L. Marcucci, T. Yanagida
To elucidate the physical properties of the force generation mechanism in molecular motors, we have obtained an analytical solution of the bidimensional Fokker-Plank equation which describes a common setup used in single molecule experiments. As a first application of this general result, we have shown that the size of the trapping system affects the dwell time of a multistable particle linearly. A quantitative application to skeletal actomyosin complex, using direct observation of force generation dynamics in the literature, shows that the size of the trapping system used was important for increasing the dwell time of the myosin head stable states to an observable time scale.
DOI
Sunday, June 30, 2013
Monitoring Trehalose Uptake and Conversion by Single Bacteria using Laser Tweezers Raman Spectroscopy
Anna Avetisyan , John Beck Jensen , and Thomas Huser
Having the ability to monitor metabolic activity at the scale of single bacterial cells non-invasively would enable us to follow changes in the distribution of activity in bacterial systems which is of major importance for topics such as integration of metabolism and development, metabolic engineering, microbial activity and drug resistance, cell-cell interactions, and quorum sensing. Here, we used laser tweezers Raman spectroscopy to monitor the in vivo real-time uptake and conversion of trehalose by single bacterial cells. This approach can be used for the quantitative determination of sugar uptake by a single bacterium and its metabolic response to the sugar application with time. We show that uptake of trehalose can be quantified in single living bacterial cells held in place by an optical trap while simultaneously collecting Raman spectra upon application of sugar to the medium. This technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of toxicity of the isotopic probes common in studying these transport processes. It can substitute the laborious and time-consuming analytical evaluation. Although the single-cell Raman spectroscopy method demonstrated here is focused on the study of trehalose uptake by Sinorhizobium meliloti, the demonstrated approach is applicable to many different organisms and carbohydrates in general.
DOI
Having the ability to monitor metabolic activity at the scale of single bacterial cells non-invasively would enable us to follow changes in the distribution of activity in bacterial systems which is of major importance for topics such as integration of metabolism and development, metabolic engineering, microbial activity and drug resistance, cell-cell interactions, and quorum sensing. Here, we used laser tweezers Raman spectroscopy to monitor the in vivo real-time uptake and conversion of trehalose by single bacterial cells. This approach can be used for the quantitative determination of sugar uptake by a single bacterium and its metabolic response to the sugar application with time. We show that uptake of trehalose can be quantified in single living bacterial cells held in place by an optical trap while simultaneously collecting Raman spectra upon application of sugar to the medium. This technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of toxicity of the isotopic probes common in studying these transport processes. It can substitute the laborious and time-consuming analytical evaluation. Although the single-cell Raman spectroscopy method demonstrated here is focused on the study of trehalose uptake by Sinorhizobium meliloti, the demonstrated approach is applicable to many different organisms and carbohydrates in general.
DOI
Measurements of the electrokinetic forces on dielectric microparticles in nematic liquid crystals using optical trapping
A. V. Ryzhkova, F. V. Podgornov, A. Gaebler, R. Jakoby, and W. Haase
We have studied the dynamics of dielectric microparticles dispersed in a nematic liquid crystal (NLC) in the presence of an external AC electric field. Investigations were performed using optical trapping technique in the cell with in-plane electrodes. It was shown that the main driving force in the bulk of the material has electrophoretic nature. It was demonstrated that the microparticle behavior strongly depends on the distance with respect to the electrode and is influenced by the dielectrophoretic force. The model, which enables estimation of the electrokinetic forces, is proposed. The forces are found from the balance with the optical trapping force. The microparticle surface charge q ≈ 2.1×10−17 C, linear electrophoretic mobilities μ∥ ≈ 10−11 m2/(V⋅s),μ⊥ ≈ 7×10−12 m2/(V⋅s), and the NLC viscosity η ≈ (21.2±4.7)×10−3 Pa⋅s at T = 40 °C are evaluated.
DOI
We have studied the dynamics of dielectric microparticles dispersed in a nematic liquid crystal (NLC) in the presence of an external AC electric field. Investigations were performed using optical trapping technique in the cell with in-plane electrodes. It was shown that the main driving force in the bulk of the material has electrophoretic nature. It was demonstrated that the microparticle behavior strongly depends on the distance with respect to the electrode and is influenced by the dielectrophoretic force. The model, which enables estimation of the electrokinetic forces, is proposed. The forces are found from the balance with the optical trapping force. The microparticle surface charge q ≈ 2.1×10−17 C, linear electrophoretic mobilities μ∥ ≈ 10−11 m2/(V⋅s),μ⊥ ≈ 7×10−12 m2/(V⋅s), and the NLC viscosity η ≈ (21.2±4.7)×10−3 Pa⋅s at T = 40 °C are evaluated.
DOI
Speed Switching of Gonococcal Surface Motility Correlates with Proton Motive Force
Rainer Kurre, Nadzeya Kouzel, Kanimozhi Ramakrishnan, Enno R. Oldewurtel, Berenike Maier
Bacterial type IV pili are essential for adhesion to surfaces, motility, microcolony formation, and horizontal gene transfer in many bacterial species. These polymers are strong molecular motors that can retract at two different speeds. In the human pathogen Neisseria gonorrhoeae speed switching of single pili from 2 µm/s to 1 µm/s can be triggered by oxygen depletion. Here, we address the question how proton motive force (PMF) influences motor speed. Using pHluorin expression in combination with dyes that are sensitive to transmembrane ΔpH gradient or transmembrane potential ΔΨ, we measured both components of the PMF at varying external pH. Depletion of PMF using uncouplers reversibly triggered switching into the low speed mode. Reduction of the PMF by ≈ 35 mV was enough to trigger speed switching. Reducing ATP levels by inhibition of the ATP synthase did not induce speed switching. Furthermore, we showed that the strictly aerobic Myxococcus xanthus failed to move upon depletion of PMF or oxygen, indicating that although the mechanical properties of the motor are conserved, its regulatory inputs have evolved differently. We conclude that depletion of PMF triggers speed switching of gonococcal pili. Although ATP is required for gonococcal pilus retraction, our data indicate that PMF is an independent additional energy source driving the high speed mode.
