Wednesday, March 22, 2017

Combining FRET and optical tweezers to study RhoGTPases spatio-temporal dynamics upon local stimulation

Federico Iseppon, Luisa MR Napolitano, Vincent Torre, Dan Cojoc

Local stimulation with optical tweezers has been used to mimic natural stimuli that occur in biological processes such as cell migration or differentiation. Carriers (beads and lipid vesicles) with sizes down to 30 nm can be manipulated with a high spatial and temporal resolution: they are positioned with a sub-micrometric precision on a specific cell compartment and the beginning of the stimulation can be triggered with millisecond precision. RhoGTPases are a Ras-related family of proteins that regulate many different functions including cell polarity, microtubule dynamics and membrane transport pathways. Here we combine local stimulation with FRET microscopy to study RhoGTPases spatial and temporal activation following guidance cue local stimulation. We used two different vectors for local delivery: silica micro-beads and micro-sized lipid vesicles. The experimental methods associated with neuronal growth cone local stimulation are discussed in detail, as well as the analysis methods. Here we present a protocol that enables to study neuronal growth cone cytoskeleton rearrangements in response to a gradient of molecules in a way that better mimics physiological conditions, and it can be similarly applied to each secreted molecule involved in cell signaling.


Laser-accelerated self-assembly of colloidal particles at the water–air interface

Mincheng Zhong, Ziqiang Wang, and Yinmei Li

We experimentally demonstrate that optical tweezers can be used to accelerate the self-assembly of colloidal particles at a water–air interface in this Letter. The thermal flow induced by optical tweezers dominates the growth acceleration at the interface. Furthermore, optical tweezers are used to create a local growth peak at the growing front, which is used to study the preferential incorporation positions of incoming particles. The results show that the particles surfed with a strong Marangoni flow tend to fill the gap and smoothen the steep peaks. When the peak is smooth, the incoming particles incorporate the crystal homogeneously at the growing front.


On the motion control of microparticles by means of an electromagnetic field increasing with time for spectroscopic applications

A. Ch. Izmailov

The possibility of controlling the motion of microparticles by means of external electromagnetic fields (nonresonance laser radiation, in particular) that induce potential wells for such particles, which are characterized by fixed spatial distribution but deepen over time to a certain level, are analyzed. It is assumed that the particles are located in high vacuum and are affected by nondissipative external forces. Slowing down of relatively fast particles when they pass through the discussed potential wells is shown. Such slowing down of particles is demonstrated using a nonresonance laser beam with intensity increasing over time as an example. Specific features of particle dynamics in the electromagnetic fields under consideration in the case of a one-dimensional rectangular potential well are studied in detail based on simple analytical relations derived from the fundamental equations of classical mechanics. The methods of particle cooling and localization demonstrated in the present work can substantially increase spectroscopy resolution of various microparticles, including, under certain conditions, atoms and molecules.


Single-molecule mechanochemical characterization of E. coli pol III core catalytic activity

M. Nabuan Naufer, David A. Murison, Ioulia Rouzina, Penny J. Beuning, Mark C. Williams

Pol III core is the three-subunit subassembly of the E. coli replicative DNA polymerase III holoenzyme. It contains the catalytic polymerase subunit α, the 3′ → 5′ proofreading exonuclease ε, and a subunit of unknown function, θ. We employ optical tweezers to characterize pol III core activity on a single DNA substrate. We observe polymerization at applied template forces F < 25 pN and exonucleolysis at F > 30 pN. Both polymerization and exonucleolysis occur as a series of short bursts separated by pauses. For polymerization, the initiation rate after pausing is independent of force. In contrast, the exonucleolysis initiation rate depends strongly on force. The measured force and concentration dependence of exonucleolysis initiation fits well to a two-step reaction scheme in which pol III core binds bimolecularly to the primer-template junction, then converts at rate k2 into an exo-competent conformation. Fits to the force dependence of kinit show that exo initiation requires fluctuational opening of two base pairs, in agreement with temperature- and mismatch-dependent bulk biochemical assays. Taken together, our results support a model in which the pol and exo activities of pol III core are effectively independent, and in which recognition of the 3′ end of the primer by either α or ε is governed by the primer stability. Thus, binding to an unstable primer is the primary mechanism for mismatch recognition during proofreading, rather than an alternative model of duplex defect recognition.


Optical methods for measuring DNA folding

Adam D. Smith, Obinna A. Ukogu, Luka M. Devenica, Elizabeth D. White, Ashley R. Carter

One of the most important biological processes is the dynamic folding and unfolding of deoxyribonucleic acid (DNA). The folding process is crucial for DNA to fit within the boundaries of the cell, while the unfolding process is essential for DNA replication and transcription. To accommodate both processes, the cell employs a highly active folding mechanism that has been the subject of intense study over the last few decades. Still, many open questions remain. What are the pathways for folding or unfolding? How does the folding equilibrium shift? And, what is the energy landscape for a particular process? Here, we review these emerging questions and the in vitro, optical methods that have provided answers, introducing the topic for those physicists seeking to step into biology. Specifically, we discuss two iconic experiments for DNA folding, the tethered particle motion (TPM) experiment and the optical tweezers experiment.

