficult to be measured by available instrumentation. We investigated the capability of plasmonic force spectroscopy of measuring the optical response at the nanoscale. The proposed technique is based on force measurements performed by combining Atomic Force Microscopy, or optical tweezers, and adiabatic compression of surface plasmon polaritons. We show that the optical forces, caused by the plasmonic field, depend on the local response of the substrates and, in principle, allow probing both the real and the imaginary part of the local permittivity with a spatial resolution of few nanometers.

Angular orientation of nanorods using nanophotonic tweezers

Pilgyu Kang, Xavier Serey, Yih-Fan Chen, and David Erickson

Near-field optical techniques have enabled the trapping, transport, and handling of nanoscopic materials much smaller than what can be manipulated with traditional optical tweezers. Here we extend the scope of what is possible by demonstrating angular orientation and rotational control of both biological and nonbiological nanoscale rods using photonic crystal nanotweezers. In our experiments, single microtubules (diameter 25 nm, length 8 μm) and multiwalled carbon nanotubes (outer diameter 110–170 nm, length 5 μm) are rotated by the optical torque resulting from their interaction with the evanescent field emanating from these devices. An angular trap stiffness of κ = 92.8 pN·nm/rad2·mW is demonstrated for the microtubules, and a torsional spring constant of 22.8 pN·nm/rad2·mW is measured for the nanotubes. We expect that this new capability will facilitate the development of high precision nanoassembly schemes and biophysical studies of bending strains of biomolecules.

Thursday, December 20, 2012

Trapping volume control in optical tweezers using cylindrical vector beams

S. E. Skelton, M. Sergides, R. Saija, M. A. Iatì, O. M. Maragó, and P. H. Jones
We present the result of an investigation into the optical trapping of spherical microparticles using laser beams with a spatially inhomogeneous polarization direction [cylindrical vector beams (CVBs)]. We perform three-dimensional tracking of the Brownian fluctuations in the position of a trapped particle and extract the trap spring constants. We characterize the trap geometry by the aspect ratio of spring constants in the directions transverse and parallel to the beam propagation direction and evaluate this figure of merit as a function of polarization angle. We show that the additional degree of freedom present in CVBs allows us to control the optical trap strength and geometry by adjusting only the polarization of the trapping beam. Experimental results are compared with a theoretical model of optical trapping using CVBs derived from electromagnetic scattering theory in the T-matrix framework.


Flow-dependent double-nanohole optical trapping of 20 nm polystyrene nanospheres

Ana Zehtabi-Oskuie, Jarrah Gerald Bergeron, and Reuven Gordon

We study the influence of fluid flow on the ability to trap optically a 20 nm polystyrene particle from a stationary microfluidic environment and then hold it against flow. Increased laser power is required to hold nanoparticles as the flow rate is increased, with an empirical linear dependence of 1 μl/(min×mW). This is promising for the delivery of additional nanoparticles to interact with a trapped nanoparticle; for example, to study protein-protein interactions, and for the ability to move the trapped particle in solution from one location to another.

Mapping 3D Focal Intensity Exposes the Stable Trapping Positions of Single Nanoparticles

Anders Kyrsting , Poul M. Bendix , and Lene B. Oddershede
The photonic interactions between a focused Gaussian laser beam and a nanoscopic particle are highly dependent on exact particle location and focal intensity distribution. So far, the 3D focal intensity distribution and the preferred position of a nanoparticle confined within the focal region were only theoretically predicted. Here, we directly map the three-dimensional focal intensity distribution, quantify stable trapping positions, and prove that certain sizes of nanoparticles stably trap in front of the focus.


Wednesday, December 19, 2012

Antibody Binding to Cryptococcus neoformans Impairs Budding by Altering Capsular Mechanical Properties

Radames J. B. Cordero, Bruno Pontes, Susana Frases, Antonio S. Nakouzi, Leonardo Nimrichter, Marcio L. Rodrigues, Nathan B. Viana and Arturo Casadevall

Abs to microbial capsules are critical for host defense against encapsulated pathogens, but very little is known about the effects of Ab binding on the capsule, apart from producing qualitative capsular reactions (“quellung” effects). A problem in studying Ab–capsule interactions is the lack of experimental methodology, given that capsules are fragile, highly hydrated structures. In this study, we pioneered the use of optical tweezers microscopy to study Ab–capsule interactions. Binding of protective mAbs to the capsule of the fungal pathogen Cryptococcus neoformans impaired yeast budding by trapping newly emerging buds inside the parental capsule. This effect is due to profound mAb-mediated changes in capsular mechanical properties, demonstrated by a concentration-dependent increase in capsule stiffness. This increase involved mAb-mediated cross-linking of capsular polysaccharide molecules. These results provide new insights into Ab-mediated immunity, while suggesting a new nonclassical mechanism of Ab function, which may apply to other encapsulated pathogens. Our findings add to the growing body of evidence that Abs have direct antimicrobial functions independent of other components of the immune system.