Bacterial type IV pili are essential for adhesion to surfaces, motility, microcolony formation, and horizontal gene transfer in many bacterial species. These polymers are strong molecular motors that can retract at two different speeds. In the human pathogen Neisseria gonorrhoeae speed switching of single pili from 2 µm/s to 1 µm/s can be triggered by oxygen depletion. Here, we address the question how proton motive force (PMF) influences motor speed. Using pHluorin expression in combination with dyes that are sensitive to transmembrane ΔpH gradient or transmembrane potential ΔΨ, we measured both components of the PMF at varying external pH. Depletion of PMF using uncouplers reversibly triggered switching into the low speed mode. Reduction of the PMF by ≈ 35 mV was enough to trigger speed switching. Reducing ATP levels by inhibition of the ATP synthase did not induce speed switching. Furthermore, we showed that the strictly aerobic Myxococcus xanthus failed to move upon depletion of PMF or oxygen, indicating that although the mechanical properties of the motor are conserved, its regulatory inputs have evolved differently. We conclude that depletion of PMF triggers speed switching of gonococcal pili. Although ATP is required for gonococcal pilus retraction, our data indicate that PMF is an independent additional energy source driving the high speed mode.
Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function
Ruth F. Sommese, Jongmin Sung, Suman Nag, Shirley Sutton, John C. Deacon, Elizabeth Choe, Leslie A. Leinwand, Kathleen Ruppel, and James A. Spudich
Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including β-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human β-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human β-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human β-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.
Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including β-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human β-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human β-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human β-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.
Kinect the dots: 3D control of optical tweezers
Lucy Shaw, Daryl Preece and Halina Rubinsztein-Dunlop
Holographically generated optical traps confine micron- and sub-micron sized particles close to the center of focused light beams. They also provide a way of trapping multiple particles and moving them in three dimensions. However, in many systems the user interface is not always advantageous or intuitive especially for collaborative work and when depth information is required. We discuss and evaluate a set of multi-beam optical tweezers that utilize off the shelf gaming technology to facilitate user interaction. We use the Microsoft Kinect sensor bar as a way of getting the user input required to generate arbitrary optical force fields and control optically trapped particles. We demonstrate that the system can also be used for dynamic light control.
DOI
Holographically generated optical traps confine micron- and sub-micron sized particles close to the center of focused light beams. They also provide a way of trapping multiple particles and moving them in three dimensions. However, in many systems the user interface is not always advantageous or intuitive especially for collaborative work and when depth information is required. We discuss and evaluate a set of multi-beam optical tweezers that utilize off the shelf gaming technology to facilitate user interaction. We use the Microsoft Kinect sensor bar as a way of getting the user input required to generate arbitrary optical force fields and control optically trapped particles. We demonstrate that the system can also be used for dynamic light control.
DOI
Saturday, June 29, 2013
Escherichia coli-Based Biophotonic Waveguides
Hongbao Xin , Yayi Li , Xiaoshuai Liu , and Baojun Li
The rapid progresses in biological and biomedical applications with optical interfaces have motivated an ever-increasing demand for biocompatible and disposable photonic components. Generally, these biophotonic components are first integrated with biocompatible materials and then interfaced with biological samples, such as living cells, for biological use. Therefore, direct formation of biophotonic components using living cells is greatly desired because the cells would serve simultaneously as samples and optical elements for signal sensing and detection. Here, we report an optical strategy for direct formation of biophotonic waveguides (bio-WGs) with Escherichia coli. The experiments demonstrate that this facile optical strategy enables forming bio-WGs with different lengths and good light propagation performances while the propagating signal can be detected in real-time. This strategy offers a seamless interface between optical and biological worlds with natural materials and provides a new opportunity for direct sensing and detection of biological signal and information in biocompatible microenvironments.
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The rapid progresses in biological and biomedical applications with optical interfaces have motivated an ever-increasing demand for biocompatible and disposable photonic components. Generally, these biophotonic components are first integrated with biocompatible materials and then interfaced with biological samples, such as living cells, for biological use. Therefore, direct formation of biophotonic components using living cells is greatly desired because the cells would serve simultaneously as samples and optical elements for signal sensing and detection. Here, we report an optical strategy for direct formation of biophotonic waveguides (bio-WGs) with Escherichia coli. The experiments demonstrate that this facile optical strategy enables forming bio-WGs with different lengths and good light propagation performances while the propagating signal can be detected in real-time. This strategy offers a seamless interface between optical and biological worlds with natural materials and provides a new opportunity for direct sensing and detection of biological signal and information in biocompatible microenvironments.
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Using Optical Tweezers for the characterization of very low viscoelastic polyelectrolytes solutions
Angelo Pommella , Valentina Preziosi , Sergio Caserta , Jonathan M. Cooper , Stefano Guido , and Manlio Tassieri
Recently, optical tweezing has been used to provide a method for microrheology addressed to measure the rheological properties of small volumes of samples. In this work, we corroborate this emerging field of microrheology by using these optical methods for the characterization of very low viscoelastic polyelectrolyte solutions. The influence of polyelectrolyte (i.e. polyacrylamide, PAM) concentration, its aging and of salt concentration are shown. The close agreement of the technique with classical bulk rheological measurements is demonstrated, illustrating the advantages of the technique.