Tuesday, March 21, 2017

Manipulating particles with light: radiation and gradient forces

David S Bradshaw and David L Andrews
The manipulation of matter with electromagnetic radiation is a capacity that has been known for over a century. However, the prominence of such optical effects only grew rapidly following the invention of optical tweezers in the 1980s. While both the original theory and the early trapping techniques are based on the radiation force, optical tweezing uses the gradient force. This paper aims to differentiate between these two clearly distinct types of optical forces, which are sometimes confused in the literature. We also discuss three completely separate forms of optical torque that can be applied to a particle, also due to an electromagnetic field. These involve the transfer of either spin or orbital angular momentum from the beam to the particle, depending on the character of the light, or the often overlooked alignment effect that can act on a cylindrical particle due to a gradient force.


Visual guide to optical tweezers

Isaac C D Lenton, Alexander B Stilgoe, Halina Rubinsztein-Dunlop and Timo A Nieminen

It is common to introduce optical tweezers using either geometric optics for large particles or the Rayleigh approximation for very small particles. These approaches are successful at conveying the key ideas behind optical tweezers in their respective regimes. However, they are insufficient for modelling particles of intermediate size and large particles with small features. For this, a full field approach provides greater insight into the mechanisms involved in trapping. The advances in computational capability over the last decade have led to better modelling and understanding of optical tweezers. Problems that were previously difficult to model computationally can now be solved using a variety of methods on modern systems. These advances in computational power allow for full field solutions to be visualised, leading to increased understanding of the fields and behaviour in various scenarios. In this paper we describe the operation of optical tweezers using full field simulations calculated using the finite difference time domain method. We use these simulations to visually illustrate various situations relevant to optical tweezers, from the basic operation of optical tweezers, to engineered particles and evanescent fields.


Controllable generation and manipulation of micro-bubbles in water with absorptive colloid particles by CW laser radiation

O. V. Angelsky, A. Ya. Bekshaev, P. P. Maksimyak, A. P. Maksimyak, S. G. Hanson, and S. M. Kontush

Micrometer-sized vapor-gas bubbles are formed due to local heating of a water suspension containing absorptive pigment particles of 100 nm diameter. The heating is performed by CW near-infrared (980 nm) laser radiation with controllable power, focused into a 100 μm spot within a 2 mm suspension layer. By changing the laser power, four regimes are realized: (1) bubble generation; (2) stable growth of the existing bubbles; (3) stationary existence of the bubbles and (4) the bubbles’ shrinkage and collapse. This behavior is interpreted based on the temperature conditions. The generation and evolution of single bubbles and ensembles of bubbles with controllable sizes and numbers is demonstrated. The bubbles are grouped within the laser-illuminated region and form quasi-ordered structures. They can easily be moved and transported controlled by the focal spot. The results are useful for applications associated with the precise manipulation, sorting and specific delivery in nano- and micro-engineering problems.


Strong plasmonic confinement and optical force in phosphorene pairs

Hua Lu, Yongkang Gong, Dong Mao, Xuetao Gan, and Jianlin Zhao

The plasmonic responses in the spatially separated phosphorene (single-layer black phosphorus) pairs are investigated, mainly containing the field enhancement, light confinement, and optical force. It is found that the strong anisotropic dispersion of black phosphorus gives rise to the direction-dependent symmetric and anti-symmetric plasmonic modes. Our results demonstrate that the symmetrical modes possess stronger field enhancement, higher light confinement, and larger optical force than the anti-symmetric modes in the nanoscale structures. Especially, the light confinement ratio and optical force for the symmetric mode along the armchair direction of black phosphorus can reach as high as >90% and >3000 pN/mW, respectively. These results may open a new door for the light manipulation at nanoscale and the design of black phosphorus based photonic devices.


Methods for Elucidation of DNA-Anticancer Drug Interactions and their Applications in the Development of New Drugs

Majus Misiak, Francesco Mantegazza, Giovanni L. Beretta

DNA damaging agents including anthracyclines, camptothecins and platinum drugs are among most frequently used drugs in the chemotherapeutic routine. Due to their relatively low selectivity for cancer cells, administration of these drugs is associated with adverse side effects, inherent genotoxicity with risk of developing secondary cancers. Development of new drugs, which could be spared of these drawbacks and characterize by improved antitumor efficacy, remains challenging yet vitally important task. These properties are in large part dictated by the selectivity of interaction between the drug and DNA and in this way the studies aimed at elucidating the complex interactions between ligand and DNA represent a key step in the drug development. Studies of the drug-DNA interactions encompass determination of DNA sequence specificity and mode of DNA binding as well as kinetic, dynamic and structural parameters of binding. Here, we consider the types of interactions between small molecule ligands and polynucleotides, how they are affected by DNA sequence and structure, and what is their significance for the antitumor activity. Based on this knowledge, we discuss the wide array of experimental techniques available to researchers for studying drug-DNA interactions, which include absorption and emission spectroscopies, NMR, magnetic and optical tweezers or atomic force microscopy. We show, using the clinical and experimental anticancer drugs as examples, how these methods provide various types of information and at the same time complement each other to provide full picture of drug- DNA interaction and aid in the development of new drugs.


