Friday, January 31, 2014

Folding a stable RNA pseudoknot through rearrangement of two hairpin structures

Yi-Ju Wu, Cheng-Han Wu, Athena Yi-Chun Yeh and Jin-Der Wen

Folding messenger RNA into specific structures is a common regulatory mechanism involved in translation. In Escherichia coli, the operator of the rpsO gene transcript folds into a pseudoknot or double-hairpin conformation. S15, the gene product, binds only to the pseudoknot, thereby repressing its own synthesis when it is present in excess in the cell. The two RNA conformations have been proposed to exist in equilibrium. However, it remained unclear how structural changes can be achieved between these two topologically distinct conformations. We used optical tweezers to study the structural dynamics and rearrangements of the rpsO operator RNA at the single-molecule level. We discovered that the two RNA structures can be interchanged spontaneously and the pseudoknot can exist in conformations that exhibit various levels of stability. Conversion from the double hairpin to a pseudoknot through potential hairpin–hairpin interactions favoured the high-stability conformation. By contrast, mutations that blocked the formation of a hairpin typically resulted in alternative low-stability pseudoknots. These results demonstrate that specific tertiary interactions of RNA can be established and modulated based on the interactions and rearrangements between secondary structural components. Our findings provide new insight into the RNA folding pathway that leads to a regulatory conformation for target protein binding.


Optimal beam diameter for lateral optical forces on microspheres at a water-air interface

Mincheng Zhong, Xi Wang, Jinhua Zhou, Ziqiang Wang, and Yinmei Li

Optical tweezers with a low numerical aperture microscope objective is used to manipulate the microspheres at the water-air interface. In this letter, we determine the optimal optical trap for the lateral manipulation of microspheres at a water-air interface. The experimental results show that the trapping force is influenced by the expansion of the trapping beam at the back aperture of the objective. The optimal filling ratio of 0.65 is suggested for lateral optical manipulation at the water-air interface. The lateral trapping forces at the water-air interface are theoretically investigated with the ray-optics model. The numerical results show that the lateral trapping forces can be changed by shrinking the diameter of the trapping laser beam. The numerical results are in accordance with the experimental results.


Size-dependent position of a single aerosol droplet in a Bessel beam trap

Thomas C Preston, Bernard J Mason, Jonathan P Reid, David Luckhaus and Ruth Signorell
The equilibrium position of an aerosol droplet trapped in a counter-propagating Bessel beam and gas flow is studied both experimentally and theoretically. This provides an aerosol analogue to the separation of particles of differing size and refractive index in counter-propagating laser beam and liquid flow, referred to as optical chromatography. Using the model system of a pure glycerol droplet it is found that, as evaporation takes place and the size of the droplet decreases, the size-dependent equilibrium position does not change in a simple monotonic fashion. Instead, the position of the droplet is greatly affected by the excitation of whispering gallery modes. This leads to sharp peaks in the equilibrium position curve, not unlike those that occur in single particle spectroscopy. The conditions necessary to excite whispering gallery modes are thoroughly investigated.


Single-Molecule Fluorescence and in Vivo Optical Traps: How Multiple Dyneins and Kinesins Interact

Benjamin H. Blehm and Paul R. Selvin

In this review, we describe experimental systems at multiple levels of complexity, including single-motor-type in vitro assays, multimotor in vitro assays, purified-organelle in vitro assays, and finally in vivo cellular assays (Figure 1). This spread of experiments allows an unprecedented view of the transport complex, as kinesin and dynein can be observed with differing components of the transport complex (i.e., different levels of accessory proteins) and in different environments. Through the combination of measurements at all of these levels of complexity, the ability to parse out the function of parts of the transport complex, and reconstitute it in vitro, becomes a real possibility.


Tuesday, January 28, 2014

Reconstruction and Identification of DNA Sequence Landscapes from Unzipping Experiments at Equilibrium

Carlo Barbieri, Simona Cocco, Thomas Jorg, Rémi Monasson

Two methods for reconstructing the free-energy landscape of a DNA molecule from the knowledge of the equilibrium unzipping force versus extension signal are introduced: a simple and fast procedure, based on a parametric representation of the experimental force signal, and a maximum-likelihood inference of coarse-grained free-energy parameters. In addition, we propose a force alignment procedure to correct for the drift in the experimental measure of the opening position, a major source of error. For unzipping data obtained by Huguet et al., the reconstructed basepair (bp) free energies agree with the running average of the true free energies on a 20–50 bp scale, depending on the region in the sequence. Features of the landscape at a smaller scale (5–10 bp) could be recovered in favorable regions at the beginning of the molecule. Based on the analysis of synthetic data corresponding to the 16S rDNA gene of bacteria, we show that our approach could be used to identify specific DNA sequences among thousands of homologous sequences in a database.


Anti-frameshifting ligand reduces the conformational plasticity of the SARS virus pseudoknot

Dustin B Ritchie , Jingchyuan Soong , William Sikkema , and Michael T. Woodside

Programmed −1 ribosomal frameshifting (−1 PRF) stimulated by mRNA pseudoknots regulates gene expression in many viruses, making pseudoknots potential targets for anti-viral drugs. The mechanism by which pseudoknots trigger −1 PRF, however, remains controversial, with several competing models. Recent work showed that high −1 PRF efficiency was linked to high pseudoknot conformational plasticity via the formation of alternate conformers. We tested whether pseudoknots bound with an anti-frameshifting ligand exhibited a similar correlation between conformational plasticity and −1 PRF efficiency, by measuring the effects of a ligand which was found to inhibit −1 PRF in the SARS coronavirus on the conformational dynamics of the SARS pseudoknot. Using single-molecule force spectroscopy to unfold pseudoknots mechanically, we found that the ligand binding effectively abolished the formation of alternate conformers. This result extends the connection between −1 PRF and conformational dynamics, and moreover suggests that targeting the conformational dynamics of pseudoknots may be an effective strategy for anti-viral drug design.