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Recently, optical tweezing has been used to provide a method for microrheology addressed to measure the rheological properties of small volumes of samples. In this work, we corroborate this emerging field of microrheology by using these optical methods for the characterization of very low viscoelastic polyelectrolyte solutions. The influence of polyelectrolyte (i.e. polyacrylamide, PAM) concentration, its aging and of salt concentration are shown. The close agreement of the technique with classical bulk rheological measurements is demonstrated, illustrating the advantages of the technique.
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Non-resonant and non-enhanced Raman Correlation Spectroscopy
A. Barbara, F. Dubois, P. Quémerais, and L. Eng
We present the first non-resonant and non-enhanced Raman correlation spectroscopy experiments. They are conducted on a confocal microscope combined with a Raman spectrometer. The thermal fluctuations of the Raman intensities scattered by dispersions of polystyrene particles of sub-micrometric diameters are measured and analysed by deriving the autocorrelation functions (ACFs) of the intensities. We show that for particles of diameter down to 200 nm, RCS measurements are successfully obtained in spite of the absence of any source of amplification of the Raman signal. For particles of diameter ranging from 200 to 750 nm, the ACFs present a time-decay behaviour in accordance with the model of free Brownian particles. For particles of 1000 nm in diameter, the AFCs present a different behaviour with a much smaller characteristic time. This results from the dynamics of a single-Brownian particle trapped in the confocal volume by the optical forces of the focus spot.
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We present the first non-resonant and non-enhanced Raman correlation spectroscopy experiments. They are conducted on a confocal microscope combined with a Raman spectrometer. The thermal fluctuations of the Raman intensities scattered by dispersions of polystyrene particles of sub-micrometric diameters are measured and analysed by deriving the autocorrelation functions (ACFs) of the intensities. We show that for particles of diameter down to 200 nm, RCS measurements are successfully obtained in spite of the absence of any source of amplification of the Raman signal. For particles of diameter ranging from 200 to 750 nm, the ACFs present a time-decay behaviour in accordance with the model of free Brownian particles. For particles of 1000 nm in diameter, the AFCs present a different behaviour with a much smaller characteristic time. This results from the dynamics of a single-Brownian particle trapped in the confocal volume by the optical forces of the focus spot.
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Probing active forces via a fluctuation-dissipation relation: Application to living cells
P. Bohec, F. Gallet, C. Maes, S. Safaverdi, P. Visco and F. van Wijland
We derive a new fluctuation-dissipation relation for non-equilibrium systems with long-term memory. We show how this relation allows one to access new experimental information regarding active forces in living cells that cannot otherwise be accessed. For a silica bead attached to the wall of a living cell, we identify a crossover time between thermally controlled fluctuations and those produced by the active forces. We show that the probe position is eventually slaved to the underlying random drive produced by the so-called active forces.
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We derive a new fluctuation-dissipation relation for non-equilibrium systems with long-term memory. We show how this relation allows one to access new experimental information regarding active forces in living cells that cannot otherwise be accessed. For a silica bead attached to the wall of a living cell, we identify a crossover time between thermally controlled fluctuations and those produced by the active forces. We show that the probe position is eventually slaved to the underlying random drive produced by the so-called active forces.
DOI
Probing DNA clamps with single-molecule force spectroscopy
Lin Wang, Xiaojun Xu, Ravindra Kumar, Buddhadev Maiti, C. Tony Liu, Ivaylo Ivanov, Tae-Hee Lee, and Stephen J. Benkovic
Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms.
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Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms.
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Interparticle force between different types of nematic colloids
Kuniyoshi Izaki and Yasuyuki Kimura
We have studied the interparticle force between colloidal particles with three different types of defects in nematic liquid crystal by dual-beam optical tweezers. The force between a dipole (D)- and a Saturn-ring (S)-type particle at large interparticle distance R is proportional to R−4.95±0.05. The force between a D- and a planar (P)-type particle and that between an S- and a P-type particle are, respectively, proportional to R−5.04±0.08 and R−5.78±0.13. The observed dependence of the interparticle force on R at large R is in agreement with that predicted by electrostatic analogy. The topological quadrupole moments for S and P particles are evaluated from experimental data. We have also studied the force curves in oblique arrangement against the far-field director for respective pairs. The experimental force curves at large R quantitatively agree with those predicted by electrostatic analogy, but they always become attractive at small R due to the reorientation and deformation of defects. The force profiles for the S-P pair are also compared with those obtained by the recent numerical simulation.
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We have studied the interparticle force between colloidal particles with three different types of defects in nematic liquid crystal by dual-beam optical tweezers. The force between a dipole (D)- and a Saturn-ring (S)-type particle at large interparticle distance R is proportional to R−4.95±0.05. The force between a D- and a planar (P)-type particle and that between an S- and a P-type particle are, respectively, proportional to R−5.04±0.08 and R−5.78±0.13. The observed dependence of the interparticle force on R at large R is in agreement with that predicted by electrostatic analogy. The topological quadrupole moments for S and P particles are evaluated from experimental data. We have also studied the force curves in oblique arrangement against the far-field director for respective pairs. The experimental force curves at large R quantitatively agree with those predicted by electrostatic analogy, but they always become attractive at small R due to the reorientation and deformation of defects. The force profiles for the S-P pair are also compared with those obtained by the recent numerical simulation.
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Rigid DNA Beams for High-Resolution Single-Molecule Mechanics
Emanuel Pfitzner, Christian Wachauf, Fabian Kilchherr, Benjamin Pelz, William M. Shih, Matthias Rief, Prof. Dr. Hendrik Dietz
Bridging the gap: Rigid DNA linkers (blue, see picture) between microspheres (green) for high-resolution single-molecule mechanical experiments were constructed using DNA origami. The resulting DNA helical bundles greatly reduce the noise generated in studies of conformation changes using optical tweezers and were applied to study small DNA secondary structures.
