Thursday, March 28, 2013

Using molecular tweezers to move and image nanoparticles

Haimei Zheng

The ability to manipulate nanoparticles is significant in nanoscale science and technology. As sizes of the objects scale down to sub-10 nm regime, it imposes a great challenge for the conventional optical tweezers. There has been much effort to explore the alternative manipulation methods and the approaches include using plasmonic nanostructures, electron beams, scanning probes, etc. In this paper, an overview of the latest advances in trapping and manipulation of nanoparticles with the focus on the emergent electron tweezers is provided.

Observation of Backaction and Self-Induced Trapping in a Planar Hollow Photonic Crystal Cavity

Nicolas Descharmes, Ulagalandha Perumal Dharanipathy, Zhaolu Diao, Mario Tonin, and Romuald Houdré
The optomechanical coupling between a resonant optical field and a nanoparticle through trapping forces is demonstrated. Resonant optical trapping, when achieved in a hollow photonic crystal cavity is accompanied by cavity backaction effects that result from two mechanisms. First, the effect of the particle on the resonant field is measured as a shift in the cavity eigenfrequency. Second, the effect of the resonant field on the particle is shown as a wavelength-dependent trapping strength. The existence of two distinct trapping regimes, intrinsically particle specific, is also revealed. Long optical trapping (>10  min) of 500 nm dielectric particles is achieved with very low intracavity powers (<120  μW).

Unexpected Membrane Dynamics Unveiled by Membrane Nanotube Extrusion

Clément Campillo, Pierre Sens, Darius Köster, Léa-Laetitia Pontani, Daniel Lévy, Patricia Bassereau, Pierre Nassoy, Cécile Sykes
In cell mechanics, distinguishing the respective roles of the plasma membrane and of the cytoskeleton is a challenge. The difference in the behavior of cellular and pure lipid membranes is usually attributed to the presence of the cytoskeleton as explored by membrane nanotube extrusion. Here we revisit this prevalent picture by unveiling unexpected force responses of plasma membrane spheres devoid of cytoskeleton and synthetic liposomes. We show that a tiny variation in the content of synthetic membranes does not affect their static mechanical properties, but is enough to reproduce the dynamic behavior of their cellular counterparts. This effect is attributed to an amplified intramembrane friction. Reconstituted actin cortices inside liposomes induce an additional, but not dominant, contribution to the effective membrane friction. Our work underlines the necessity of a careful consideration of the role of membrane proteins on cell membrane rheology in addition to the role of the cytoskeleton.

Integrin-dependent force transmission to the extracellular matrix by α-actinin triggers adhesion maturation

Pere Roca-Cusachs, Armando del Rio, Eileen Puklin-Faucher, Nils C. Gauthier, Nicolas Biais, and Michael P. Sheetz
Focal adhesions are mechanosensitive elements that enable mechanical communication between cells and the extracellular matrix. Here, we demonstrate a major mechanosensitive pathway in which α-actinin triggers adhesion maturation by linking integrins to actin in nascent adhesions. We show that depletion of the focal adhesion protein α-actinin enhances force generation in initial adhesions on fibronectin, but impairs mechanotransduction in a subsequent step, preventing adhesion maturation. Expression of an α-actinin fragment containing the integrin binding domain, however, dramatically reduces force generation in depleted cells. This behavior can be explained by a competition between talin (which mediates initial adhesion and force generation) and α-actinin for integrin binding. Indeed, we show in an in vitro assay that talin and α-actinin compete for binding to β3 integrins, but cooperate in binding to β1 integrins. Consistently, we find opposite effects of α-actinin depletion and expression of mutants on substrates that bind β3 integrins (fibronectin and vitronectin) versus substrates that only bind β1 integrins (collagen). We thus suggest that nascent adhesions composed of β3 integrins are initially linked to the actin cytoskeleton by talin, and then α-actinin competes with talin to bind β3 integrins. Force transmitted through α-actinin then triggers adhesion maturation. Once adhesions have matured, α-actinin recruitment correlates with force generation, suggesting that α-actinin is the main link transmitting force between integrins and the cytoskeleton in mature adhesions. Such a multistep process enables cells to adjust forces on matrices, unveiling a role of α-actinin that is different from its well-studied function as an actin cross-linker.

Differential interferometric particle tracking on the subnanometer- and submillisecond-scale

Dennis Müller, Dieter R. Klopfenstein, and Rainer G. Ulbrich

We describe an interferometric method to measure the movement of a subwavelength probe particle relative to an immobilized reference particle with high spatial (Δx = 0.9nm) and temporal (Δt = 200μs) resolution. The differential method eliminates microscope stage drift. An upright microscope is equipped with laser dark field illumination (λ0 = 532nm, P0 = 30mW) and a compact modified Mach-Zehnder interferometer is mounted on the camera exit of the microscope, where the beams of scattered light of both particles are combined. The resulting interferograms provide in two channels subnanometer information about the motion of the probe particle relative to the reference particle. The interferograms are probed with two avalanche photodiodes. We applied this method to measuring the movement of kinesin along microtubules and were able to resolve the generic 8-nm steps at high ATP concentrations without external forces.