Friday, March 17, 2017

On-chip laser processing for the development of multifunctional microfluidic chips

Huan Wang, Yong-Lai Zhang, Wei Wang, Hong Ding, Hong-Bo Sun

In the development of microfluidic chips, conventional 2D processing technologies contribute to the manufacturing of basic microchannel networks. Nevertheless, in the pursuit of versatile microfluidic chips, flexible integration of multifunctional components within a tiny chip is still challenging because a chip containing micro-channels is a non-flat substrate. Recently, on-chip laser processing (OCLP) technology has emerged as an appealing alternative to achieve chip functionalization through in situ fabrication of 3D microstructures. Here, the recent development of OCLP-enabled multifunctional microfluidic chips, including several accessible photochemical/photophysical schemes, and photosensitive materials permiting OCLP, is reviewed. To demonstrate the capability of OCLP technology, a series of typical micro-components fabricated using OCLP are introduced. The prospects and current challenges of this field are discussed.


Integrating Optical Tweezers with Up-converting Luminescence: A Non-amplification Analytical Platform for Quantitative Detection of MicroRNA-21 Sequences

Cheng-Yu Li, Di Cao, Chong-Yang Song, Chun-Miao Xu, Yu-Yan Ma, Zhi-Ling Zhang, Dai-Wen Pang and Hongwu Tang

Sensitive non-amplification detection of biomolecules is a major concern in analytical science. Here, we report a single-microsphere based imaging assay method by integrating up-converting luminescence with optical tweezers (OT) for detecting microRNAs. By taking advantages of the anti-Stokes luminescence and a minimal three-dimensional excitation region formed by OT, there exist a very low background signal around a trapped sandwich structure complex microsphere enriched with targets (miRNA-21). This effect is further enhanced by combining it with a sensitive imaging detector (EMCCD) and thus achieves a competitive detection limit of 12 fM with quite sound selectivity and no complicated signal amplification designs. As a proof-of-concept study, this analytical methodology can also be employed to quantify the amount of miRNA-21 sequences from as low as 100 cancer cells, making it a promising new means for biomedical applications.


Integrated Optofluidic Chip for Low-Volume Fluid Viscosity Measurement

Tie Yang, Giovanni Nava, Valerio Vitali, Francesca Bragheri, Roberto Osellame, Tommaso Bellini, Ilaria Cristiani and Paolo Minzioni

In the present work, an integrated optofluidic chip for fluid viscosity measurements in the range from 1 mPa·s to 100 mPa·s is proposed. The device allows the use of small sample volumes (<1 µL) and the measurement of viscosity as a function of temperature. Thanks to the precise control of the force exerted on dielectric spheres by optical beams, the viscosity of fluids is assessed by comparing the experimentally observed movement of dielectric beads produced by the optical forces with that expected by numerical calculations. The chip and the developed technique are validated by analyzing several fluids, such as Milli-Q water, ethanol and water–glycerol mixtures. The results show a good agreement between the experimental values and those reported in the literature. The extremely reduced volume of the sample required and the high flexibility of this technique make it a good candidate for measuring a wide range of viscosity values as well as for the analysis of nonlinear viscosity in complex fluids.


Vitamin E nanoemulsion activity on stored red blood cells

C. A. L. Silva, C. A. Azevedo Filho, G. Pereira, D. C. N. Silva, M. C. A. B. Castro, A. F. Almeida, S. C. A. Lucena, B. S. Santos, M. L. Barjas-Castro, A. Fontes

Stored red blood cells (RBCs) undergo numerous changes that have been termed RBC storage lesion, which can be related to oxidative damage. Vitamin E is an important antioxidant, acting on cell lipids. Thus, this study aimed to investigate vitamin E activity on stored RBCs.
We prepared a vitamin E nanoemulsion that was added to RBC units and stored at 4 °C. Controls, without vitamin E, were kept under the same conditions. Reactive oxygen species (ROS) production was monitored for up to 35 days of storage. RBC elasticity was also evaluated using an optical tweezer system.
Vitamin E-treated samples presented a significant decrease in ROS production. Additionally, the elastic constant for vitamin E-treated RBCs did not differ from the control.
Vitamin E decreased the amount of ROS in stored RBCs. Because vitamin E acts on lipid oxidation, results suggest that protein oxidation should also be considered a key factor for erythrocyte elastic properties. Thus, further studies combining vitamin E with protein antioxidants deserve attention, aiming to better preserve overall stored RBC properties.