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Bridging the gap: Rigid DNA linkers (blue, see picture) between microspheres (green) for high-resolution single-molecule mechanical experiments were constructed using DNA origami. The resulting DNA helical bundles greatly reduce the noise generated in studies of conformation changes using optical tweezers and were applied to study small DNA secondary structures.
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Protein folding and unfolding under force
Bharat Jagannathan, Susan Marqusee
The recent revolution in optics and instrumentation has enabled the study of protein folding using extremely low mechanical forces as the denaturant. This exciting development has led to the observation of the protein folding process at single molecule resolution and its response to mechanical force. Here, we describe the principles and experimental details of force spectroscopy on proteins, with a focus on the optical tweezers instrument. Several recent results will be discussed to highlight the importance of this technique in addressing a variety of questions in the protein folding field.
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The recent revolution in optics and instrumentation has enabled the study of protein folding using extremely low mechanical forces as the denaturant. This exciting development has led to the observation of the protein folding process at single molecule resolution and its response to mechanical force. Here, we describe the principles and experimental details of force spectroscopy on proteins, with a focus on the optical tweezers instrument. Several recent results will be discussed to highlight the importance of this technique in addressing a variety of questions in the protein folding field.
DOI
Monday, June 24, 2013
Optofluidic realization and retaining of cell–cell contact using an abrupt tapered optical fibre
Hongbao Xin, Yao Zhang, Hongxiang Lei, Yayi Li, Huixian Zhang & Baojun Li
Studies reveal that there exists much interaction and communication between bacterial cells, with parts of these social behaviors depending on cell–cell contacts. The cell–cell contact has proved to be crucial for determining various biochemical processes. However, for cell culture with relatively low cell concentration, it is difficult to precisely control and retain the contact of a small group of cells. Particularly, the retaining of cell–cell contact is difficult when flows occur in the medium. Here, we report an optofluidic method for realization and retaining of Escherichia coli cell–cell contact in a microfluidic channel using an abrupt tapered optical fibre. The contact process is based on launching a 980-nm wavelength laser into the fibre, E. coli cells were trapped onto the fibre tip one after another, retaining cell–cell contact and forming a highly organized cell chain. The formed chains further show the ability as bio-optical waveguides.
DOI
Studies reveal that there exists much interaction and communication between bacterial cells, with parts of these social behaviors depending on cell–cell contacts. The cell–cell contact has proved to be crucial for determining various biochemical processes. However, for cell culture with relatively low cell concentration, it is difficult to precisely control and retain the contact of a small group of cells. Particularly, the retaining of cell–cell contact is difficult when flows occur in the medium. Here, we report an optofluidic method for realization and retaining of Escherichia coli cell–cell contact in a microfluidic channel using an abrupt tapered optical fibre. The contact process is based on launching a 980-nm wavelength laser into the fibre, E. coli cells were trapped onto the fibre tip one after another, retaining cell–cell contact and forming a highly organized cell chain. The formed chains further show the ability as bio-optical waveguides.
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Ultrafast spinning of gold nanoparticles in water using circularly polarized light
Anni Lehmuskero , Robin Ogier , Tina Gschneidtner , Peter Johansson , and Mikael KällControlling the position and movement of small objects with light is an appealing way to manipulate delicate samples, such as living cells or nanoparticles. It is well-known that optical gradient and radiation pressure forces caused by a focused laser beam enables trapping and manipulation of objects with strength that is dependent on the particle’s optical properties. However, by utilizing transfer of photon spin angular momentum, it is also possible set objects into rotational motion simply by targeting them with a beam of circularly polarized light. Here we show that this effect can set ~200 nm radii gold particles trapped in water in 2D by a laser tweezers into rotation at frequencies that reach several kilohertz, much higher than any previously reported light driven rotation of a microscopic object. We derive a theory for the fluctuations in light scattering from a rotating particle and we argue that the high rotation frequencies observed experimentally is the combined result of favorable optical particle properties and a low local viscosity due to substantial heating of the particles surface layer. The high rotation speed suggests possible applications in nanofluidics, optical sensing and micro-tooling of soft matter.
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Kinect 4 ... holographic optical tweezers
C Muhiddin, D B Phillips, M J Miles, L Picco and D M Carberry
The 3D position and orientation of a microtool confined in multiple optical traps needs to be controlled in order for one to perform modern, challenging experiments; for example, in order to utilize it as a scanning probe and investigate the surface of optically sensitive cells. The control interface has traditionally used the keyboard/mouse combination—limiting manipulations to a series of 1D/2D transforms. In this paper we demonstrate how the Kinect can be utilized to control the position and orientation of a microtool utilizing macroscopic models.
DOI
The 3D position and orientation of a microtool confined in multiple optical traps needs to be controlled in order for one to perform modern, challenging experiments; for example, in order to utilize it as a scanning probe and investigate the surface of optically sensitive cells. The control interface has traditionally used the keyboard/mouse combination—limiting manipulations to a series of 1D/2D transforms. In this paper we demonstrate how the Kinect can be utilized to control the position and orientation of a microtool utilizing macroscopic models.
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Neuronal beacon
Black, A. Mondal, Y. Kim, and S. K. Mohanty
The controlled navigation of the axonal growth cone of a neuron toward the dendrite of its synaptic partner neuron is the fundamental process in forming neuronal circuitry. While a number of technologies have been pursued for axonal guidance over the past decades, they are either invasive or not controllable with high spatial and temporal resolution and are often limited by low guidance efficacy. Here, we report a neuronal beacon based on light for highly efficient and controlled guidance of cortical primary neurons.