Analysis of multiple physical parameters for mechanical phenotyping of living cells

T. R. Kießling, M. Herrera, K. D. Nnetu, E. M. Balzer, M. Girvan, A. W. Fritsch, S. S. Martin, J. A. Käs, W. Losert
Since the cytoskeleton is known to regulate many cell functions, an increasing amount of effort to characterize cells by their mechanical properties has occured. Despite the structural complexity and dynamics of the multicomponent cytoskeleton, mechanical measurements on single cells are often fit to simple models with two to three parameters, and those parameters are recorded and reported. However, different simple models are likely needed to capture the distinct mechanical cell states, and additional parameters may be needed to capture the ability of cells to actively deform. Our new approach is to capture a much larger set of possibly redundant parameters from cells’ mechanical measurement using multiple rheological models as well as dynamic deformation and image data. Principal component analysis and network-based approaches are used to group parameters to reduce redundancies and develop robust biomechanical phenotyping. Network representation of parameters allows for visual exploration of cells’ complex mechanical system, and highlights unexpected connections between parameters. To demonstrate that our biomechanical phenotyping approach can detect subtle mechanical differences, we used a Microfluidic Optical Cell Stretcher to mechanically stretch circulating human breast tumor cells bearing genetically-engineered alterations in c-src tyrosine kinase activation, which is known to influence reattachment and invasion during metastasis.


Wednesday, March 27, 2013

Two-color laser manipulation of single organic molecules based on nonlinear optical response

Tetsuhiro Kudo, Hajime Ishihara
We theoretically propose two-color laser manipulation with greatly improved efficiency to mechanically manipulate single organic molecules. The present method is based on the nonlinear resonant laser manipulation proposed in a recent study. In the first part, we describe a method to trap single organic molecules that can be more effective than ever before utilizing two-color beams. In the second part, we demonstrate the possibility to selectively “pull” single organic molecules with a particular type of electronic-level scheme by using single-side illumination of traveling light.


3D Manipulation of Protein Microcrystals with Optical Tweezers for X-ray Crystallography

T Hikima, K Hashimoto, H Murakami, G Ueno, Y Kawano, K Hirata, K Hasegawa, T Kumasaka and M Yamamoto
In some synchrotron facilities such as SPring-8, X-ray microbeams have been utilized for protein crystallography, allowing users to collect diffraction data from a protein microcrystal. Usually, a protein crystal is picked up manually from a crystallization droplet. However it is very difficult to manipulate the protein microcrystals which are very small and fragile against a shock and changes of temperature and solvent condition. We have been developing an automatic system applying the optical tweezers with two lensed fiber probes to manipulate the fragile protein microcrystal. The system succeeded in trapping a crystal and levitating it onto the cryoloop in the solvent. X-ray diffraction measurement for the manipulated protein microcrystals indicated that laser irradiation and trap with 1064nm wavelength hardly affected the result of X-ray structural analysis.


Purple sea urchin Strongylocentrotus purpuratus gamete manipulation using optical trapping and microfluidics

Charlie Chandsawangbhuwana ; Linda Z. Shi ; Qingyuan Zhu ; Michael W. Berns
A system has been developed that allows for optical and fluidic manipulation of gametes. The optical manipulation is performed by using a single-point gradient trap with a 40× oil immersion PH3 1.3 NA objective on a Zeiss inverted microscope. The fluidic manipulation is performed by using a custom microfluidic chamber designed to fit into the short working distance between the condenser and objective. The system is validated using purple sea urchin Strongylocentrotus purpuratus gametes and has the potential to be used for mammalian in vitro fertilization and animal husbandry.

Optical generation, templating, and polymerization of three-dimensional arrays of liquid-crystal defects decorated by plasmonic nanoparticles

Julian S. Evans, Paul J. Ackerman, Dirk J. Broer, Jao van de Lagemaat, and Ivan I. Smalyukh
Defects in liquid crystals are used to model topological entities ranging from Skyrmions in high-energy physics to early-universe cosmic strings, as well as find practical applications in self-assembly of diffraction gratings and in scaffolding of plasmonic nanoparticles, but they are hard to control and organize into three-dimensional lattices. We laterally scan focused laser beams to produce periodic arrays of twist-stabilized defects forming either linear (fingers) or axially symmetric (torons) configurations in partially polymerizable liquid crystal films. Polymerization allows for stabilization of these structures and the formation of three-dimensional arrays of defects by stacking of the thin cholesteric films on top of each other. In the process of fabrication of such arrays, we polymerize the liquid crystal film with an array of torons or fingers and then sequentially produce and photopolymerize new liquid crystal layers on top of it, thus obtaining a three-dimensional structure of twist-stabilized defects in a layer-by-layer fashion. Templating by the polymerized layer spontaneously yields ordered organization of fingers and torons in the new cholesteric layer, thus enabling a three-dimensional ordered structure of defects. Nondestructive three-dimensional imaging of director fields by use of three-photon excitation fluorescence polarizing microscopy reveals the nature of topological singularities and physical underpinnings behind the observed templating effect. Three-dimensional patterning of defects templates the self-assembly of plasmonic nanoparticles into individual singularities and their arrays, laying the groundwork for potential applications in nanophotonics, plasmonics, metamaterial fabrication, and nanoscale energy conversion.


Biomechanics of haemostasis and thrombosis in health and disease: from the macro- to molecular scale

Reginald Tran, David R. Myers, Jordan Ciciliano, Elaissa L. Trybus Hardy, Yumiko Sakurai, Byungwook Ahn, Yongzhi Qiu, Robert G. Mannino, Meredith E. Fay, Wilbur A. Lam
Although the processes of haemostasis and thrombosis have been studied extensively in the past several decades, much of the effort has been spent characterizing the biological and biochemical aspects of clotting. More recently, researchers have discovered that the function and physiology of blood cells and plasma proteins relevant in haematologic processes are mechanically, as well as biologically, regulated. This is not entirely surprising considering the extremely dynamic fluidic environment that these blood components exist in. Other cells in the body such as fibroblasts and endothelial cells have been found to biologically respond to their physical and mechanical environments, affecting aspects of cellular physiology as diverse as cytoskeletal architecture to gene expression to alterations of vital signalling pathways. In the circulation, blood cells and plasma proteins are constantly exposed to forces while they, in turn, also exert forces to regulate clot formation. These mechanical factors lead to biochemical and biomechanical changes on the macro- to molecular scale. Likewise, biochemical and biomechanical alterations in the microenvironment can ultimately impact the mechanical regulation of clot formation. The ways in which these factors all balance each other can be the difference between haemostasis and thrombosis. Here, we review how the biomechanics of blood cells intimately interact with the cellular and molecular biology to regulate haemostasis and thrombosis in the context of health and disease from the macro- to molecular scale. We will also show how these biomechanical forces in the context of haemostasis and thrombosis have been replicated or measured in vitro.