Lateral migration of an elastic capsule by optical force in a uniform flow

Cheong Bong Chang, Wei-Xi Huang, and Hyung Jin Sung

The lateral migration of an elastic capsule under an optical force in a uniform flow was studied to show the separation characteristics according to the elastic properties in the cross-type optical separator. The initially spherical capsule was moved through the fluid flow using a laser beam with a Gaussian distribution focused along the direction normal to the flow device surface. To simulate such a system, a penalty immersed boundary method was adopted to enable fluid-membrane coupling, and a dynamic ray tracing method was applied to the optical force calculation. The effects of the elastic properties of the capsule membrane (the surface Young's modulus and the bending modulus) on the lateral migration were studied. By increasing the surface Young's modulus, the capsule deformed less and the migration distance increased; however, buckling occurred in the capsule with a high surface Young's modulus. Buckling could be suppressed by increasing the bending rigidity. The effects of the flow velocity and the laser beam power were also examined. In the simulation, the S number, i.e., the ratio of the optical force to the viscous force, was adjusted by decreasing the flow velocity or increasing the laser beam power. The migration distance increased as the S number increased, and a constant lateral migration distance was obtained for a rigid particle for a given S number. An elastic capsule under conditions intermediate between a fixed flow velocity and a fixed laser beam power, however, did not yield a constant lateral migration distance due to the extent of the deformation in the different situations. To predict the lateral migration distance of an elastic capsule, a nondimensional parameter, Se, was defined to include the effects of the optical force, the elastic force, and the fluid viscous force. A unified tendency of the lateral migration distance with Se was obtained for capsules with intermediate elasticity, by varying either the flow velocity or the laser beam power.

Quantitative Assessment of the Interplay Between DNA Elasticity and Cooperative Binding of Ligands

L. Siman, I. S. S. Carrasco, J. K. L. da Silva, M. C. de Oliveira, M. S. Rocha, and O. N. Mesquita
Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We propose a methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation. We obtain an expression for the effective persistence length as a function of the total ligand concentration, which we apply to our data of the DNA-cationic β-cyclodextrin and to the DNA-HU protein data available in the literature, determining the values of the local persistence lengths, the dissociation constant, and the degree of cooperativity for each set of data. In both cases the persistence length behaves nonmonotonically as a function of ligand concentration and based on the results obtained we discuss some physical aspects of the interplay between DNA elasticity and cooperative binding of ligands.


Dynamical instabilities of a Brownian particle in weak adhesion

Deepak Kumar, Shankar Ghosh, and S. Bhattacharya

Dynamical processes involved in weak adhesion are explored through a single cycle of an optically trapped Brownian colloidal silica particle detaching from, and reattaching to, a glass substrate immersed in a fluid in the presence of an externally applied force. Micro-rheology, video-microscopy, and Nyquist noise measurements reveal both stochastic and deterministic dynamics of the process. When analyzed in terms of the viscoelastic response of the stress coupling medium between the objects, the unsticking instability shows remarkable similarities with yielding and fracture-mechanics of macro-scale solids. The resticking dynamics demonstrates stochastic instabilities through a spatiotemporally punctuated descent of the particle down an energy landscape with a hierarchy of metastable minima.


Future Challenges in Single-Molecule Fluorescence and Laser Trap Approaches to Studies of Molecular Motors

Mary Williard Elting, James A. Spudich

Single-molecule analysis is a powerful modern form of biochemistry, in which individual kinetic steps of a catalytic cycle of an enzyme can be explored in exquisite detail. Both single-molecule fluorescence and single-molecule force techniques have been widely used to characterize a number of protein systems. We focus here on molecular motors as a paradigm. We describe two areas where we expect to see exciting developments in the near future: first, characterizing the coupling of force production to chemical and mechanical changes in motors, and second, understanding how multiple motors work together in the environment of the cell.