DOI
The controlled navigation of the axonal growth cone of a neuron toward the dendrite of its synaptic partner neuron is the fundamental process in forming neuronal circuitry. While a number of technologies have been pursued for axonal guidance over the past decades, they are either invasive or not controllable with high spatial and temporal resolution and are often limited by low guidance efficacy. Here, we report a neuronal beacon based on light for highly efficient and controlled guidance of cortical primary neurons.
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Simulation of longitudinal string waves through a single polymer molecule
Anders Korsbäck, Anders E. Wallin, Edward HæggströmThe capability of an individual polymer molecule to carry an acoustic wave along its length was investigated by a Brownian dynamics simulation using the algorithm of Ermak and McCammon and the discrete worm-like chain model. A 3 μμm long DNA strand featuring 50 nm persistence length was subjected to longitudinal oscillations (2 kHz to 25 kHz) at one end, and the properties of the resulting propagating interaction were studied and shown to be wave-like. A proof of concept experiment is proposed, where an optical tweezers dumbbell experiment is conducted to induce and receive the wave, and the result is compared to that of a control experiment with the DNA strand absent. Simulations were conducted to show what one might expect to see in such an experiment.
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Thursday, June 20, 2013
FACS-sorted particles reduce the data variance in optical tweezers-assisted dynamic force spectroscopy measurements
T Stangner, D Singer, C Wagner, C Gutsche, O Ueberschär, R Hoffmann and F Kremer
By combining optical tweezers-assisted dynamic force spectroscopy experiments with fluorescence activated cell sorting (FACS), we demonstrate a new approach to reducing the data variance in measuring receptor–ligand interactions on a single molecule level by ensuring similar coating densities. Therefore, the carboxyfluorescein-labelled monophosphorylated peptide tau226–240[pThr231] is anchored on melamine resin beads and these beads are sorted by FACS to achieve a homogeneous surface coverage. To quantify the impact of the fluorescence dye on the bond parameters between the phosphorylated peptide and the corresponding phosphorylation specific anti-human tau monoclonal antibody HPT-104, we perform dynamic force spectroscopy and compare the results to data using unsorted beads covered with the non-fluorescence peptide analogue. Finally, we demonstrate that the data variance of the relative binding frequency is significantly decreased by a factor of 3.4 using pre-sorted colloids with a homogeneous ligand coating compared to using unsorted colloids.
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By combining optical tweezers-assisted dynamic force spectroscopy experiments with fluorescence activated cell sorting (FACS), we demonstrate a new approach to reducing the data variance in measuring receptor–ligand interactions on a single molecule level by ensuring similar coating densities. Therefore, the carboxyfluorescein-labelled monophosphorylated peptide tau226–240[pThr231] is anchored on melamine resin beads and these beads are sorted by FACS to achieve a homogeneous surface coverage. To quantify the impact of the fluorescence dye on the bond parameters between the phosphorylated peptide and the corresponding phosphorylation specific anti-human tau monoclonal antibody HPT-104, we perform dynamic force spectroscopy and compare the results to data using unsorted beads covered with the non-fluorescence peptide analogue. Finally, we demonstrate that the data variance of the relative binding frequency is significantly decreased by a factor of 3.4 using pre-sorted colloids with a homogeneous ligand coating compared to using unsorted colloids.
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Multiphoton polymerization using optical trap assisted nanopatterning
Karl-Heinz Leitz, Yu-Cheng Tsai, Florian Flad, Eike Schäffer, Ulf Quentin, Ilya Alexeev, Romain Fardel, Craig B. Arnold, and Michael Schmidt
In this letter, we show the combination of multiphoton polymerization and optical trap assisted nanopatterning (OTAN) for the additive manufacturing of structures with nanometer resolution. User-defined patterns of polymer nanostructures are deposited on a glass substrate by a 3.5 μm polystyrene sphere focusing IR femtosecond laser pulses, showing minimum feature sizes of λ/10. Feature size depends on the applied laser fluence and the bead surface spacing. A finite element model describes the intensity enhancement in the microbead focus. The results presented suggest that OTAN in combination with multiphoton processing is a viable technique for additive nanomanufacturing with sub-diffraction-limited resolution.
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In this letter, we show the combination of multiphoton polymerization and optical trap assisted nanopatterning (OTAN) for the additive manufacturing of structures with nanometer resolution. User-defined patterns of polymer nanostructures are deposited on a glass substrate by a 3.5 μm polystyrene sphere focusing IR femtosecond laser pulses, showing minimum feature sizes of λ/10. Feature size depends on the applied laser fluence and the bead surface spacing. A finite element model describes the intensity enhancement in the microbead focus. The results presented suggest that OTAN in combination with multiphoton processing is a viable technique for additive nanomanufacturing with sub-diffraction-limited resolution.
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Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms
Krishna K. Sarangapani, Bungo Akiyoshi, Nicole M. Duggan, Sue Biggins, and Charles L. Asbury
During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser-trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms: by weakening the kinetochore-microtubule interface and also by destabilizing the kinetochore-attached microtubule tip.
DOI
During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser-trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms: by weakening the kinetochore-microtubule interface and also by destabilizing the kinetochore-attached microtubule tip.
DOI
Tuesday, June 18, 2013
Optically trapped and driven paddle-wheel
Theodor Asavei, Timo A Nieminen, Vincent L Y Loke, Alexander B Stilgoe, Richard Bowman, Daryl Preece, Miles J Padgett, Norman R Heckenberg and Halina Rubinsztein-Dunlop
We demonstrate the control and rotation of an optically trapped object, an optical paddle-wheel, with the rotation direction normal to the beam axis. This is in contrast to the usual situation where the rotation is about the beam axis. The paddle-wheel can be optically driven and moved to any position in the field of view of the microscope, which can be of interest for various biological applications where controlled application of a fluid flow is needed in a particular location and in a specific direction. This is of particular interest in signal transduction studies in cells, especially when a cell is flat and spread out on a surface.