Photoclickable Dendritic Molecular Glue: Noncovalent-to-Covalent Photochemical Transformation of Protein Hybrids

Noriyuki Uchida, Kou Okuro, Yamato Niitani, Xiao Ling, Takayuki Ariga, Michio Tomishige, and Takuzo Aida
A water-soluble dendron with a fluorescein isothiocyanate (FITC) fluorescent label and bearing nine pendant guanidinium ion (Gu+)/benzophenone (BP) pairs at its periphery (GlueBP-FITC) serves as a “photoclickable molecular glue”. By multivalent salt-bridge formation between Gu+ ions and oxyanions, GlueBP-FITC temporarily adheres to a kinesin/microtubule hybrid. Upon subsequent exposure to UV light, this noncovalent binding is made permanent via a cross-linking reaction mediated by carbon radicals derived from the photoexcited BP units. This temporal-to-permanent transformation by light occurs quickly and efficiently in this preorganized state, allowing the movements of microtubules on a kinesin-coated glass plate to be photochemically controlled. A fundamental difference between such temporal and permanent bindings was visualized by the use of “optical tweezers”.

Friday, March 22, 2013

Surface-Enhanced Phosphorescence Measurement by an Optically Trapped Colloidal Ag Nanoaggregate on Anionic Thiacarbocyanine H-Aggregate

Yasutaka Kitahama, Masato Kashihara, Tamitake Itoh, and Yukihiro Ozaki
A citrate-reduced Ag nanoaggregate was optically trapped on a fiber-shaped H-aggregate of an anionic thiacarbocyanine dye against Coulomb repulsion by focusing a near-infrared (NIR) laser beam. As the NIR laser power increased, namely, as the Ag nanoaggregate approaches the H-aggregate, phosphorescence from the H-aggregate with the Ag nanoaggregate excited moderately at 514 and 647 nm was strengthened, although that at 568 nm was weakened. By excitation at 568 nm, which was close to a surface plasmon resonance peak of the Ag nanoaggregate, surface-plasmon-enhanced optical trapping potential well might have deepened, and then the Ag nanoaggregate might have approached the H-aggregate too closely to enhance the phosphorescence because of energy transfer to the metal. As the excitation laser intensity increased, namely, as the surface-plasmon-enhanced optical trapping potential well was deepened, the phosphorescence enhancement factor trended upward and then downward by enhancement due to plasmon at a close distance from the Ag surface and the energy transfer at the closer distance, respectively.

Non-B DNA Structures Show Diverse Conformations and Complex Transition Kinetics Comparable to RNA or Proteins—A Perspective from Mechanical Unfolding and Refolding Experiments

Zhongbo Yu, Hanbin Mao

With the firm demonstration of the in vivo presence and biological functions of many non-B DNA structures, it is of great significance to understand their physiological roles from the perspective of structural conformation, stability, and transition kinetics. Although relatively simple in primary sequences compared to proteins, non-B DNA species show rather versatile conformations and dynamic transitions. As the most-studied non-B DNA species, the G-quadruplex displays a myriad of conformations that can interconvert between each other in different solutions. These features impose challenges for ensemble-average techniques, such as X-ray crystallography, NMR spectroscopy, and circular dichroism (CD), but leave room for single-molecular approaches to illustrate the structure, stability, and transition kinetics of individual non-B DNA species in a solution mixture. Deconvolution of the mixture can be further facilitated by statistical data treatment, such as iPoDNano (integrated population deconvolution with nanometer resolution), which resolves populations with subnanometer size differences. This Personal Account summarizes current mechanical unfolding and refolding methods to interrogate single non-B DNA species, with an emphasis on DNA G-quadruplexes and i-motifs. These single-molecule studies start to demonstrate that structures and transitions in non-B DNA species can approach the complexity of those in RNA or proteins, which provides solid justification for the biological functions carried out by non-B DNA species.


α-Hemolysin membrane pore density measured on liposomes

Joël Lemière , Karine Guevorkian , Clément Campillo , Cécile Sykes and Timo Betz
Membrane pore proteins are powerful tools that allow manipulation of the inside composition of micron sized bioreactors such as artificial liposomes. While the pores self-assemble very reliably on phospholipid bilayers, the determination of the number of pores in situ for liposomes remains difficult. Here we present three independent methods to establish the number of pores on different types of liposomes: (A) the loss of refractive index due to equilibration of the inside and outside buffer conditions, and the loss of volume by (B) membrane aspiration and by (C) membrane tether pulling experiments. With these three methods we are able to determine the pore density on the membrane, and all measurements give similar values; an average pore distance is found on the order of 100 nm.

Three dimensional force detection of gold nanoparticles using backscattered light detection

Lu Huang, Honglian Guo, Kunlong Li, Yuhui Chen, Baohua Feng, and Zhi-Yuan Li
We demonstrate three-dimensional position and force detection of single gold nanosphere (GNP) and gold nanorod (GNR) particles in optical trap by combining backscattered light detection and dark field imaging. The trapping stiffness of the GNPs and GNRs for all three dimensions is measured. The results show that the spring constants in the propagation direction of the trapping laser are somewhat weaker than in other two directions for GNPs. While for GNRs, the spring constants in the polarization direction of the trapping laser are a little weaker than in other two directions. The effect of trapping laser polarization on the particles yields different spring constants in the transverse plane which is perpendicular to the propagation direction. And this effect is larger on GNRs than GNPs.