Multi-Dimensional Manipulation of Yeast Cells Using a LP_11 Mode Beam

Zhang, Y.; Liang, P. ; Lei, J. ; Wang, L. ; Liu, Z. ; Yang, J. ; Yuan, L.

We report a new method for constructing a single fiber optical tweezers, which can realize multi-dimensional manipulation of trapped yeast cells by using a LP$_{bf 11}$ mode beam excited in a normal communication single core optical fiber. This allows a simple and convenient orientation control on the trapped yeast cells. The LP$_{bf 11}$ mode beam, both for generating trap and orientation manipulation, has been modulated by using the tension and twisting loaded on the fiber. We present experimental results of controllable deflection and orientation manipulation of the yeast cells. To the best of our knowledge, it is the first report about the trapped yeast cells being driven by the normal single fiber optical tweezers in multi dimensions, and it constitutes a new development for single fiber optical trapping and makes possible of more practical applications in the biomedical research fields.


Dynamically reconfigurable Fibre Optical Spanner

Thorsten Kolb, Sahradha Albert, Michael Haug and Graeme Whyte

In this paper we describe a pneumatically actuated fibre-optic spanner integrated into a microfluidic Lab-on-a-Chip device for the controlled trapping and rotation of living cells. The dynamic nature of the system allows interactive control over the rotation speed with the same optical power. The use of a multi-layer device makes it possible to rotate a cell both in the imaging plane and also in a perpendicular plane allowing tomographic imaging of the trapped living cell. The integrated device allows easy operation and by combining it with high-resolution confocal microscopy we show for the first time that the pattern of rotation can give information regarding the sub-cellular composition of a rotated cell.


Controlled Particle Collision Leads to Direct Observation of Docking and Fusion of Lipid Droplets in an Optical Trap

Chiran Ghimire , Deepak Koirala , Malcom B. Mathis , Edgar Eduard Kooijman , and Hanbin Mao

As an intracellular organelle, phospholipid coated lipid droplets have shown increasing importance due to their expanding biological functions other than the lipid storage. The growing biological significance necessitates a close scrutiny on lipid droplets, which have been proposed to mature in a cell through processes such as fusions. Unlike phospholipid vesicles that are well known to fuse through docking and hemifusion steps, little is known on the fusion of lipid droplets. Herein, we used laser tweezers to capture two micrometer-sized 1,2,3-trioleoylglycerol (triolein) droplets coated with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) that closely resemble intracellular lipid droplets. We started the fusion processes by a well-controlled collision between the two lipid droplets in phosphate buffers at pH 7.4. By monitoring the change in the pathway of a trapping laser that captures the collided lipid droplets, docking and fusion events were clearly distinguished for the first time and their lifetimes were determined with a resolution of 10 microseconds after post-synchronization analyses. Our method revealed that the rate limiting docking process is affected by anions according to a Hofmeister series, which sheds light on the important role of interfacial water shedding during the process. During the actual fusion, the kinetics between bare triolein droplets is faster than lipid droplets, suggesting that breaking of phospholipid coating is involved in the process. This scenario was further supported by direct observation of a short-lived hemifusion state with ~ 46 milliseconds lifetime in POPC coated lipid droplets, but not in bare triolein droplets.


Optical tweezers assisted imaging of the Z-ring in Escherichia coli: measuring its radial width

G Carmon, P Kumar and M Feingold

Using single-beam, oscillating optical tweezers we can trap and rotate rod-shaped bacterial cells with respect to the optical axis. This technique allows imaging fluorescently labeled three-dimensional sub-cellular structures from different, optimized viewpoints. To illustrate our method we measure D, the radial width of the Z-ring in unconstricted Escherichia coli. We use cells that express FtsZ-GFP and have their cytoplasmic membrane stained with FM4-64. In a vertically oriented cell, both the Z-ring and the cytoplasmic membrane images appear as symmetric circular structures that lend themselves to quantitative analysis. We found that D cong 100 nm, much larger than expected.


Thursday, January 23, 2014

Tapered nanofiber trapping of high-refractive-index nanoparticles

Jon D. Swaim, Joachim Knittel and Warwick P. Bowen

A nanofiber-based optical tweezer is demonstrated. Trapping is achieved by combining attractive near-field optical gradient forces with repulsive electrostatic forces. Silica-coated Fe2O3 nanospheres of 300 diameter are trapped as close as 50 nm away from the surface with 810 μW of optical power, with a maximum trap stiffness of 2.7 pN μm−1. Electrostatic trapping forces up to 0.5 pN are achieved, a factor of 50 larger than those achievable for the same optical power in conventional optical tweezers. Efficient collection of the optical field directly into the nanofiber enables ultra-sensitive tracking of nanoparticle motion and extraction of its characteristic Brownian motion spectrum, with a minimum position sensitivity of 3.4 Å/√Hz .