DOI
We demonstrate the control and rotation of an optically trapped object, an optical paddle-wheel, with the rotation direction normal to the beam axis. This is in contrast to the usual situation where the rotation is about the beam axis. The paddle-wheel can be optically driven and moved to any position in the field of view of the microscope, which can be of interest for various biological applications where controlled application of a fluid flow is needed in a particular location and in a specific direction. This is of particular interest in signal transduction studies in cells, especially when a cell is flat and spread out on a surface.
DOI
Microcrystal manipulation with laser tweezers
A. Wagner, R. Duman, B. Stevens and A. Ward
X-ray crystallography is the method of choice to deduce atomic resolution structural information from macromolecules. In recent years, significant investments in structural genomics initiatives have been undertaken to automate all steps in X-ray crystallography from protein expression to structure solution. Robotic systems are widely used to prepare crystallization screens and change samples on synchrotron beamlines for macromolecular crystallography. The only remaining manual handling step is the transfer of the crystal from the mother liquor onto the crystal holder. Manual mounting is relatively straightforward for crystals with dimensions of >25 µm; however, this step is nontrivial for smaller crystals. The mounting of microcrystals is becoming increasingly important as advances in microfocus synchrotron beamlines now allow data collection from crystals with dimensions of only a few micrometres. To make optimal usage of these beamlines, new approaches have to be taken to facilitate and automate this last manual handling step. Optical tweezers, which are routinely used for the manipulation of micrometre-sized objects, have successfully been applied to sort and mount macromolecular crystals on newly designed crystal holders. Diffraction data from CPV type 1 polyhedrin microcrystals mounted with laser tweezers are presented.
DOI
X-ray crystallography is the method of choice to deduce atomic resolution structural information from macromolecules. In recent years, significant investments in structural genomics initiatives have been undertaken to automate all steps in X-ray crystallography from protein expression to structure solution. Robotic systems are widely used to prepare crystallization screens and change samples on synchrotron beamlines for macromolecular crystallography. The only remaining manual handling step is the transfer of the crystal from the mother liquor onto the crystal holder. Manual mounting is relatively straightforward for crystals with dimensions of >25 µm; however, this step is nontrivial for smaller crystals. The mounting of microcrystals is becoming increasingly important as advances in microfocus synchrotron beamlines now allow data collection from crystals with dimensions of only a few micrometres. To make optimal usage of these beamlines, new approaches have to be taken to facilitate and automate this last manual handling step. Optical tweezers, which are routinely used for the manipulation of micrometre-sized objects, have successfully been applied to sort and mount macromolecular crystals on newly designed crystal holders. Diffraction data from CPV type 1 polyhedrin microcrystals mounted with laser tweezers are presented.
DOI
Self-Organized Chiral Microspheres
A. Mazzulla, G. Cipparrone, R. J. Hernandez, A. Pane & R. Bartolino
A variety of chiral microspheres, each one possessing distinctive configurations of the molecular director, are studied. The process to obtain the solid particles is illustrated and some of their optical features is discussed.The optical properties of these objects depend from the emulsion preparation standards. The self-organized structures have been tailored by means of both the chiral dopant concentration and the molecular anchoring at the surface. The microspheres reveal exciting optical properties, i.e. they show specific kinds of selective reflection that can be sorted as radial, conical and cylindrical. The features of these micro-photonic devices are confirmed through experiments of polarized optical manipulation and laser emission.
These complex structures constitute very peculiar micro-systems, inspiring attractive technological applications.
DOI
A variety of chiral microspheres, each one possessing distinctive configurations of the molecular director, are studied. The process to obtain the solid particles is illustrated and some of their optical features is discussed.The optical properties of these objects depend from the emulsion preparation standards. The self-organized structures have been tailored by means of both the chiral dopant concentration and the molecular anchoring at the surface. The microspheres reveal exciting optical properties, i.e. they show specific kinds of selective reflection that can be sorted as radial, conical and cylindrical. The features of these micro-photonic devices are confirmed through experiments of polarized optical manipulation and laser emission.
These complex structures constitute very peculiar micro-systems, inspiring attractive technological applications.
DOI
Assessment of the Elasticity of Erythrocytes in Different Physiological Fluids by Laser Traps
Taylor Barnes, Adam Shulman, Anthony Farone, Mary Farone, Daniel Erenso
In the study of the mechanical properties of the erythrocytes (red blood cells-RBCs) the blood sample is commonly diluted in fluids that do not compromise the integrity of the cells. Fetal bovine serum (FBS), newborn bovine serum (NBBS), and phosphate buffer (PBS) solution with a concentration that can provide the right osmotic pressure are fluids commonly used to dilute the blood samples in such studies. Here we have presented the effect of these fluids on the elastic properties of the RBCs that we studied using laser traps. Two laser traps are directly used to trap and deform the cell by exerting a force distributed on the entire cell. The relative changes in size of the cell are studied as a function of the applied force to investigate any effects on the mechanical deformability of RBCs when the cells are suspended in these fluids. The results have shown that the elasticity of the RBCs in the NBBS is not statistically different from the elasticity of the cells in the PBS solution; however the results for the elasticity of the cells in FBS are found to be significantly higher.