Thursday, March 21, 2013

Modified Hybrid Plasmonic Waveguides as Tunable Optical Tweezers

Zhang Lu and Yang Shu
We propose a series of modified hybrid plasmonic waveguide systems. It is found that their propagation distances and mode areas depend on their shapes notably. The optical trapping forces exerting on the dielectric nanoparticles are calculated, and the strength and range of the forces can be adjusted by altering the shapes of the waveguides. These features demonstrate the possibility of using the modified hybrid waveguide systems to design tunable nanoscale optical tweezers.

Measurements of forces produced by the mitotic spindle using optical tweezers

Jessica Ferraro-Gideon, Rozhan Sheykhani, Qingyuan Zhu, Michelle L. Duquette, Michael W. Berns, and Arthur Forer

We used a trapping laser to stop chromosome movements in Mesostoma and crane-fly spermatocytes and to stop inward movements of spindle poles after laser cuts across PtK2 cell half-spindles. Mesostoma spermatocyte kinetochores execute oscillatory movements to and away from the spindle pole for 1-2 h so we could trap kinetochores multiple times in the same spermatocyte. The trap was focussed to a single point using a 63x oil immersion objective. Trap powers of 15 mW to 23 mW caused kinetochore oscillations to stop or decrease. Kinetochore oscillations resumed when the trap was released. In crane-fly spermatocytes trap powers of 56 mW to 85 mW stopped or slowed poleward chromosome movement. In PtK2 cells 8 mW trap power stopped the spindle pole from moving toward the equator. Forces in the traps were calculated using the equation F = Q’P/c, where P is the laser power and c is the speed of light. Using appropriate Q’ coefficients, the forces for stopping pole movements were 0.3-2.3 pN, and for stopping chromosome movements in Mesostoma spermatocytes and crane-fly spermatocytes were 2 pN-3pN and 6-10pN, respectively. These forces are close to theoretical calculations of forces causing chromosome movements but 100 times lower than the 700pN measured in grasshopper spermatocytes (Nicklas, 1983).

Holographic optical tweezers obtained by using the three-dimensional Gerchberg–Saxton algorithm

Hao Chen, Yunfeng Guo, Zhaozhong Chen, Jingjing Hao, Ji Xu, Hui-Tian Wang and Jianping Ding
An extension of the Gerchberg–Saxton algorithm from two dimensions to three is used to configure a continuous optical trap geometry. Intensity tailoring in a continuous, three-dimensional (3D) volume rather than in multiple discrete two-dimensional planes yields flexible 3D holographic optical tweezers. A numerical simulation and optical demonstrations of continuous 3D beam shaping and particle trapping confirm the capabilities of the method.

Experimental study of the Stokes-Einstein relation by using oscillating optical tweezers and a position tracking method

Chungil Ha, Sung-Jin Kim, Hyuk Kyu Pak
Transportation and delivery of microscopic materials in very small and complex systems such as biological organisms are mainly done by physical diffusion. This phenomenon in a fluid system with a low Reynolds number can be explained using the Stokes-Einstein relation D = k B T/β, where D is the diffusion coefficient, T is the temperature of the system, and β is the viscous friction coefficient of the background fluid. For a spherical particle with radius a in a fluid of viscosity η, β = 6πηa. As far as we know, all the experimental tests of this relation before ours measured only D, η, a, and T due to the experimental difficulties in measuring β directly. In this research, we tested this relation from a different perspective. The diffusion coefficient D and the viscous friction coefficient β were experimentally measured in the same system by using a position tracking method and an oscillating optical tweezers technique, respectively. We found that our experimental results supported the Stokes-Einstein relation very well.

Single-cell discrimination based on optical tweezers Raman spectroscopy

HongFei Ma, Yong Zhang, AnPei Ye
The ability to discriminate between single cells in a label-free and noninvasive fashion is important for the classification of cells, and for the identification of similar cells from different origins. In this paper, we present the Raman spectroscopy-based identification of different types of single cells in aqueous media, and discrimination between the same types of cells from different donors using a novel Laser Tweezers Raman Spectroscopy (LTRS) technique, which combines laser trapping and micro-Raman spectroscopy. First, we measured the spectra of individual living human erythrocytes, i.e. red blood cells, and leucocytes (U937 cancer cells). High-quality Raman spectra with low fluorescence were obtained using a home-LTRS apparatus and 20 cells were measured for each cell type. The smoothing, baseline subtraction, and normalization of the data were followed by a principal components analysis (PCA). The PCA loading plots showed that the two different types of cells could be completely separated based only on the first component (PC1) (i.e. the peaks at 1300 cm−1); the discrimination accuracy could therefore reach 100%. More than 50 spectra were taken for each erythrocyte obtained from the four healthy volunteers. The average discrimination accuracy was 84.5% for two random individuals taken from the four volunteers, according to the first and second PCs. This work demonstrates that LTRS is a powerful tool for the accurate identification and discrimination of single cells, and it has the potential to be applied for the highly sensitive identification of cells in clinical diagnosis and medical jurisprudence.