Wednesday, January 22, 2014

Accurate measurement of stiffness of leukemia cells and leukocytes using optical trap by rate-jump method

Z.L. Zhou, T.H. Hui, Bin Tang and A.H.W. Ngan

Accurate measurement of the elastic modulus of soft biological cells in micro/nano scale range is still challenging. Tests involving constant-rate loading often yield results which are rate dependent, due to the viscous component of the deformation. In this work, a rate-jump indentation method was employed on an optical tweezers system to measure the stiffness of non-adherent blood cells, which are the softest types of cells. Compared to the traditional Hertzian method of indentation, the rate-jump method is found to be able to yield invariant elastic modulus from K562 myelogenous leukemia cells. The optical tweezers indentation method proposed can therefore serve as a standard protocol for obtaining the intrinsic elastic modulus of extremely soft cells, with applied forces in the pico-newton range. This method is also found to be effective in grading the stiffness values of myelogenous leukemia cell lines (K562 and HL60) and normal leukocytes, indicating that it can be used to identify normal cells from diseased counterparts without biochemical analysis.


Optical Tweezers Analysis of DNA–Protein Complexes

Iddo Heller , Tjalle P. Hoekstra , Graeme A. King , Erwin J. G. Peterman , and Gijs J. L. Wuite

Cellular DNA is continuously engaged in a molecular dance with many accessory proteins. These proteins are responsible for orchestrating the storage, maintenance, and transfer of genetic information. Together, the genomic operations make up the various metabolic pathways of DNA, which cascade into all aspects of cellular life. Over the past two decades, optical tweezers analyses of DNA–protein complexes have provided unique mechanistic insights into this molecular dance. Numerous assays have been devised to probe and elucidate DNA–protein interactions spanning the full range of DNA metabolic processes using optical tweezers. In this review, we provide an overview of the field, highlighting the unique opportunities that optical tweezers provide for DNA–protein analysis. We will describe the different experimental assays used and their limitations and advantages and review the many contributions that optical tweezers have made to our fundamental understanding of DNA transactions.


Rapid 3D fluorescence imaging of individual optically trapped living immune cells

Deanna Wolfson, Michael Steck, Martin Persson, Gregory McNerney, Ana Popovich, Mattias Goksör, Thomas Huser

We demonstrate an approach to rapidly characterize living suspension cells in 4 dimensions while they are immobilized and manipulated within optical traps. A single, high numerical aperture objective lens is used to separate the imaging plane from the trapping plane. This facilitates full control over the position and orientation of multiple trapped cells using a spatial light modulator, including directed motion and object rotation, while also allowing rapid 4D imaging. This system is particularly useful in the handling and investigation of the behavior of non-adherent immune cells. We demonstrate these capabilities by imaging and manipulating living, fluorescently stained Jurkat T cells.


Imprintable membranes from incomplete chiral coalescence

Mark J. Zakhary, Thomas Gibaud, C. Nadir Kaplan, Edward Barry, Rudolf Oldenbourg, Robert B. Meyer & Zvonimir Dogic

Coalescence is an essential phenomenon that governs the equilibrium behaviour in a variety of systems from intercellular transport to planetary formation. In this report, we study coalescence pathways of circularly shaped two-dimensional colloidal membranes, which are one rod-length-thick liquid-like monolayers of aligned rods. The chirality of the constituent rods leads to three atypical coalescence pathways that are not found in other simple or complex fluids. In particular, we characterize two pathways that do not proceed to completion but instead produce partially joined membranes connected by line defects—π-wall defects or alternating arrays of twisted bridges and pores. We elucidate the structure and energetics of these defects and ascribe their stability to a geometrical frustration inherently present in chiral colloidal membranes. Furthermore, we induce the coalescence process with optical forces, leading to a robust on-demand method for imprinting networks of channels and pores into colloidal membranes.


Monday, January 20, 2014

Making a big thing of a small cell – recent advances in single cell analysis

Kerstin Galler, Christina Große, Katharina Bräutigam, Jürgen Popp and Ute Neugebauer

Single cell analysis is an emerging field requiring a high level interdisciplinary collaboration to provide detailed insights into the complex organisation, function and heterogeneity of life. This review is addressed to life science researchers as well as researchers developing novel technologies. It covers all aspects of the characterisation of single cells (with a special focus on mammalian cells) from morphology to genetics and different omics-techniques to physiological, mechanical and electrical methods. In recent years, tremendous advances have been achieved in all fields of single cell analysis: 1. improved spatial and temporal resolution of imaging techniques to enable the tracking of single molecule dynamics within single cells; 2. increased throughput to reveal unexpected heterogeneity between different individual cells raising the question what characterizes a cell type and what is just natural biological variation; and 3. emerging multimodal approaches try to bring together information from complementary techniques paving the way for a deeper understanding of the complexity of biological processes. This review also covers the first successful translations of single cell analysis methods to diagnostic applications in the field of tumour research (especially circulating tumour cells), regenerative medicine, drug discovery and immunology.