DOI
In the study of the mechanical properties of the erythrocytes (red blood cells-RBCs) the blood sample is commonly diluted in fluids that do not compromise the integrity of the cells. Fetal bovine serum (FBS), newborn bovine serum (NBBS), and phosphate buffer (PBS) solution with a concentration that can provide the right osmotic pressure are fluids commonly used to dilute the blood samples in such studies. Here we have presented the effect of these fluids on the elastic properties of the RBCs that we studied using laser traps. Two laser traps are directly used to trap and deform the cell by exerting a force distributed on the entire cell. The relative changes in size of the cell are studied as a function of the applied force to investigate any effects on the mechanical deformability of RBCs when the cells are suspended in these fluids. The results have shown that the elasticity of the RBCs in the NBBS is not statistically different from the elasticity of the cells in the PBS solution; however the results for the elasticity of the cells in FBS are found to be significantly higher.
DOI
Exploring how infrared radiation enhances the attractive interaction between a cell pair by its electromagnetic nature
Bor-Wen Yang, Chu Yeh, Po-Cheng Lin, Chi-Tse Chao
Electromagnetic radiation can be categorized into ionizing and non-ionizing varieties. To determine the mechanism how non-ionizing radiation affects biological cells, we analyzed the difference between its thermal and electromagnetic effects. Two-beam optical tweezers were designed to demonstrate that infrared radiation could enhance the cellular interaction between red blood cells by its electromagnetic nature. An IR spot in the optical tweezers was irradiated on two RBCs to polarize them and induce electromagnetic attraction, while the other focused visible spot was used to quantify the intensity of the intercellular interaction. It was found that 0.1 mW/μm2 infrared radiation was adequate to cause pN-scale interaction between a cell pair, which was only 1/1000 of the power density used in a CD-R drive. We then set up a model to describe how non-ionizing radiation affected a cell assembly by deriving electromagnetic micro-stress transverse to its propagation axis.
DOI
Electromagnetic radiation can be categorized into ionizing and non-ionizing varieties. To determine the mechanism how non-ionizing radiation affects biological cells, we analyzed the difference between its thermal and electromagnetic effects. Two-beam optical tweezers were designed to demonstrate that infrared radiation could enhance the cellular interaction between red blood cells by its electromagnetic nature. An IR spot in the optical tweezers was irradiated on two RBCs to polarize them and induce electromagnetic attraction, while the other focused visible spot was used to quantify the intensity of the intercellular interaction. It was found that 0.1 mW/μm2 infrared radiation was adequate to cause pN-scale interaction between a cell pair, which was only 1/1000 of the power density used in a CD-R drive. We then set up a model to describe how non-ionizing radiation affected a cell assembly by deriving electromagnetic micro-stress transverse to its propagation axis.
DOI
Sunday, June 16, 2013
Laser guidance–based cell detection in a microfluidic biochip
Wan Qin; Lucas Schmidt; Xiaoqi Yang; Lina Wei; Ting Huang; Julie X. Yuan; Xiang Peng; Xiaocong Yuan; Bruce Z. Gao
We developed a microfluidic biochip to perform laser guidance on two cell types, chick embryonic forebrain neurons and spinal cord neurons. Observation of neurons under a high-magnification microscope, which we obtained from these two cell types, showed no difference in morphology. However, when flowing in the microfluidic channel and simultaneously being laser guided, the two cell types gained quite different guidance speeds under the same experimental conditions. The results demonstrate that different cell types with the same morphology (e.g., size, shape, etc.) can be effectively distinguished from each other by measuring the difference in guidance speeds (the maximum flow speed minus the initial flow speed). This technique is expected to provide a new approach to high-throughput, label-free cell sorting with high sensitivity.
DOI
We developed a microfluidic biochip to perform laser guidance on two cell types, chick embryonic forebrain neurons and spinal cord neurons. Observation of neurons under a high-magnification microscope, which we obtained from these two cell types, showed no difference in morphology. However, when flowing in the microfluidic channel and simultaneously being laser guided, the two cell types gained quite different guidance speeds under the same experimental conditions. The results demonstrate that different cell types with the same morphology (e.g., size, shape, etc.) can be effectively distinguished from each other by measuring the difference in guidance speeds (the maximum flow speed minus the initial flow speed). This technique is expected to provide a new approach to high-throughput, label-free cell sorting with high sensitivity.
DOI
Dynamical and phase-diagram study on stable optical pulling force in Bessel beams
Neng Wang, Jun Chen, Shiyang Liu, and Zhifang Lin
Based on the generalized Lorenz-Mie theory and Maxwell stress tensor formulism, we calculate the transverse force constant matrix and perform a linear stability analysis on a spherical particle that is subject to negative longitudinal optical force (NLOF) under the illumination of Bessel beams. Phase diagrams with respect to the material parameters are presented, which exhibit the possibility of the appearance of NLOF. From dynamical simulations of the particle performed both in the transverse plane and along the longitudinal direction, an even clearer picture of the realization of stable NLOF is presented. It is shown that, due to rotation induced by the orbital angular-momentum of light, higher order Bessel beams cannot stably confine a particle to the beam center where NLOF occurs in the absence of ambient damping, which largely limits their applications for long-distance, stable, backward particle transportation. On the other hand, zero-order Bessel beams can achieve stable transverse confinement of the manipulated particle and act as an optical tractor beam per se. In addition, for a nonmagnetic particle with relative permeability μ=1, a Bessel beam with transverse electric polarization is more favorable for the realization of NLOF than a transverse magnetic beam. Finally, a brief discussion is also presented of the conditions under which an off-beam-axis particle could be suitable for backward transportation using NLOF.