Thermophoretic Tweezers for Low-Power and Versatile Manipulation of Biological Cells

Linhan Lin, Xiaolei Peng, Xiaoling Wei, Zhangming Mao, Chong Xie, and Yuebing Zheng

Optical manipulation of biological cells and nanoparticles is significantly important in life sciences, early disease diagnosis, and nanomanufacturing. However, low-power and versatile all-optical manipulation has remained elusive. Herein, we have achieved light-directed versatile thermophoretic manipulation of biological cells at an optical power 100–1000 times lower than that of optical tweezers. By harnessing the permittivity gradient in the electric double layer of the charged surface of the cell membrane, we succeed at the low-power trapping of suspended biological cells within a light-controlled temperature gradient field. Furthermore, through dynamic control of optothermal potentials using a digital micromirror device, we have achieved arbitrary spatial arrangements of cells at a resolution of ∼100 nm and precise rotation of both single and assemblies of cells. Our thermophoretic tweezers will find applications in cellular biology, nanomedicine, and tissue engineering.


Thursday, March 16, 2017

Aberration compensation for optical trapping of cells within living mice

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

Optical tweezers have been used to trap and manipulate microparticles within living animals. When the optical trap is constructed with an oil-immersion objective, it suffers from spherical aberration. There have been many investigations on the influence of spherical aberration when the particles are trapped in a water medium. However, the dependence of optical force on trapping depth is still ambiguous when the trapped particles are immersed in a high refractive index medium (such as biological tissue, refractive index solution) in experiments. In this paper, the microparticles are immersed in an aqueous solution of glycerol to mimic the cells within biological tissue. As the trapping laser is focused into the specimen, spherical aberration is introduced, degrading the optical trapping performance. It is similar to trapping in water; altering the effective tube length can also compensate for the spherical aberration of the optical trap in a high refractive index medium. Finally, the cells in living mice are trapped by the optical tweezers with the help of spherical aberration compensation.


Optical tweezers studies of transcription by eukaryotic RNA polymerases

Ana Lisica, Stephan W. Grill

Transcription is the first step in the expression of genetic information and it is carried out by large macromolecular enzymes called RNA polymerases. Transcription has been studied for many years and with a myriad of experimental techniques, ranging from bulk studies to high-resolution transcript sequencing. In this review, we emphasise the advantages of using single-molecule techniques, particularly optical tweezers, to study transcription dynamics. We give an overview of the latest results in the single-molecule transcription field, focusing on transcription by eukaryotic RNA polymerases. Finally, we evaluate recent quantitative models that describe the biophysics of RNA polymerase translocation and backtracking dynamics.


Plasmonic Particles with Unique Optical Interaction and Mechanical Motion Properties

Jiafang Li, Jing Liu, Ximin Tian, Zhi-Yuan Li

Metal nanoparticles have unique localized surface plasmon resonance (SPR) properties due to the strong interaction of localized surface plasmon polariton (SPP) with incident light. This review will cover some of our recent theoretical and experimental studies on exploring the unique optical interaction and mechanical motion properties of plasmonic particles that originate from SPR enhanced light-matter interaction. Firstly, the efficient enhancement of both the fluorescence excitation and emission process of dye molecules by the double SPR modes (longitudinal and transverse modes) in gold nanorods, and surface plasmon amplification in metal nanoparticles with gain is discussed. Secondly, it is theoretically demonstrated that two basic physical processes of molecules interacting with light, i.e., the elastic Rayleigh scattering and inelastic Raman scattering, will strongly intertwine and correlate with each other in many plasmonic surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) nanosystems. Thirdly, it is experimentally shown that SPR can enhance the optical force and torque of nanoparticles embedded within non-intrusive optical tweezers. The work presented in this review shows that plasmonic particles can possess unique optical interaction and mechanical motion properties when their geometries are deliberately controlled.


Hidden topological constellations and polyvalent charges in chiral nematic droplets

Gregor Posnjak, Simon Čopar & Igor Muševič

Topology has an increasingly important role in the physics of condensed matter, quantum systems, material science, photonics and biology, with spectacular realizations of topological concepts in liquid crystals. Here we report on long-lived hidden topological states in thermally quenched, chiral nematic droplets, formed from string-like, triangular and polyhedral constellations of monovalent and polyvalent singular point defects. These topological defects are regularly packed into a spherical liquid volume and stabilized by the elastic energy barrier due to the helical structure and confinement of the liquid crystal in the micro-sphere. We observe, for the first time, topological three-dimensional point defects of the quantized hedgehog charge q=−2, −3. These higher-charge defects act as ideal polyvalent artificial atoms, binding the defects into polyhedral constellations representing topological molecules.