Cytoskeleton Modification and Cholesterol Depletion Affect Membrane Properties and Caveolae Positioning of CHO Cells

Maja Grundner, Špela Zemljič Jokhadar

The formation of protrusions is necessary for numerous biological processes. It involves extension of the plasma membrane, and the force needed for this is provided by the actin cytoskeleton. Tether pulling with optical tweezers can mimic the formation of a protrusion, so we used this method to investigate the effects of modifying not only actin (with latrunculin A) but also microtubules (with nocodazole) and the plasma membrane itself (with methyl-β-cyclodextrin) on the Chinese hamster ovary cell membrane. After these modifications, the membrane reservoir was supposed to redistribute. Caveolae constitute a small part of the reservoir, so the redistribution of caveolar proteins such as caveolin-1 and cavin-1 that represents caveolae per se was assessed. The main findings concerning protrusion force and membrane reservoir availability were as follows: (1) they correlated inversely, (2) their values underwent the greatest change after microtubule disruption, and (3) membrane composition had a major influence on the parameters studied. F-actin disruption and cholesterol depletion decreased, and microtubule disruption increased the amount of the caveolar proteins (caveolae). Caveolae presented just an example of the membrane reservoir, and from our findings, we suppose that the perturbations caused were too large to be related to caveolae redistribution alone. The integrity of the cytoskeleton and plasma membrane composition are important factors in the formation of protrusions and in determining the availability and distribution of the membrane reservoir.


Optical Mirror from Laser-Trapped Mesoscopic Particles

Tomasz M. Grzegorczyk, Johann Rohner and Jean-Marc Fournier

Trapping of mesoscopic particles by optical forces usually relies on the gradient force, whereby particles are attracted into optical wells formed by landscaping the intensity of an optical field. This is most often achieved by optical Gaussian beams, interference patterns, general phase contrast methods, or other mechanisms. Hence, although the simultaneous trapping of several hundreds of particles can be achieved, these particles remain mostly independent with negligible interaction. Optical matter, however, relies on close packing and binding forces, with fundamentally different electrodynamic properties. In this Letter, we build ensembles of optically bound particles to realize a reflective surface that can be used to image an object or to focus a light beam. To our knowledge, this is the first experimental proof of the creation of a mirror by optical matter, and represents an important step toward the realization of a laser-trapped mirror (LTM) in space. From a theoretical point of view, optically bound close packing requires an exact solver of Maxwell’s equations in order to precisely compute the field scattered by the collection of particles. Such rigorous calculations have been developed and are used here to study the focusing and resolving power of an LTM.


Probing the Mechanisms of Translation with Force

Christian M. Kaiser and Ignacio Tinoco , Jr.

In this Review, we will describe the application of single-molecule approaches to study the translation of mRNA by single ribosomes, focusing on mechanical manipulation with optical tweezers. Single-molecule methods have distinct advantages over traditional ensemble (or bulk) experiments in identifying the molecular mechanisms underlying this complex biological process. This has particularly important implications for elucidating the kinetics of processive molecular machines, such as ribosomes. The kinetics contain abundant information about how molecular machines act in a coordinated fashion to accomplish a complicated task, such as deciphering a nucleic acid sequence and synthesizing the encoded protein. However, kinetics are governed by stochastic processes; thus, each molecule in a reaction takes a different amount of time to react. It is impossible to maintain synchronicity over several steps of a sequential reaction. At any time in an ensemble reaction of many molecules, there will be reactants, products, and all of the intermediates. For a large number of ribosomes that start translating a particular mRNA at the same time, some will be decoding the first codon, while others are reading the second, third, fourth, etc. In contrast, at any time in a single-molecule reaction, there is only one species. The characteristics of each single species can be determined.


Friday, January 17, 2014

Sensitivity analysis of light force accelerometer based on optical trapping Mie microsphere

Li-shuang Feng, Hong-chen Jiao, Bao-yin Yao

For achieving the greatest sensitivity, numerical simulation is proposed to optimize the parameters of light force accelerometer (LFA). Firstly, the basic principle of the LFA is analyzed, and the mathematical model is established to describe the axial and radial optical trapping forces on microspheres in the fundamental mode Gaussian laser beam. Secondly, the axial and radial optical trapping forces applied on Mie spheres are simulated to obtain the optimal parameters of open-loop LFA. Results show that the sensitivity can reach 103 μm/g. Finally, the LFA system is proposed based on the closed-loop scheme with dual beams. This can provide reference for the design and fabrication of LFA in future.


Tweezing and manipulating micro- and nanoparticles by optical nonlinear endoscopy

Min Gu, Hongchun Bao, Xiaosong Gan, Nicholas Stokes and Jingzhi Wu

The precise control and manipulation of micro- and nanoparticles using an optical endoscope are potentially important in biomedical studies, bedside diagnosis and treatment in an aquatic internal organ environment, but they have not yet been achieved. Here, for the first time, we demonstrate optical nonlinear endoscopic tweezers (ONETs) for directly controlling and manipulating aquatic micro- and nanobeads as well as gold nanorods. It is found that two-photon absorption can enhance the trapping force on fluorescent nanobeads by up to four orders of magnitude compared with dielectric nanobeads of the same size. More importantly, two-photon excitation leads to a plasmon-mediated optothermal attracting force on nanorods, which can extend far beyond the focal spot. This new phenomenon facilitates a snowball effect that allows the fast uploading of nanorods to a targeted cell followed by thermal treatment within 1 min. As two-photon absorption allows an operation wavelength at the center of the transmission window of human tissue, our work demonstrates that ONET is potentially an unprecedented tool for precisely specifying the location and dosage of drug particles and for rapidly uploading metallic nanoparticles to individual cancer cells for treatment.