Friday, December 14, 2012

Mechanical Characteristics of Human Red Blood Cell Membrane Changing due to C60 Nano-particles Infiltration

Yue Zheng

The mechanical characteristics of human red blood cell (RBC) membrane changing due to C60 nano-particles (NPs) infiltration have been investigated in present work. Using experimental approaches including optical tweezers (OT) stretching and atomic force microscopy (AFM) indentation, we find that RBCs with C60 NPs presence are softer than normal RBCs. The stress-strain relations of both normal and C60 infiltrated RBC membrane are extracted from the data of AFM indentation, from which we proved that C60 NPs infiltration can affect the mechanical properties of RBC membrane and tend to weaken the tensile resistance of lipids bilayers. In order to explain experimental phenomenon, a mechanical model has been developed. Based on this model, the stress-strain relations of both normal and C60 infiltrated lipids bilayers are calculated with considering intermolecular interaction. The theoretical results are in great agreement with the experimental results. The influence of C60 NP’s concentration on mechanical properties of RBC membrane is successfully predicted. Higher concentrations of C60 NPs in the lipids bilayers will bring more damage to the cell membrane, implying that the dosage of C60 NPs should be controlled in medical applications.

Mechanical identities of RNA and DNA double helices unveiled at the single-molecule level

Elías Herrero-Galán , Maria Eugenia Fuentes-Perez ,Carolina Carrasco , Jose M Valpuesta , Jose L Carrascosa , Fernando Moreno-Herrero , and J. Ricardo Arias-Gonzalez
Double-stranded (ds) RNA is the genetic material of a variety of viruses and has been recently recognized as a relevant molecule in cells for its regulatory role. Despite the elastic response of dsDNA has been thoroughly characterized in recent years in single-molecule stretching experiments, an equivalent study with dsRNA is still lacking. Here, we have engineered long dsRNA molecules for their individual characterization contrasting information with dsDNA molecules of the same sequence. It is known that dsRNA is an A-form molecule unlike dsDNA which exhibits B-form in physiological conditions. These structural types are distinguished at the single-molecule level with Atomic Force Microscopy (AFM) and are the basis to understand their different elastic response. Force-extension curves of dsRNA with optical and magnetic tweezers manifest two main regimes of elasticity, an entropic regime whose end is marked by the A-form contour-length and an intrinsic regime that ends in a low-cooperative overstretching transition in which the molecule extends to 1.7 times its A-form contour-length. DsRNA does not switch between the A and B conformations in the presence of force. Finally, dsRNA presents both a lower stretch modulus and overstretching transition force than dsDNA, whereas the electrostatic and intrinsic contributions to the persistence length are larger.

Cytoskeletal Strains in Modeled Optohydrodynamically Stressed Healthy and Diseased Biological Cells

Sean S. Kohles, Yu Liang, and Asit K. Saha

Controlled external chemomechanical stimuli have been shown to influence cellular and tissue regeneration/degeneration, especially with regards to distinct disease sequelae or health maintenance. Recently, a unique three-dimensional stress state was mathematically derived to describe the experimental stresses applied to isolated living cells suspended in an optohydrodynamic trap (optical tweezers combined with microfluidics). These formulae were previously developed in two and three dimensions from the fundamental equations describing creeping flows past a suspended sphere. The objective of the current study is to determine the full-field cellular strain response due to the applied three-dimensional stress environment through a multiphysics computational simulation. In this investigation, the multiscale cytoskeletal structures are modeled as homogeneous, isotropic, and linearly elastic. The resulting computational biophysics can be directly compared with experimental strain measurements, other modeling interpretations of cellular mechanics including the liquid drop theory, and biokinetic models of biomolecule dynamics. The described multiphysics computational framework will facilitate more realistic cytoskeletal model interpretations, whose intracellular structures can be distinctly defined, including the cellular membrane substructures, nucleus, and organelles.