Extending calibration-free force measurements to optically-trapped rod-shaped samples

Frederic Català, Ferran Marsà, Mario Montes-Usategui, Arnau Farré & Estela Martín-Badosa

Optical trapping has become an optimal choice for biological research at the microscale due to its non-invasive performance and accessibility for quantitative studies, especially on the forces involved in biological processes. However, reliable force measurements depend on the calibration of the optical traps, which is different for each experiment and hence requires high control of the local variables, especially of the trapped object geometry. Many biological samples have an elongated, rod-like shape, such as chromosomes, intracellular organelles (e.g., peroxisomes), membrane tubules, certain microalgae, and a wide variety of bacteria and parasites. This type of samples often requires several optical traps to stabilize and orient them in the correct spatial direction, making it more difficult to determine the total force applied. Here, we manipulate glass microcylinders with holographic optical tweezers and show the accurate measurement of drag forces by calibration-free direct detection of beam momentum. The agreement between our results and slender-body hydrodynamic theoretical calculations indicates potential for this force-sensing method in studying protracted, rod-shaped specimens.


Wednesday, March 15, 2017

Fiber-Based Optical Gun for Particle Shooting

Hongchang Deng, Yaxun Zhang, Tinging Yuan, Xiaotong Zhang, Yu Zhang, Zhihai Liu, and Libo Yuan

We proposed and fabricated a fiber-based optical gun for both particle trapping and shooting. The all-fiber device is made of a coaxial core optical fiber with a center core and a coaxial circular core. The fiber has a cone-frustum-shaped tip to enable the circular core to generate a focused ring light as a trapping beam, providing a stable 3D trapping potential well. When a small particle is trapped, a Gaussian beam is launched as a shooting light at the fiber center core to push the particle away from the fiber tip along the propagation direction of the beam. Here, we find that (1) the highly focused ring field with considerably lowered focusing intensity can generate a very stable particle-trapping potential well in three dimensions and the photothermal effect is also greatly reduced due to the lower optical power requirement for trapping and (2) the shooting light with a Gaussian profile not only supplies a radiation pushing force on the small particle, but also has restrictions and guiding effects as a gun barrel to propel the small particle out of the trapping well at a high speed along the beam propagation direction. The particle shooting distance can reach several hundreds of micrometers. Transverse deviation from the optical axis can be controlled within several micrometers under disturbances of ambient fluid flow. Our proposed method extends the potential applications of fiber-based optical manipulation, e.g., microparticle sorting in biology, accurate delivery of microparticles of a drug to the target cells, and observation of drug synergism.


Resonance optical trapping of individual dye-doped polystyrene particles with blue- and red-detuned lasers

Tetsuhiro Kudo, Hajime Ishihara, and Hiroshi Masuhara

We demonstrate resonance optical trapping of individual dye-doped polystyrene particles with blue- and red-detuned lasers whose energy are higher and lower compared to electronic transition of the dye molecules, respectively. Through the measurement on how long individual particles are trapped at the focus, we here show that immobilization time of dye-doped particles becomes longer than that of bare ones. We directly confirm that the immobilization time of dye-doped particles trapped by the blue-detuned laser becomes longer than that by the red-detuned one. These findings are well interpreted by our previous theoretical proposal based on nonlinear optical response under intense laser field. It is discussed that the present result is an important step toward efficient and selective manipulation of molecules, quantum dots, nanoparticles, and various nanomaterials based on their quantum mechanical properties.


Optical tug-of-war tweezers: shaping light for dynamic control of bacterial cells

Joshua Lamstein, Anna Bezryadina, Daryl Preece, Joseph C. Chen, and Zhigang Chen

We design and demonstrate new types of optical tweezers with lateral pulling forces that allow full control of biological samples with complex geometric shapes. With appropriate beam shaping, the dual tug-of-war tweezers effectively hold and stretch elongated biological objects of different sizes, and the triangular tug-of-war tweezers with threefold rotational symmetry steadily hold asymmetric objects in the plane of observation and exert stretching forces along three directions. We successfully apply these tweezers to manipulate microparticles and bacterial cells in aqueous media.


Interactions of mucins with the Tn or Sialyl Tn cancer antigens including MUC1 are due to GalNAc–GalNAc interactions

Kristin E Haugstad,  Soosan Hadjialirezaei,  Bjørn T Stokke,  C Fred Brewer, Thomas A Gerken,  Joy Burchell,  Gianfranco Picco,  Marit Sletmoen