Wednesday, January 15, 2014

Optical particle sorting on an optofluidic chip

Kaelyn D. Leake, Brian S. Phillips, Thomas D. Yuzvinsky, Aaron R. Hawkins, and Holger Schmidt

We report size-based sorting of micro- and sub-micron particles using optical forces on a planar optofluidic chip. Two different combinations of fluid flow and optical beam directions in liquid-core waveguides are demonstrated. These methods allow for tunability of size selection and sorting with efficiencies as high as 100%. Very good agreement between experimental results and calculated particle trajectories in the presence of flow and optical forces is found.


Dynamics of microscopic objects in optical tweezers: experimental determination of underdamped regime and numerical simulation using multiscale analysis

Mahdi Haghshenas-Jaryani, Bryan Black, Sarvenaz Ghaffari, James Drake, Alan Bowling, Samarendra Mohanty

This article presents new experimental observations and numerical simulations to investigate the dynamic behavior of micro–nano-sized objects under the influence of optical tweezers (OTs). OTs are scientific tools that can apply forces and moments to small particles using a focused laser beam. The motions of three polystyrene microspheres of different diameters, 1,950, 990, and 500 nm, are examined. The results show a transition from the overdamped motion of the largest bead to the underdamped motion of the smallest bead. The experiments are verified using a dynamic model of a microbead under the influence of Gaussian beam OTs that is modeled using ray-optics. The time required to numerically integrate the classic Newton–Euler model is quite long because a picosecond step size must be used. This run time can be reduced using a first-order model, and greatly reduced using a new multiscale model. The difference between these two models is the underdamped behavior predicted by the multiscale model. The experimentally observed underdamped behavior proves that the multiscale model predicts the actual physics of a nano-sized particle moving in a fluid environment characterized by a low Reynolds number.


Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

Barbara Hissa, Bruno Pontes, Paula Magda S. Roma, Ana Paula Alves, Carolina D. Rocha, Thalita M. Valverde, Pedro Henrique N. Aguiar, Fernando P. Almeida, Allan J. Guimarães, Cristina Guatimosim, Aristóbolo M. Silva, Maria C. Fernandes, Norma W. Andrews, Nathan B. Viana, Oscar N. Mesquita, Ubirajara Agero, Luciana O. Andrade

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.


Monday, January 13, 2014

Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics

Lorenzo Bongini, Luca Melli, Vincenzo Lombardi and Pasquale Bianco

Under a tension of ∼65 pN, double-stranded DNA undergoes an overstretching transition from its basic (B-form) conformation to a 1.7 times longer conformation whose nature is only recently starting to be understood. Here we provide a structural and thermodynamic characterization of the transition by recording the length transient following force steps imposed on the λ-phage DNA with different melting degrees and temperatures (10–25°C). The shortening transient following a 20–35 pN force drop from the overstretching force shows a sequence of fast shortenings of double-stranded extended (S-form) segments and pauses owing to reannealing of melted segments. The lengthening transients following a 2–35 pN stretch to the overstretching force show the kinetics of a two-state reaction and indicate that the whole 70% extension is a B-S transition that precedes and is independent of melting. The temperature dependence of the lengthening transient shows that the entropic contribution to the B-S transition is one-third of the entropy change of thermal melting, reinforcing the evidence for a double-stranded S-form that maintains a significant fraction of the interstrand bonds. The cooperativity of the unitary elongation (22 bp) is independent of temperature, suggesting that structural factors, such as the nucleic acid sequence, control the transition.


Low-force transitions in single titin molecules reflect a memory of contractile history

Zsolt Mártonfalvi, Pasquale Bianco, Marco Linari, Marco Caremani, Attila Nagy, Vincenzo Lombardi and Miklós Kellermayer

Titin, a giant elastomeric muscle protein has been implicated to function as a sensor of sarcomeric stress and strain but with unresolved mechanisms. To gain insight into titin's mechanosensory function here we manipulated single molecules with high-resolution optical tweezers. Discrete, stepwise transitions, with rates faster than canonical Ig-domain unfolding occurred during stretch at forces as low as 5 pN. Multiple mechanisms and molecular regions (PEVK, proximal tandem-Ig, N2A) are likely to be involved. The pattern of transitions is sensitive to the history of contractile events. Monte-Carlo simulations recovered our experimental results and predicted that structural transitions may begin prior to the complete extension of the PEVK domain. High-resolution AFM of titin extended with meniscus forces supported this prediction. Addition of glutamate-rich PEVK-domain fragments competitively inhibited the viscoelastic response in both single titin molecules and muscle fibers, indicating that intra-PEVK-domain interactions contribute significantly to sarcomere mechanics. Thus, under non-equilibrium conditions across the physiological force range, titin extends via a complex pattern of history-dependent discrete conformational transitions which, by dynamically exposing ligand-binding sites, may set the stage for the biochemical sensing of the sarcomeric mechanical status.


Manipulation and assembly of ZnO nanowires with single holographic optical tweezers system

Jing Li and Gang Du

ZnO nanowires, characterized with high melting points, are hard to assemble together with laser fusion. In order to build micro–nano structures with ZnO nanowires, a polymer film with a low melting point and high optical transparency is introduced as a substrate for ZnO nanowires to be deposited. A holographic optical tweezers system is used not only to manipulate ZnO nanowires, but also to melt the polymer film for the fixation of ZnO nanowires. By this method, micro–nano structures composed of ZnO nanowires are produced, which can be utilized as subwavelength optical waveguides.