Thursday, December 13, 2012

Acousto-optically generated potential energy landscapes: Potential mapping using colloids under flow

Michael P. N. Juniper, Rut Besseling, Dirk G. A. L. Aarts, and Roel P. A. Dullens
Optical potential energy landscapes created using acousto-optical deflectors are characterized via solvent-driven colloidal particles. The full potential energy of both single optical traps and complex landscapes composed of multiple overlapping traps are determined using a simple force balance argument. The potential of a single trap is shown to be well described by a Gaussian trap with stiffness found to be consistent with those obtained by a thermal equilibrium method. We also obtain directly the depth of the well, which (as with stiffness) varies with laser power. Finally, various complex systems ranging from double-well potentials to random landscapes are generated from individually controlled optical traps. Predictions of these landscapes as a sum of single Gaussian wells are shown to be a good description of experimental results, offering the potential for fully controlled design of optical landscapes, constructed from single optical traps.

Friday, December 7, 2012

Attractive-repulsive dynamics on light-responsive chiral microparticles induced by polarized tweezers

Raul Josue Hernandez , Alfredo Mazzulla , Alfredo Pane , Karen Volke Sepulveda and Gabriella Cipparrone
Multifunctional colloidal micro and nano-particles with controlled architectures have very promising properties for applications in bio and nanotechnologies. Here we report on the unique dichotomous dynamical behaviour of chiral spherical microparticles, either fluid or solid, manipulated by polarized optical tweezers. The particles are created using a reactive mesogen mixed with a chiral dopant to form cholesteric droplets in water emulsion. The photopolymerization enables to freeze the chiral supramolecular configurations in solid particles. Different internal architectures in the supramolecular structures, guided by the interfacial chemistry, enable to obtain global-optically isotropic or anisotropic spherical objects. Their optical manipulation reveals particular features. We show that the light can exert a repulsive or attractive force on the same particle depending on the handedness of its circular polarization, for particles having radial configuration of the cholesteric helices. We demonstrate that the unique selective reflection property of the cholesteric phase is responsible of the observed effect. On the other hand very exotic dynamics is observed in case of anisotropic chiral particles. Depending on the light handedness, they behave like Janus spherical particles with dissimilar optical properties that mean dielectric particles having a surface that is in part transparent and in part reflecting. The combination of optical manipulation with other control parameters (electric or magnetic), as well as the integration of other functionalities suggest exciting developments for applications of these micro-devices in the micro and optofluidics, microphotonics and materials science.

Thursday, December 6, 2012

Cell elongation via intrinsic antipodal stretching forces

T. Sawetzki, C. D. Eggleton, and D. W. M. Marr
To probe the mechanical properties of cells, we investigate a technique to perform deformability-based cytometry that inherently induces normal antipodal surface forces using a single line-shaped optical trap. We show theoretically that these opposing forces are generated simultaneously over curved microscopic object surfaces with optimal magnitude at low numerical apertures, allowing the directed stretching of elastic cells with a single, weakly focused laser source. Matching these findings with concomitant experimental observations, we elongate red blood cells, effectively stretching them within the narrow confines of a steep, optically induced potential well.

Confocal Raman Microscopy Probing of Temperature-Controlled Release from Individual, Optically-Trapped Phospholipid Vesiclesan

Jonathan J. Schaefer , Chaoxiong Ma , and Joel M. Harris
Control of permeability of phospholipid vesicle (liposome) membranes is critical to their applications in analytical sensing, in fundamental studies of chemistry in small volumes, and in encapsulation and release of payloads for site-directed drug delivery. Applications of liposome formulations in drug delivery often take advantage of the enhanced permeability of phospholipid membranes at their gel-to-fluid phase transition, where the release of encapsulated molecules can be initiated by an increase in temperature. Despite numerous successful liposome formulations for encapsulation and release methods to study the kinetics, this process has been limited to investigations of bulk vesicle dispersions, which provide little or no information about the vesicle membrane structure and its relationship to the kinetics of trans-membrane transport. In this work, confocal Raman microscopy is adapted to study temperature-dependent release of a model compound, 3-nitrobenzene sulfonate (3-NBS), from individual optically trapped phospholipid vesicles, while simultaneously monitoring structural changes in the vesicle membrane reported by vibrational modes of phospholipid acyl chains and the local environment of the encapsulated compound. The confocal geometry allows efficient excitation and collection of Raman scattering from a single vesicle, while optical trapping allows more than hour-long observations of the same vesicle. With window factor analysis to resolve component spectra, temperature-controlled release of 3-NBS through vesicle membranes composed of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was measured and compared to transport through a lysolipid-containing membrane specifically formulated for efficient drug delivery.