The molecular mechanism(s) underlying the enhanced self-interactions of mucins possessing the Tn (GalNAcα1-Ser/Thr) or STn (NeuNAcα2-6GalNAcα1-Ser/Thr) cancer markers were investigated using optical tweezers (OT). The mucins examined included modified porcine submaxillary mucin containing the Tn epitope (Tn-PSM), ovine submaxillary mucin with the STn epitope (STn-OSM), and recombinant MUC1 analogs with either the Tn and STn epitope. OT experiments in which the mucins were immobilized onto polystyrene beads revealed identical self-interaction characteristics for all mucins. Identical binding strength and energy landscape characteristics were also observed for synthetic polymers displaying multiple GalNAc decorations. Polystyrene beads without immobilized mucins showed no self-interactions and also no interactions with mucin-decorated polystyrene beads. Taken together, the experimental data suggest that in these molecules, the GalNAc residue mediates interactions independent of the anchoring polymer backbone. Furthermore, GalNAc–GalNAc interactions appear to be responsible for self-interactions of mucins decorated with the STn epitope. Hence, Tn-MUC1 and STn-MUC1 undergo self-interactions mediated by the GalNAc residue in both epitopes, suggesting a possible molecular role in cancer. MUC1 possessing the T (Galβ1-3GalNAcα1-Ser/Thr) or ST antigen (NeuNAcα2-3Galβ1-3GalNAcα1-Ser/Thr) failed to show self-interactions. However, in the case of ST-MUC1, self-interactions were observed after subsequent treatment with neuraminidase and β-galactosidase. This enzymatic treatment is expected to introduce Tn-epitopes and these observations thus further strengthen the conclusion that the observed interactions are mediated by the GalNAc groups.


Effective computational modeling of erythrocyte electro-deformation

Nicola A. Nodargi, Paolo Bisegna, Federica Caselli

Due to its crucial role in pathophysiology, erythrocyte deformability represents a subject of intense experimental and modeling research. Here a computational approach to electro-deformation for erythrocyte mechanical characterization is presented. Strong points of the proposed strategy are: (1) an accurate computation of the mechanical actions induced on the cell by the electric field, (2) a microstructurally-based continuum model of the erythrocyte mechanical behavior, (3) an original rotation-free shell finite element, especially suited to the application in hand. As proved by the numerical results, the developed tool is effective and sound, and can foster the role of electro-deformation in single-cell mechanical phenotyping.


Friday, March 3, 2017

Focal adhesion kinase activity is required for actomyosin contractility-based invasion of cells into dense 3D matrices

Claudia T. Mierke, Tony Fischer, Stefanie Puder, Tom Kunschmann, Birga Soetje & Wolfgang H. Ziegler

The focal adhesion kinase (FAK) regulates the dynamics of integrin-based cell adhesions important for motility. FAK’s activity regulation is involved in stress-sensing and focal-adhesion turnover. The effect of FAK on 3D migration and cellular mechanics is unclear. We analyzed FAK knock-out mouse embryonic fibroblasts and cells expressing a kinase-dead FAK mutant, R454-FAK, in comparison to FAK wild-type cells. FAK knock-out and FAKR454/R454 cells invade dense 3D matrices less efficiently. These results are supported by FAK knock-down in wild-type fibroblasts and MDA-MB-231 human breast cancer cells showing reduced invasiveness. Pharmacological interventions indicate that in 3D matrices, cells deficient in FAK or kinase-activity behave similarly to wild-type cells treated with inhibitors of Src-activity or actomyosin-contractility. Using magnetic tweezers experiments, FAKR454/R454 cells are shown to be softer and exhibit impaired adhesion to fibronectin and collagen, which is consistent with their reduced 3D invasiveness. In line with this, FAKR454/R454 cells cannot contract the matrix in contrast to FAK wild-type cells. Finally, our findings demonstrate that active FAK facilitates 3D matrix invasion through increased cellular stiffness and transmission of actomyosin-dependent contractile force in dense 3D extracellular matrices.


Anaphase A: Disassembling Microtubules Move Chromosomes toward Spindle Poles

Charles L. Asbury

The separation of sister chromatids during anaphase is the culmination of mitosis and one of the most strikingly beautiful examples of cellular movement. It consists of two distinct processes: Anaphase A, the movement of chromosomes toward spindle poles via shortening of the connecting fibers, and anaphase B, separation of the two poles from one another via spindle elongation. I focus here on anaphase A chromosome-to-pole movement. The chapter begins by summarizing classical observations of chromosome movements, which support the current understanding of anaphase mechanisms. Live cell fluorescence microscopy studies showed that poleward chromosome movement is associated with disassembly of the kinetochore-attached microtubule fibers that link chromosomes to poles. Microtubule-marking techniques established that kinetochore-fiber disassembly often occurs through loss of tubulin subunits from the kinetochore-attached plus ends. In addition, kinetochore-fiber disassembly in many cells occurs partly through ‘flux’, where the microtubules flow continuously toward the poles and tubulin subunits are lost from minus ends. Molecular mechanistic models for how load-bearing attachments are maintained to disassembling microtubule ends, and how the forces are generated to drive these disassembly-coupled movements, are discussed.