Monday, March 18, 2013

Analytical Techniques for Single-Liposome Characterization

Xiaomei Yan , Chaoxing Chen , Shaobin Zhu , Tianxun Huang and Shuo Wang
Liposomes or phospholipid vesicles are one of the most versatile nanoparticles used to convey drugs, vaccines, genes, enzymes, or other substances to target cells and as a model to mimic biological membranes. To fulfill their roles in drug delivery and biotechnology, physical and chemical properties of liposomes, such as size, shape, chemical composition, lamellarity, encapsulation efficiency of cargo molecules, and the density of proteins reconstituted in the membrane need to be characterized to ensure a reproducible preparation of vesicles. Compared to bulk analysis, techniques focusing on the individual analysis of liposomes can reveal the heterogeneity that is otherwise masked by ensemble averaging. Herein, we review the recent advancement in techniques for single-liposome characterization.

Long-Distance Axial Trapping with Focused Annular Laser Beams

Ming Lei, Ze Li, Shaohui Yan, Baoli Yao, Dan Dan, Yujiao Qi, Jia Qian, Yanlong Yang, Peng Gao, Tong Ye
Focusing an annular laser beam can improve the axial trapping efficiency due to the reduction of the scattering force, which enables the use of a lower numerical aperture (NA) objective lens with a long working distance to trap particles in deeper aqueous medium. In this paper, we present an axicon-to-axicon scheme for producing parallel annular beams with the advantages of higher efficiency compared with the obstructed beam approach. The validity of the scheme is verified by the observation of a stable trapping of silica microspheres with relatively low NA microscope objective lenses (NA = 0.6 and 0.45), and the axial trapping depth of 5 mm is demonstrated in experiment.

Guiding Spatial Arrangements of Silver Nanoparticles by Optical Binding Interactions in Shaped Light Fields

Zijie Yan, Raman A. Shah, Garrett Chado, Stephen K. Gray, Matthew Pelton, and Norbert F. Scherer

We demonstrate assembly of spheroidal Ag nanoparticle clusters, chains and arrays induced by optical binding. Particles with diameters of 40 nm formed ordered clusters and chains in aqueous solution when illuminated by shaped optical fields with a wavelength of 800 nm; specifically, close-packed clusters were formed in cylindrically symmetric optical traps, and linear chains were formed in line traps. We developed a coupled-dipole model to calculate the optical forces between an arbitrary number of particles and successfully predicted the experimentally observed particle separations and arrangements as well as their dependence on the polarization of the incident light. This demonstrates that the interaction between these small Ag particles and light is well described by approximating the particles as point dipoles, showing that these experiments extend optical binding into the Rayleigh regime. For larger Ag nanoparticles, with diameters of approximately 100 nm, the optical-binding forces become comparable to the largest gradient forces in the optical trap, and the particles can arrange themselves into regular arrays or synthetic photonic lattices. Finally, we discuss the differences between our experimental observations and the point dipole theory and suggest factors that prevent the Ag nanoparticles from aggregating as expected from the theory.

Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy

Graeme A. Kinga, Peter Grossa, Ulrich Bockelmann, Mauro Modesti, Gijs J. L. Wuitea, and Erwin J. G. Peterman
Mechanical stress plays a key role in many genomic processes, such as DNA replication and transcription. The ability to predict the response of double-stranded (ds) DNA to tension is a cornerstone of understanding DNA mechanics. It is widely appreciated that torsionally relaxed dsDNA exhibits a structural transition at forces of ∼65 pN, known as overstretching, whereby the contour length of the molecule increases by ∼70%. Despite extensive investigation, the structural changes occurring in DNA during overstretching are still generating considerable debate. Three mechanisms have been proposed to account for the increase in DNA contour length during overstretching: strand unpeeling, localized base-pair breaking (yielding melting bubbles), and formation of S-DNA (strand unwinding, while base pairing is maintained). Here we show, using a combination of fluorescence microscopy and optical tweezers, that all three structures can exist, uniting the often contradictory dogmas of DNA overstretching. We visualize and distinguish strand unpeeling and melting-bubble formation using an appropriate combination of fluorescently labeled proteins, whereas remaining B-form DNA is accounted for by using specific fluorescent molecular markers. Regions of S-DNA are associated with domains where fluorescent probes do not bind. We demonstrate that the balance between the three structures of overstretched DNA is governed by both DNA topology and local DNA stability. These findings enhance our knowledge of DNA mechanics and stability, which are of fundamental importance to understanding how proteins modify the physical state of DNA.

Friday, March 15, 2013

Refractive optical wing oscillators with one reflective surface

Alexandra B. Artusio-Glimpse, Timothy J. Peterson, and Grover A. Swartzlander
An optical wing is a cambered rod that experiences a force and torque owing to the reflection and transmission of light from the surface. Here we address how such a wing may be designed to maintain an efficient thrust from radiation pressure (RP) while also providing a torque that returns the wing to a source facing orientation. The torsional stiffness of two different wing cross-sections is determined from numerical ray-tracing analyses. These results demonstrate the potential to construct a passive sun-tracking, space flight system or a microscopic surface measurement device based on RP force and torque.

Mie scattering and optical forces from evanescent fields: A complex-angle approach

Aleksandr Y. Bekshaev, Konstantin Y. Bliokh, and Franco Nori
Mie theory is one of the main tools describing scattering of propagating electromagnetic waves by spherical particles. Evanescent optical fields are also scattered by particles and exert radiation forces which can be used for optical near-field manipulations. We show that the Mie theory can be naturally adopted for the scattering of evanescent waves via rotation of its standard solutions by a complex angle. This offers a simple and powerful tool for calculations of the scattered fields and radiation forces. Comparison with other, more cumbersome, approaches shows perfect agreement, thereby validating our theory. As examples of its application, we calculate angular distributions of the scattered far-field irradiance and radiation forces acting on dielectric and conducting particles immersed in an evanescent field.