Recent Advancements in Optofluidics-Based Single-cell Analysis: Optical On-Chip Cellular Manipulation, Treatment, and Property Detection

Nien-Tsu Huang, Hua-li Zhang, Meng-Ting Chung, Jung Hwan Seo and Katsuo Kurabayashi

Cellular analysis plays important roles in various biological applications, such as cell biology, drug development, and disease diagnosis. Conventional cellular analysis usually measures the average response from a whole cell group. However, the bulk measurement may cause misleading interpretations due to cell heterogeneity. Another problem is that current cellular analysis may not be able to differentiate various subsets of cell population, each exhibiting a different behavior than others. Single-cell analysis techniques are developed to analyze cellular properties, conditions, or functional responses in a large cell population at the individual cell level. Integrated optics with microfluidics platform provides a well-controlled microenvironment to precisely control single cell conditions and perform non-invasive high-throughput analysis. This paper reviews recent developments of optofluidics technology for various optics-based single-cell analyses, which involve single cell manipulation, treatment, and property detection. Finally, we provide our views on the future development of integrated optics with microfluidics for single-cell analysis and discuss potential challenges and opportunities of this emerging research field in biological applications.


Photodamage in a Mitochondrial Membrane Model Modulated by the Topology of Cationic and Anionic Meso-Tetrakis Porphyrin Free Bases

Cintia Kawai, Juliana C. Araújo-Chaves, Taciana Magrini, Camila O. C. C. Sanches, Sandra M. S. Pinto, Herculano Martinho, Nasser Daghastanli, Iseli L. Nantes

The photodynamic effects of the cationic TMPyP (meso-tetrakis [N-methyl-4-pyridyl]porphyrin) and the anionic TPPS4 (meso-tetrakis[4-sulfonatophenyl]porphyrin) against PC/CL phosphatidylcholine/cardiolipin (85/15%) membranes were probed to address the influence of phorphyrin binding on lipid damage. Electronic absorption spectroscopy and zeta potential demonstrated that only TMPyP binds to PC/CL large unilamellar vesicles (LUVs). The photodamage after irradiation with visible light was analyzed by dosages of lipid peroxides (LOOH) and thiobarbituric reactive substance and by a contrast phase image of the giant unillamelar vesicles (GUVs). Both TMPyP and TPPS4 promoted differentiated quantitative and qualitative damages on LUVs and GUVs. The damages were more extensive and faster by using the cationic porphyrin. The increase of LOOH was higher in the presence of D2O, and was impaired by sodium azide and sorbic acid. The effect of D2O was higher for TPPS4 as the photosensitizer. The use of DCFH demonstrated that liposomes prevent the photo-bleaching of TMPyP. The results are consistent with a more stable TMPyP that generates long-lived singlet oxygen preferentially partitioned in the bilayer. Conversely, TPPS4 generates singlet oxygen in the bulk whose lifetime is increased in D2O. Therefore, the affinity of the porphyrin to the membrane modulates the rate, type and degree of lipid damage.


Dynamics of a chain of optically coupled micro droplets

T. Crouzil, M. Perrin

We study a chain of fluid droplets excited by two incoherent laser beams. Such structured object is merely an array of spherical lenses, that can guide a TEMpq optical mode. Taking into account the optical forces exerted by two counterpropagating beams, we show that the droplets can be trapped and the chain auto-organizes in the optical potential. The model takes into account the possible coalescence of several droplets, and shows that the droplet size can increase before they become trapped at stable postitions. For some input beam parameters (beam waist size and position), we have observed dynamic trapping : the droplets experience collective oscillation. Meanwhile, the beam shape evolves periodically in time.


Forced protein unfolding leads to highly elastic and tough protein hydrogels

Jie Fang, Alexander Mehlich, Nobuyasu Koga, Jiqing Huang, Rie Koga, Xiaoye Gao, Chunguang Hu, Chi Jin, Matthias Rief, Juergen Kast, David Baker & Hongbin Li

Protein-based hydrogels usually do not exhibit high stretchability or toughness, significantly limiting the scope of their potential biomedical applications. Here we report the engineering of a chemically cross-linked, highly elastic and tough protein hydrogel using a mechanically extremely labile, de novo-designed protein that assumes the classical ferredoxin-like fold structure. Due to the low mechanical stability of the ferredoxin-like fold structure, swelling of hydrogels causes a significant fraction of the folded domains to unfold. Subsequent collapse and aggregation of unfolded ferredoxin-like domains leads to intertwining of physically and chemically cross-linked networks, entailing hydrogels with unusual physical and mechanical properties: a negative swelling ratio, high stretchability and toughness. These hydrogels can withstand an average strain of 450% before breaking and show massive energy dissipation. Upon relaxation, refolding of the ferredoxin-like domains enables the hydrogel to recover its massive hysteresis. This novel biomaterial may expand the scope of hydrogel applications in tissue engineering.


Multi-frequency near-field scanning optical microscopy

Dana C Kohlgraf-Owens, Léo Greusard, Sergey Sukhov, Yannick De Wilde and Aristide Dogariu

We demonstrate a new multi-frequency approach for mapping near-field optically induced forces with subwavelength spatial resolution. The concept relies on oscillating a scanning probe at two different frequencies. Oscillations at one frequency are driven electrically to provide positional feedback regulation. Modulations at another frequency are induced optically and are used to measure the mechanical action of the optical field on the probe. Because the measurement is based on locally detecting the force of the electromagnetic radiation acting on the probe, the new method does not require a photodetector to map the radiation distribution and, therefore, can provide true broadband detection of light with a single probe.