Radiation forces on a Rayleigh dielectric sphere produced by highly focused parabolic scaling Bessel beams

Mengwen Guo and Daomu Zhao

The radiation forces on a Rayleigh dielectric particle induced by a highly focused parabolic scaling Bessel beam (PSBB) are investigated. Numerical results show that the zero-order PSBB can be used to trap a high-index particle at the focus and near the focus by the first-order PSBB. For the low-index particle, it can be guided or confined in the dark core of the nonzero-order PSBB but cannot be stably trapped in this single-beam trap. Further, we analyze the condition of trapping stability. It is found that the lower limit in the particle radius for stable trapping is different for different orders.


Direct measurement of cortical force generation and polarization in a living parasite

Rachel V. Stadler, Lauren A. White, Ke Hu, Brian P. Helmke, and William H. Guilford

Apicomplexa is a large phylum of intracellular parasites that are notable for the diseases they cause, including toxoplasmosis, malaria and cryptosporidiosis. A conserved motile system is critical to their lifecycles as it drives directional gliding motility between cells, as well as invasion of and egress from host cells. However, our understanding of this system is limited by a lack of measurements of the forces driving parasite motion. We used a laser trap to measure the function of the motility apparatus of living Toxoplasma gondii by adhering a microsphere to the surface of an immobilized parasite. Motion of the microsphere reflected underlying forces exerted by the motile apparatus. We found that force generated at the parasite surface begins with no preferential directionality, but becomes directed toward the rear of the cell after a period of time. The transition from non-directional to directional force generation occurs on spatial intervals consistent with the lateral periodicity of structures associated with the membrane pellicle, and is influenced by the kinetics of actin filament polymerization and cytoplasmic calcium. A lysine methyltransferase regulates both the magnitude and polarization of the force. Our work provides a novel means to dissect the motile mechanisms of these pathogens.


Directed rotational motion of birefringent particles by randomly changing the barrier height at the threshold in a washboard potential

Basudev Roy, Erik Schaffer

This communication presents a simulation study of the directed rotational motion of a birefringent spherical particle trapped in optical tweezers with randomly varying ellipticity of a trapping light at the point of threshold. When noise is not applied, the potential barrier due to the linear component of the polarization is simulated to be sufficient to prevent directed rotation till a certain threshold value of ellipticity. Random variations to the ellipticity cause random variations in the barrier height, including instants when the barrier is lower than the threshold energy level. It is due to this that the particle exhibits directed rotational motion at ellipticity lower than the threshold value. We also examine the rotational velocity of the birefringent particle as a function of the extent of zero-mean random noise applied to the ellipticity.


Thursday, March 2, 2017

Atomization efficiency and photon yield in laser-induced breakdown spectroscopy analysis of single nanoparticles in an optical trap

Pablo Purohit, Francisco J. Fortes, J. Javier Laserna

Laser-induced breakdown spectroscopy (LIBS) was employed for investigating the influence of particle size on the dissociation efficiency and the absolute production of photons per mass unit of airborne solid graphite spheres under single-particle regime. Particles of average diameter of 400 nm were probed and compared with 2 μm particles. Samples were first catapulted into aerosol form and then secluded in an optical trap set by a 532 nm laser. Trap stability was quantified before subjecting particles to LIBS analysis. Fine alignment of the different lines comprising the optical catapulting-optical trapping-laser-induced breakdown spectroscopy instrument and tuning of excitation parameters conditioning the LIBS signal such as fluence and acquisition delay are described in detail with the ultimate goal of acquiring clear spectroscopic data on masses as low as 75 fg. The atomization efficiency and the photon yield increase as the particle size becomes smaller. Time-resolved plasma imaging studies were conducted to elucidate the mechanisms leading to particle disintegration and excitation.


Rich stochastic dynamics of co-doped Er:Yb fluorescence upconversion nanoparticles in the presence of thermal, non-conservative, harmonic and optical forces

Rene A Nome, Cecilia Sorbello, Matías Jobbágy, Beatriz C Barja, Vitor Sanches, Joyce S Cruz and Vinicius F Aguiar

The stochastic dynamics of individual co-doped Er:Yb upconversion nanoparticles (UCNP) were investigated from experiments and simulations. The UCNP were characterized by high-resolution scanning electron microscopy, dynamic light scattering, and zeta potential measurements. Single UCNP measurements were performed by fluorescence upconversion micro-spectroscopy and optical trapping. The mean-square displacement (MSD) from single UCNP exhibited a time-dependent diffusion coefficient which was compared with Brownian dynamics simulations of a viscoelastic model of harmonically bound spheres. Experimental time-dependent two-dimensional trajectories of individual UCNP revealed correlated two-dimensional nanoparticle motion. The measurements were compared with stochastic trajectories calculated in the presence of a non-conservative rotational force field. Overall, the complex interplay of UCNP adhesion, thermal fluctuations and optical forces led to a rich stochastic behavior of these nanoparticles.