Selective photodeposition of zinc nanoparticles on the core of a single-mode optical fiber

J. G. Ortega-Mendoza, F. Chávez, P. Zaca-Morán, C. Felipe, G. F. Pérez-Sánchez, G. Beltran-Pérez, O. Goiz, and R. Ramos-Garcia

An experimental and theoretical study about selective photodeposition of metallic zinc nanoparticles onto an optical fiber end is presented. It is well known that metallic nanoparticles possess a high absorption coefficient and therefore trapping and manipulation is more challenging than dielectric particles. Here, we demonstrate a novel trapping mechanism that involves laser-induced convection flow (due to heat transfer from the zinc particles) that partially compensates both absorption and scattering forces in the vicinity of the fiber end. The gradient force is too small and plays no role on the deposition process. The interplay of these forces produces selective deposition of particles whose size is directly controlled by the laser power. In addition, a novel trapping mechanism termed convective-optical trapping is demonstrated.


Thursday, March 14, 2013

The properties of gold nanospheres studied with dark field optical trapping

Lin Ling, Lu Huang, Jinxin Fu, Honglian Guo, Jiafang Li, H. Daniel Ou-Yang, and Zhi-Yuan Li
We demonstrate trapping and characterization of multiple gold nanospheres with a setup composed of dark field imaging and optical tweezers. The number of trapped nanospheres is quantified by the overall dark-field scattering intensity. The spectra of the scattering intensity show that there is no interparticle coupling among trapped nanospheres when the density of nanospheres in the trap is low enough (less than 10 particles), while the density of nanosphere increases the interparticle coupling of nanospheres becomes obvious. In addition, the trapping potential of a single gold nanosphere is obtained by trapping an ensemble of gold nanospheres.

Reversal of optical binding force by Fano resonance in plasmonic nanorod heterodimer

Q. Zhang, J. J. Xiao, X. M. Zhang, Y. Yao, and H. Liu
We present calculations of the optical force on heterodimer of two gold nanorods aligned head-to-tail, under plane wave illumination that is polarized along the dimer axis. It is found that near the dipole-quadrupole Fano resonance, the optical binding force between the nanorods reverses, indicating an attractive to repulsive transition. This is in contrast to homodimer which in similar configuration shows no negative binding force. Moreover, the force spectrum features asymmetric line shape and shifts accordingly when the Fano resonance is tuned by varying the nanorods length or their gap. We show that the force reversal is associated with the strong phase variation between the hybridized dipole and quadrupole modes near the Fano dip. The numerical results may be demonstrated by a near-field optical tweezer and shall be useful for studying “optical matters” in plasmonics.

Pushing, pulling and twisting liquid crystal systems: exploring new directions with laser manipulation

Jennifer L. Sanders, Yiming Yang, Mark R. Dickinson and Helen F. Gleeson

Optical tweezers are exciting tools with which to explore liquid crystal (LC) systems; the motion of particles held in laser traps through LCs is perhaps the only approach that allows a low Ericksen number regime to be accessed. This offers a new method of studying the microrheology associated with micrometre-sized particles suspended in LC media—and such hybrid systems are of increasing importance as novel soft-matter systems. This paper describes the microrheology experiments that are possible in nematic materials and discusses the sometimes unexpected results that ensue. It also presents observations made in the inverse system; micrometre-sized droplets of LC suspended in an isotropic medium.

Tuesday, March 12, 2013

Direct Visualization of DNA Recognition by Restriction Endonuclease EcoRI

Chiung-Fang Huang, Pei-Wen Peng, Chih-Ming Cheng, Jing-Shin Tsai, Wei-Ting Wang, Chien-Ting Hsu, Keng-Liang Ou, Tzu-Sen Yang

Restriction endonuclease is found in bacteria and can precisely cut off the DNA sequence specificity invaded from outside. Using the restriction endonuclease to cleave the double-stranded DNA at specific recognition nucleotide sequences is regarded as important biotechnology. However, traditional approaches cannot directly observe the process of specific binding of DNA by the restriction endonuclease. Single-molecule biotechnologies, including an optical tweezers system and single-molecule fluorescence detection system, were proposed to directly observe how the restriction endonuclease EcoRI found the recognition-binding site. Overall, the study showed that EcoRI conjugated quantum dots (QDs) possessed the ability to recognize its specific binding sites. In addition, the distributions of the QD-EcoRI trajectory in the transverse direction were nearly the same; however, the difference occurred in the longitudinal direction along the DNA axis during the early and late stages of EcoRI recognition. These results suggest one-dimensional sliding of EcoRI existed along the DNA contour during EcoRI recognition. In addition, when EcoRI slided along the DNA contour, it would require more than 400 seconds for EcoRI to correctly find its DNA target site.

A toolbox for generating single-stranded DNA in optical tweezers experiments

Andrea Candelli, Tjalle Hoekstra, Geraldine Farge, Peter Gross, Erwin G. Peterman, Gijs L. Wuite
Essential genomic transactions such as DNA-damage repair and DNA replication take place on single-stranded DNA (ssDNA) or require specific single-stranded/double-stranded DNA (ssDNA/dsDNA) junctions (SDSJ). A significant challenge in single-molecule studies of DNA-protein interactions using optical trapping is the design and generation of appropriate DNA templates. In contrast to double-stranded DNA (dsDNA), only a limited toolbox is available for the generation of ssDNA constructs for optical tweezers experiments. Here, we present several kinds of DNA templates suitable for single-molecule experiments requiring segments of ssDNA of several kilobases in length. These different biotinylated dsDNA templates can be tethered between optically trapped microspheres and can, by the subsequent use of force-induced DNA melting, be converted into partial or complete ssDNA molecules. We systematically investigated the time scale and efficiency of force-induced melting at different ionic strengths for DNA molecules of different sequences and lengths. Furthermore, we quantified the impact of microspheres of different sizes on the lifetime of ssDNA tethers in optical tweezers experiments. Together, these experiments provide deeper insights into the variables that impact the production of ssDNA for single molecules studies and represent a starting point for further optimization of DNA templates that permit the investigation of protein binding and kinetics on ssDNA.