Thursday, January 9, 2014

Dynamic plasmonic tweezers enabled single-particle-film-system gap-mode Surface-enhanced Raman scattering

Junfeng Shen, Jian Wang, Cuijiao Zhang, Changjun Min, Hui Fang, Luping Du, Siwei Zhu and X.-C. Yuan

Based on numerical simulation and experiment, we demonstrate a dynamic single-particle-film Surface-enhanced Raman scattering (SERS) system enabled by manipulation of a single gold nanoparticle by plasmonic nano-tweezers (PNT). A corresponding dynamic plasmonic gap-mode is induced by the hybridization of the surface plasmon polaritons (SPPs) on the film and the localized surface plasmon of the particle. This gap-mode produces an additional enhancement of ∼104 compared to the bare SPPs without the particle, reaching a final SERS enhancement factor of ∼109. Enabled by nano-manipulation with PNT, this dynamic single-particle-film-system provides a promising route to controllable SERS detection in aqueous environments.


Tuesday, January 7, 2014

Photothermal Heating of Nanowires

Paden B. Roder , Bennett E. Smith , E. James Davis , and Peter J. Pauzauskie

A theoretical model is developed here in tandem with single-beam laser trapping experiments to elucidate the effects of the numerous thermal, optical, and geometric parameters that affect internal temperature distributions within finite nanowires (NWs) during laser irradiation. Analytical solutions to the heat-transfer equation are presented to predict internal temperature distributions within individual nanowires based on numerical calculations of the internal electromagnetic heat source. Single-beam laser-trapping experiments are performed to measure photothermal heating of silicon NWs. Silicon has not been considered to date for photothermal heating applications due to its indirect bandgap and low absorption coefficient in the near-infrared tissue-transparency window. We also show here that ion-implantation may be used to increase the optical absorption of silicon nanowires (SiNWs) leading to significant heating to temperatures greater than 42°C in an aqueous environment at an irradiance of 3 MW/cm^2. Experimental observations of photothermal heating agree well with theoretical predictions. Calculations for comparison with amorphous-carbon NWs reveal significantly greater heating effects, as well as internal radial gradients not observed for SiNWs.


Optical Trapping of Nanoparticles and Quantum Dots

Poul M. Bendix, Liselotte Jauffred, Kamilla Norregaard, and Lene B. Oddershede

Optical manipulation of nanostructures offers new exciting possibilities for building new nano-architectures and for exploring the fundamental interactions between light and nanoparticles. The optical properties of nanostructures differ substantially from those of similar bulk material and exhibit an exquisite sensitivity on nanoparticle shape and composition. The plethora of particles available today expands the possibilities of optical manipulation to include control over particle temperature, luminescence, orientation, and even over the rotational optical momentum transferred to the nanoparticle. Here, we summarize recent experimental advances within optical manipulation of individual nanoparticles and quantum dots with a focus on resonant versus non-resonant trapping, optically induced heating, spherical aberration, and orientation control. Also, we present novel quantitative data on the photonic interaction between gold nanoshells and a focused laser beam. Lastly, promising applications of the biophotonical properties of nanoparticles within nanoscience and biophysics are pointed out.


Flexibility within the heads of muscle myosin-2 molecules

Neil Billington, Derek J. Revill, Stan A. Burgess, Peter D. Chantler, Peter J. Knight

We show that negative stain electron microscopy and image processing of nucleotide-free (apo) striated muscle myosin-2 subfragment-1 (S1), possessing one or both light chains, is capable of resolving significant amounts of structural detail. The overall appearance of the motor and the lever is similar in rabbit, scallop and chicken S1. Projection matching of class averages of the different S1 types to projection views of two different crystal structures of apo S1 shows that all types most commonly closely resemble the appearance of the scallop S1 structure rather than the methylated chicken S1 structure. Methylation of chicken S1 has no effect on the structure of the molecule at this resolution: it too resembles the scallop S1 crystal structure. The lever is found to vary in its angle of attachment to the motor domain, with a hinge point located in the so-called pliant region between the converter and essential light chain. The chicken S1 crystal structure lies near one end of the range of flexion observed. The Gaussian spread of angles of flexion suggests that flexibility is driven thermally, from which a torsional spring constant of ~ 23 pN · nm/rad2 is estimated on average for all S1 types, similar to myosin-5. This translates to an apparent cantilever-type stiffness at the tip of the lever of 0.37 pN/nm. Because this stiffness is lower than recent estimates from myosin-2 heads attached to actin, we suggest that binding to actin leads to an allosteric stiffening of the motor-lever junction.