Hot-nanoparticle-mediated fusion of selected cells

Azra Bahadori, Lene B. Oddershede, Poul M. Bendix

Complete fusion of two selected cells allows for the creation of novel hybrid cells with inherited genetic properties from both original cells. Alternatively, via fusion of a selected cell with a selected vesicle, chemicals or genes can be directly delivered into the cell of interest, to control cellular reactions or gene expression. Here, we demonstrate how to perform an optically controlled fusion of two selected cells or of one cell and one vesicle. Fusion is mediated by laser irradiating plasmonic gold nanoparticles optically trapped between two cells (or a vesicle and a cell) of interest. This hot-particle-mediated fusion causes total mixing of the two cytoplasms and the two cell membranes resulting in formation of a new hybrid cell with an intact cell membrane and enzymatic activity following fusion. Similarly, fusion between a vesicle and a cell results in delivery of the vesicle cargo to the cytoplasm, and after fusion, the cell shows signs of viability. The method is an implementation of targeted drug delivery at the single-cell level and has a great potential for cellular control and design.


Stable optical trapping and sensitive characterization of nanostructures using standing-wave Raman tweezers

Mu-ying Wu, Dong-xiong Ling, Lin Ling, William Li & Yong-qing Li

Optical manipulation and label-free characterization of nanoscale structures open up new possibilities for assembly and control of nanodevices and biomolecules. Optical tweezers integrated with Raman spectroscopy allows analyzing a single trapped particle, but is generally less effective for individual nanoparticles. The main challenge is the weak gradient force on nanoparticles that is insufficient to overcome the destabilizing effect of scattering force and Brownian motion. Here, we present standing-wave Raman tweezers for stable trapping and sensitive characterization of single isolated nanostructures with a low laser power by combining a standing-wave optical trap with confocal Raman spectroscopy. This scheme has stronger intensity gradients and balanced scattering forces, and thus can be used to analyze many nanoparticles that cannot be measured with single-beam Raman tweezers, including individual single-walled carbon nanotubes (SWCNT), graphene flakes, biological particles, SERS-active metal nanoparticles, and high-refractive semiconductor nanoparticles. This would enable sorting and characterization of specific SWCNTs and other nanoparticles based on their increased Raman fingerprints.


Optical Trap Loading of Dielectric Microparticles In Air

Haesung Park, Thomas W. LeBrun

We demonstrate a method to trap a selected dielectric microparticle in air using radiation pressure from a single-beam gradient optical trap. Randomly scattered dielectric microparticles adhered to a glass substrate are momentarily detached using ultrasonic vibrations generated by a piezoelectric transducer (PZT). Then, the optical beam focused on a selected particle lifts it up to the optical trap while the vibrationally excited microparticles fall back to the substrate. A particle may be trapped at the nominal focus of the trapping beam or at a position above the focus (referred to here as the levitation position) where gravity provides the restoring force. After the measurement, the trapped particle can be placed at a desired position on the substrate in a controlled manner.
In this protocol, an experimental procedure for selective optical trap loading in air is outlined. First, the experimental setup is briefly introduced. Second, the design and fabrication of a PZT holder and a sample enclosure are illustrated in detail. The optical trap loading of a selected microparticle is then demonstrated with step-by-step instructions including sample preparation, launching into the trap, and use of electrostatic force to excite particle motion in the trap and measure charge. Finally, we present recorded particle trajectories of Brownian and ballistic motions of a trapped microparticle in air. These trajectories can be used to measure stiffness or to verify optical alignment through time domain and frequency domain analysis. Selective trap loading enables optical tweezers to track a particle and its changes over repeated trap loadings in a reversible manner, thereby enabling studies of particle-surface interaction.


Local Arp2/3-dependent actin assembly modulates applied traction force during apCAM adhesion site maturation

Kenneth B. Buck, Andrew W. Schaefer, Vincent T. Schoonderwoert, Matthew S. Creamer, Eric R. Dufresne, and Paul Forscher

Homophilic binding of immunoglobulin superfamily molecules such as the Aplysia cell adhesion molecule (apCAM) leads to actin filament assembly near nascent adhesion sites. Such actin assembly can generate significant localized forces that have not been characterized in the larger context of axon growth and guidance. We used apCAM-coated bead substrates applied to the surface of neuronal growth cones to characterize the development of forces evoked by varying stiffness of mechanical restraint. Unrestrained bead propulsion matched or exceeded rates of retrograde network flow and was dependent on Arp2/3 complex activity. Analysis of growth cone forces applied to beads at low stiffness of restraint revealed switching between two states: frictional coupling to retrograde flow and Arp2/3-dependent propulsion. Stiff mechanical restraint led to formation of an extensive actin cup matching the geometric profile of the bead target and forward growth cone translocation; pharmacological inhibition of the Arp2/3 complex or Rac attenuated F-actin assembly near bead binding sites, decreased the efficacy of growth responses, and blocked accumulation of signaling molecules associated with nascent adhesions. These studies introduce a new model for regulation of traction force in which local actin assembly forces buffer nascent adhesion sites from the mechanical effects of retrograde flow.