Trapping and Propelling Microparticles at Long Range by Using an Entirely Stripped and Slightly Tapered No-Core Optical Fiber

Fang-Wen Sheu and Yen-Si Huang

A stripped no-core optical fiber with a 125 µm diameter was transformed into a symmetric and unbroken optical fiber that tapers slightly to a 45-µm-diameter waist. The laser light can be easily launched into the no-core optical fiber. The enhanced evanescent wave of the slightly tapered no-core optical fiber can attract nearby 5-µm-diameter polystyrene microparticles onto the surface of the tapered multimode optical fiber within fast flowing fluid and propel the trapped particles in the direction of the light propagation to longer delivery range than is possible using a slightly tapered telecom single-mode optical fiber.


Saturday, March 9, 2013

Peptide-mediated desmoglein 3 crosslinking prevents pemphigus vulgaris autoantibody-induced skin blistering

Volker Spindler, Vera Rötzer, Carina Dehner, Bettina Kempf, Martin Gliem, Mariya Radeva, Eva Hartlieb, Gregory S. Harms, Enno Schmidt and Jens Waschke
In pemphigus vulgaris, a life-threatening autoimmune skin disease, epidermal blisters are caused by autoantibodies primarily targeting desmosomal cadherins desmoglein 3 (DSG3) and DSG1, leading to loss of keratinocyte cohesion. Due to limited insights into disease pathogenesis, current therapy relies primarily on nonspecific long-term immunosuppression. Both direct inhibition of DSG transinteraction and altered intracellular signaling by p38 MAPK likely contribute to the loss of cell adhesion. Here, we applied a tandem peptide (TP) consisting of 2 connected peptide sequences targeting the DSG adhesive interface that was capable of blocking autoantibody-mediated direct interference of DSG3 transinteraction, as revealed by atomic force microscopy and optical trapping. Importantly, TP abrogated autoantibody-mediated skin blistering in mice and was effective when applied topically. Mechanistically, TP inhibited both autoantibody-induced p38 MAPK activation and its association with DSG3, abrogated p38 MAPK-induced keratin filament retraction, and promoted desmosomal DSG3 oligomerization. These data indicate that p38 MAPK links autoantibody-mediated inhibition of DSG3 binding to skin blistering. By limiting loss of DSG3 transinteraction, p38 MAPK activation, and keratin filament retraction, which are hallmarks of pemphigus pathogenesis, TP may serve as a promising treatment option.

Unwinding and rewinding the nucleosome inner turn: Force dependence of the kinetic rate constants

S. G. J. Mochrie, A. H. Mack, D. J. Schlingman, R. Collins, M. Kamenetska, and L. Regan

A simple model for the force-dependent unwinding and rewinding rates of the nucleosome inner turn is constructed and quantitatively compared to the results of recent measurements [ A. H. Mack et al. J. Mol. Biol. 423 687 (2012)]. First, a coarse-grained model for the histone-DNA free-energy landscape that incorporates both an elastic free-energy barrier and specific histone-DNA bonds is developed. Next, a theoretical expression for the rate of transitions across a piecewise linear free-energy landscape with multiple minima and maxima is presented. Then, the model free-energy landscape, approximated as a piecewise linear function, and the theoretical expression for the transition rates are combined to construct a model for the force-dependent unwinding and rewinding rates of the nucleosome inner turn. Least-mean-squares fitting of the model rates to the rates observed in recent experiments rates demonstrates that this model is able to well describe the force-dependent unwinding and rewinding rates of the nucleosome inner turn, observed in the recent experiments, except at the highest forces studied, where an additional ad hoc term is required to describe the data, which may be interpreted as an indication of an alternate high-force nucleosome disassembly pathway, that bypasses simple unwinding. The good agreement between the measurements and the model at lower forces demonstrates that both specific histone-DNA contacts and an elastic free-energy barrier play essential roles for nucleosome winding and unwinding, and quantifies their relative contributions.


Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors

Ken’ya Furuta, Akane Furuta, Yoko Y. Toyoshima, Misako Amino, Kazuhiro Oiwa, and Hiroaki Kojima

Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 μm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.

Thursday, March 7, 2013

Morphology-dependent resonance of the optical forces on Mie particles in an Airy beam

Yang Yang, Wei-Ping Zang, Zi-Yu Zhao, and Jian-Guo Tian

Morphology-dependent resonance (MDR) of the optical forces for a particle illuminated by Airy beams is investigated with respect to its internal field distribution. We find the ring structures arising from the resonance transform significantly with the parametric evolution of Airy evanescent wave, and the interference of the internal waves have a great impact on the Q factor and the background of the resonant peak, but it’s not proper for Airy transmitted wave. The multiple reflections of the evanescent wave between the particle and the interface are also investigated, which show significant impacts on the region where the energy concentrate in.