Measurements of the evaporation and hygroscopic response of single fine-mode aerosol particles using a Bessel beam optical trap

Michael I. Cotterell, Bernard J. Mason, Antonia E. Carruthers, Jim S. Walker, Andrew J. Orr-Ewing and Jonathan P. Reid

A single horizontally-propagating zeroth order Bessel laser beam with a counter-propagating gas flow was used to confine single fine-mode aerosol particles over extended periods of time, during which process measurements were performed. Particle sizes were measured by the analysis of the angular variation of light scattered at 532 nm by a particle in the Bessel beam, using either a probe beam at 405 nm or 633 nm. The vapour pressures of glycerol and 1,2,6-hexanetriol particles were determined to be 7.5 ± 2.6 mPa and 0.20 ± 0.02 mPa respectively. The lower volatility of hexanetriol allowed better definition of the trapping environment relative humidity profile over the measurement time period, thus higher precision measurements were obtained compared to those for glycerol. The size evolution of a hexanetriol particle, as well as its refractive index at wavelengths 532 nm and 405 nm, were determined by modelling its position along the Bessel beam propagation length while collecting phase functions with the 405 nm probe beam. Measurements of the hygroscopic growth of sodium chloride and ammonium sulfate have been performed on particles as small as 350 nm in radius, with growth curves well described by widely used equilibrium state models. These are the smallest particles for which single-particle hygroscopicity has been measured and represent the first measurements of hygroscopicity on fine mode and near-accumulation mode aerosols, the size regimes bearing the most atmospheric relevance in terms of loading, light extinction and scattering. Finally, the technique is contrasted with other single particle and ensemble methods, and limitations are assessed.


Measurement of the Electrostatic Interaction between Polyelectrolyte Brush Surfaces by Optical Tweezers

Daiki Murakami, Ai Takenaka, Motoyasu Kobayashi, Hiroshi Jinnai, and Atsushi Takahara

We demonstrated an optical tweezers method to measure the electrostatic interaction between the strong polyelectrolyte brushes, poly(2-(methacryloyloxy)ethyltrimethylammonium chloride) (PMTAC), grafted on silica particles in aqueous media. The weak electrostatic interaction was successfully detected with a resolution of less than 0.1 μN m–1. The apparent Debye length, including the charge distribution in the polymer brush and the surface potential, decreased as the salt concentration in the medium increased. The experimentally obtained surface charge density was much smaller than that estimated from the amount of polyelectrolyte on the surface. Furthermore, the dissociation of ionic groups was enhanced by decreasing the grafting density of the polyelectrolyte brush. The results suggest that the majority of chloride counterions was immobilized in the dense polyelectrolyte brush layer to neutralize the high charge density.


Monday, January 6, 2014

Calibrating optical tweezers with Bayesian inference

Maximilian U. Richly, Silvan Türkcan, Antoine Le Gall, Nicolas Fiszman, Jean-Baptiste Masson, Nathalie Westbrook, Karen Perronet, and Antigoni Alexandrou

We present a new method for calibrating an optical-tweezer setup that does not depend on input parameters and is less affected by systematic errors like drift of the setup. It is based on an inference approach that uses Bayesian probability to infer the diffusion coefficient and the potential felt by a bead trapped in an optical or magnetic trap. It exploits a much larger amount of the information stored in the recorded bead trajectory than standard calibration approaches. We demonstrate that this method outperforms the equipartition method and the power-spectrum method in input information required (bead radius and trajectory length) and in output accuracy.


Ultrasensitive Size-Selection of Plasmonic Nanoparticles by Fano Interference Optical Force

Zhipeng Li, Shunping Zhang, Lianming Tong, Peijie Wang, Bin Dong, and Hongxing Xu

In this paper, we propose a solution for the ultrasensitive optical selection of plasmonic nanoparticles using Fano interference-induced scattering forces. Under a Gaussian beam excitation, the scattering of a plasmonic nanoparticle at its Fano resonance becomes strongly asymmetric in the lateral direction and consequently results in a net transverse scattering force, that is, Fano interference-induced force. The magnitude of this transverse scattering force is comparable with the gradient force in conventional optical manipulation experiments. More interestingly, the Fano scattering force is ultrasensitive to the particle size and excitation frequency due to the phase sensitivity of the interference between adjacent plasmon modes in the particle. Utilizing this distinct feature, we show the possibility of size-selective sorting of silver and gold nanoparticles with an accuracy of about ±10 nm and silica-gold core–shell nanoparticles with shell thickness down to several nanometers. These results would add to the toolbox of optical manipulation and fabrication.


Hollow Bessel-like beam as an optical guide for a stream of microscopic particles

Niko Eckerskorn, Li Li, Richard A. Kirian, Jochen Küpper, Daniel P. DePonte, Wieslaw Krolikowski, Woei M. Lee, Henry N. Chapman, and Andrei V. Rode

Current aerosol sample injection methods for coherent x-ray morphology suffer from excessive sample consumption due to the dispersion of the aerosol. To remedy this we propose here a high aspect ratio optical funnel by using a hollow Bessel-like beam with variable divergence, which may reduce sample consumption significantly. We present estimated optical forces exerted on the particles in the transverse plane, depending on various experimental conditions. We show that light pressure imposed by a funnel formed with 4.2 W continuous wave laser is sufficient to divert a stream of 2 µm polystyrene particles travelling ~50 m/s by ~1.5 × 10−3 rad.


Modifying the laser beam intensity distribution for obtaining improved strength characteristics of an optical trap

Mikhail Aleksandrovich Rykov and Roman Vasilevich Skidanov

In this article we study modified optical beams used as optical tweezers for guiding biological micro-objects. We mean to achieve more efficient micromanipulation by using crescent intensity distribution. During laboratory experiments to test their theoretical projections we manufactured a diffractive optical element (DOE) to generate the proposed intensity distribution. Experimental estimations are provided for DOE energy efficiency. We conduct both theoretical and experimental studies of the crescent beam trapping strength. It transpires that in some cases crescent-shaped beams are more efficient than more commonly used Gaussian beams.