Tuesday, December 4, 2012

Dynamic of the Dielectric Nano-Particle in Optical Tweezer Using Counter-Propagating Pulsed Laser Beams

Quang Quy Ho and Dinh Hai Hoang

In this article, the dynamical process of the dielectric particle in the optical tweezer using the counter-propagating Gaussian pulses is investigated by the Langevin equation concerning the Brownian motion. The temporal stabilities of particle is simulated. The influence of the duration, repetition period and delay time between pulses on stability is discussed.


Computation of Optical Force on Nanoparticles Using Locally Non-Orthogonal Overlapping Yee FDTD Method

Jinjie Liu, Moysey Brio, Jerome V. Moloney

In this paper, a locally non-orthogonal overlapping Yee (OY) FDTD method is proposed in order to accurately calculates the optical force on dielectric and dispersive nanoparticles. It extends our previous work to geometries with sharp corners and dispersive materials. In addition to consistently achieving the smallest errors in comparison to the standard FDTD method, the OY approach is a stable non-orthogonal FDTD method that attains second-order convergence when sharp corners are present.


Torturing Proteins–Useful Science or Just a Clever Experiment?

Philip Micheal Williams

Unfolding individual proteins by mechanical force has occupied biophysicists for the past 15 years or so. From the initial studies of the muscle protein titin, researchers have studied the effect of force on biomolecules ranging from small enzymes to large ribosomes. The majority of experiments use the atomic force microscope (AFM) as a means to stress proteins tethered between a surface and the probe, and elegant molecular biology approaches are used to produce constructs of repeating protein units which, when stretched and unfolded, produce a characteristic saw-toothed pattern that can be used to confirm complete or partial unfolding. In 2005 my colleague Jane Clarke and I noted that much of the work that had been done by then was to demonstrate the power of the AFM as a tool and that forced unfolding was beginning to move beyond a descriptive science towards a quantitative analysis of protein structure, stability and unfolding landscapes. We noted that the study of proteins that experience force in vivo was obvious but necessitating the use of new techniques such as optical tweezers (OT), and in discussing experiments to study the effects of solvent conditions, the direction of force, the marrying with computational simulations and the like, we were implying that the theoretical knowledge underpinning such quantification and necessary technological developments were in place. Some seven years later, it is interesting to see how the field has advanced and whether such quantitative analysis of our ‘obvious’ proteins has occurred.


Fiber-optic spanner

Bryan J. Black and Samarendra K. Mohanty

Methods of controllable, noncontact rotation of optically trapped microscopic objects have garnered significant attention for tomographic imaging and microfluidic actuation. Here, we report development of a fiber-optic spanner and demonstrate controlled rotation of smooth muscle cells. The rotation is realized by introducing a transverse offset between two counterpropagating beams emanating from single-mode optical fibers. The rotation speed and surrounding microfluidic flow could be controlled by varying balanced laser beam powers. Further, we demonstrate simultaneous translation and rotation of the fiber-optically trapped cell by varying the laser power of one fiber-optic arm.


Radiation pressure makes ellipsoidal particles tumble

B. M. Mihiretie, P. Snabre, J. C. Loudet and B. Pouligny

We report on optical levitation of dielectric particles, of prolate ellipsoidal shape, a few tens of micrometers in length, in a low-aperture laser beam. Ellipsoids of moderate aspect ratio (k < 3) are observed to be trapped on the axis of the laser beam, similarly to simple spheres. Conversely, elongated particles (k > 3) cannot be kept immobile, and rather undergo sustained oscillating motions, comprising both lateral and angular excursions around the beam axis; hence the name "tumble". The observed tumbling motion, a straightforward manifestation of the non-conservative character of radiation pressure forces, is explained through a 2-dimensional ray optics model of the interaction of light with an ellipsoid.


Modeling highly focused laser beam in optical tweezers with the vector Gaussian beam in the T-matrix method

Paul B. Bareil and Yunlong Sheng

The vector Gaussian beam with high-order corrections is used to describe accurately the laser beam up to numerical aperture NA=1.20 in the optical tweezers for trapping nanoparticles. The beam is then expanded in the T-matrix method into the vector spherical wave function (VSWF) series using the point matching method with a new selection of the matching points. The errors in the beam description and in the VSWF expansion are much lower than those that occur in the paraxial Gaussian beam model.