Optical Trapping at Gigapascal Pressures

Richard W. Bowman, Graham M. Gibson, Miles J. Padgett, Filippo Saglimbeni and Roberto Di Leonardo

Diamond anvil cells allow the behavior of materials to be studied at pressures up to hundreds of gigapascals in a small and convenient instrument. However, physical access to the sample is impossible once it is pressurized. We show that optical tweezers can be used to hold and manipulate particles in such a cell, confining micron-sized transparent beads in the focus of a laser beam. Here, we use a modified optical tweezers geometry, allowing us to trap through an objective lens with a higher working distance, overcoming the constraints imposed by the limited angular acceptance of the anvil cell. We demonstrate the effectiveness of the technique by measuring water’s viscosity at pressures of up to 1.3 GPa. In contrast to previous viscosity measurements in anvil cells, our technique measures absolute viscosity and does not require scaling to the accepted value at atmospheric pressure. This method could also measure the frequency dependence of viscosity as well as being sensitive to anisotropy in the medium’s viscosity.

Dynamic analysis of a diffusing particle in a trapping potential

Moshe Lindner, Guy Nir, Anat Vivante, Ian T. Young, and Yuval Garini
The dynamics of a diffusing particle in a potential field is ubiquitous in physics, and it plays a pivotal role in single-molecule studies. We present a formalism for analyzing the dynamics of diffusing particles in harmonic potentials at low Reynolds numbers using the time evolution of the particle probability distribution function. We demonstrate the power of the formalism by simulation and by measuring and analyzing a nanobead tethered to a single DNA molecule. It allows one to simultaneously extract all the parameters that describe the system, namely, the diffusion coefficient and the restoring-force constant.

A Minimal Optical Trapping and Imaging Microscopy System

Carmen Noemí Hernández Candia, Sara Tafoya Martínez, Braulio Gutiérrez-Medina
We report the construction and testing of a simple and versatile optical trapping apparatus, suitable for visualizing individual microtubules (~25 nm in diameter) and performing single-molecule studies, using a minimal set of components. This design is based on a conventional, inverted microscope, operating under plain bright field illumination. A single laser beam enables standard optical trapping and the measurement of molecular displacements and forces, whereas digital image processing affords real-time sample visualization with reduced noise and enhanced contrast. We have tested our trapping and imaging instrument by measuring the persistence length of individual double-stranded DNA molecules, and by following the stepping of single kinesin motor proteins along clearly imaged microtubules. The approach presented here provides a straightforward alternative for studies of biomaterials and individual biomolecules.

Wednesday, March 6, 2013

Reversible Photoinduced Formation and Manipulation of a Two-Dimensional Closely Packed Assembly of Polystyrene Nanospheres on a Metallic Nanostructure

Tatsuya Shoji, Michiko Shibata, Noboru Kitamura, Fumika Nagasawa, Mai Takase, Kei Murakoshi, Atsushi Nobuhiro, Yoshihiko Mizumoto, Hajime Ishihara and Yasuyuki Tsuboi

Nanostructure-enhanced optical trapping of polymer beads was investigated by means of fluorescence microspectroscopy. It was found that trapping behavior was quite sensitive to the particle size as well as excitation light intensity. We present a 2D closely packed assembly of polystyrene nanospheres on a gold nanostructure that is triggered by gap-mode localized surface plasmon (LSP) excitation. We discuss the trapping mechanism from the viewpoints of not only the radiation force but also of the thermal force (thermophoresis and thermal convection) induced by near-infrared laser irradiation. Thermophoresis worked as a repulsive force whose direction was opposed to that of the radiation force. On the other hand, thermal convection acted in favor of trapping: It supplied nanospheres toward the LSP excitation area. By suppressing the repulsive force, the assembled trapped nanospheres took the form of hexagonal shapes on a gold nanostructure. By optimizing irradiation parameters, we achieved 2D manipulation of nanospheres on a substrate. Our method has advantages over the conventional optical tweezers technique because of its weak light intensity, and could be a promising method of creating and manipulating a 2D colloidal crystal on a plasmonic substrate.

The bystander effect in optically trapped red blood cells due to Plasmodium falciparum infection

Apurba Paul, Rani Pallavi, Utpal S. Tatu and Vasant Natarajan

In a previous study of the properties of red blood cells (RBC) trapped in an optical tweezers trap, an increase in the spectrum of Brownian fluctuations for RBCs from a Plasmodium falciparum culture (due to increased rigidity) compared with normal RBCs was measured. A bystander effect was observed, whereby RBCs actually hosting the parasite had an effect on the physical properties of remaining non-hosting RBCs.

Light-driven three-dimensional rotational motion of dandelion-shaped microparticles

Hagay Shpaisman, David B. Ruffner, and David G. Grier
Chemically synthesized colloidal particles featuring large-scale surface asperities can be trapped and manipulated in fluid media through holographic optical trapping. Light scattering by these particles' surface features provides a mechanism for holographic optical traps also to exert torques on them, thereby setting them in steady rotation about arbitrary axes in three dimensions. When pairs of rotating particles are brought close enough that their surface features mesh, they form microscopic gear trains. These micro-opto-mechanical systems can be arranged in any desired three-dimensional configuration.


Optical Trapping of Microalgae at 735-1064 nm: Photodamage Assessment

Z. Pilát, J. Ježek, M. Šerý, M. Trtílek, L. Nedbal, P. Zemánek

Living microalgal cells differ from other cells that are used as objects for optical micromanipulation, in that they have strong light absorption in the visible range, and by the fact that their reaction centers are susceptible to photodamage. We trapped cells of the microalga Trachydiscus minutus using optical tweezers with laser wavelengths in the range from 735 nm to 1064 nm. The exposure to high photon flux density caused photodamage that was strongly wavelength dependent. The photochemical activity before and after exposure was assessed using a pulse amplitude modulation (PAM) technique. The photochemical activity was significantly and irreversibly suppressed by a 30s exposure to incident radiation at 735, 785, and 835 nm at a power of 25 mW. Irradiance at 885, 935 and 1064 nm had negligible effect at the same power. At a wavelength 1064 nm, a trapping power up to 218 mW caused no observable photodamage.