DOI
Based on the generalized Lorenz-Mie theory and Maxwell stress tensor formulism, we calculate the transverse force constant matrix and perform a linear stability analysis on a spherical particle that is subject to negative longitudinal optical force (NLOF) under the illumination of Bessel beams. Phase diagrams with respect to the material parameters are presented, which exhibit the possibility of the appearance of NLOF. From dynamical simulations of the particle performed both in the transverse plane and along the longitudinal direction, an even clearer picture of the realization of stable NLOF is presented. It is shown that, due to rotation induced by the orbital angular-momentum of light, higher order Bessel beams cannot stably confine a particle to the beam center where NLOF occurs in the absence of ambient damping, which largely limits their applications for long-distance, stable, backward particle transportation. On the other hand, zero-order Bessel beams can achieve stable transverse confinement of the manipulated particle and act as an optical tractor beam per se. In addition, for a nonmagnetic particle with relative permeability μ=1, a Bessel beam with transverse electric polarization is more favorable for the realization of NLOF than a transverse magnetic beam. Finally, a brief discussion is also presented of the conditions under which an off-beam-axis particle could be suitable for backward transportation using NLOF.
DOI
Intraflagellar transport drives flagellar surface motility
Sheng Min Shih, Benjamin D Engel, Fatih Kocabas, Thomas Bilyard, Arne Gennerich, Wallace F Marshall, Ahmet Yildiz
The assembly and maintenance of all cilia and flagella require intraflagellar transport (IFT) along the axoneme. IFT has been implicated in sensory and motile ciliary functions, but the mechanisms of this relationship remain unclear. Here, we used Chlamydomonas flagellar surface motility (FSM) as a model to test whether IFT provides force for gliding of cells across solid surfaces. We show that IFT trains are coupled to flagellar membrane glycoproteins (FMGs) in a Ca2+-dependent manner. IFT trains transiently pause through surface adhesion of their FMG cargos, and dynein-1b motors pull the cell towards the distal tip of the axoneme. Each train is transported by at least four motors, with only one type of motor active at a time. Our results demonstrate the mechanism of Chlamydomonas gliding motility and suggest that IFT plays a major role in adhesion-induced ciliary signaling pathways.
DOI
The assembly and maintenance of all cilia and flagella require intraflagellar transport (IFT) along the axoneme. IFT has been implicated in sensory and motile ciliary functions, but the mechanisms of this relationship remain unclear. Here, we used Chlamydomonas flagellar surface motility (FSM) as a model to test whether IFT provides force for gliding of cells across solid surfaces. We show that IFT trains are coupled to flagellar membrane glycoproteins (FMGs) in a Ca2+-dependent manner. IFT trains transiently pause through surface adhesion of their FMG cargos, and dynein-1b motors pull the cell towards the distal tip of the axoneme. Each train is transported by at least four motors, with only one type of motor active at a time. Our results demonstrate the mechanism of Chlamydomonas gliding motility and suggest that IFT plays a major role in adhesion-induced ciliary signaling pathways.
DOI
Geometrical edgeactants control interfacial bending rigidity of colloidal membranes
Mark J Zakhary, Prerna Sharma, Andrew Ward, Sevim Yardimici and Zvonimir Dogic
Colloidal membranes are a one rod-length thick monolayer of aligned rods which spontaneously assembles in a mixture of rod-like viruses and non-adsorbing polymer. The complex structure of the monolayer edge modifies its fluctuations effectively smoothing out the interface. We demonstrate that long filaments such as F-actin or flagella preferentially dissolve in the membrane's edge and can be used to control its interfacial properties. This effect is not driven by energetic interactions, but is rather a direct consequence of the intrinsic geometry of the constituent particles; hence we call such filaments geometrical edgeactants. Using optical manipulation techniques we adsorb individual filaments onto the edge of the colloidal membrane and measure their influence on the edge fluctuations. Edgeactants stiffen the interface, increasing its bending rigidity by up to an order of magnitude. Furthermore, they also locally suppress a polymorphic transition into twisted ribbons inherent to colloidal membranes. These results demonstrate new ways to control soft materials in which the final structure is only determined by the geometry of the constituent objects.
DOI
Colloidal membranes are a one rod-length thick monolayer of aligned rods which spontaneously assembles in a mixture of rod-like viruses and non-adsorbing polymer. The complex structure of the monolayer edge modifies its fluctuations effectively smoothing out the interface. We demonstrate that long filaments such as F-actin or flagella preferentially dissolve in the membrane's edge and can be used to control its interfacial properties. This effect is not driven by energetic interactions, but is rather a direct consequence of the intrinsic geometry of the constituent particles; hence we call such filaments geometrical edgeactants. Using optical manipulation techniques we adsorb individual filaments onto the edge of the colloidal membrane and measure their influence on the edge fluctuations. Edgeactants stiffen the interface, increasing its bending rigidity by up to an order of magnitude. Furthermore, they also locally suppress a polymorphic transition into twisted ribbons inherent to colloidal membranes. These results demonstrate new ways to control soft materials in which the final structure is only determined by the geometry of the constituent objects.
DOI
Gold nanorods as probes in two-photon fluorescence correlation spectroscopy: Emerging applications and potential artifacts
Da-Shin Wang, Shih-Chung Wei, Shih-Chu Liao, Chii-Wann Lin
Owing to the highly efficient two-photon fluorescence of gold nanorods and very short fluorescence lifetime compared with the rotational correlation time, the rotation and diffusion of a single gold nanorod can be easily observed by two-photon fluorescence correlation spectroscopy (TP-FCS). This property, along with the previous successful use as a contrast agent in two-photon fluorescence imaging, suggests a potential application in TP-FCS as well. Although the FCS measurement becomes highly efficient with gold nanorods as probes, the amplitude and temporal decay of the measured correlation functions depend critically on excitation power. Here, we investigate various photophysical processes of gold nanorods to determine the cause of such a sensitive power dependency. This understanding provides a basis for choosing appropriate FCS models to recover reasonable physical parameters. Although the correlation function amplitude G(0) is 32 times lower when the excitation power increases from 20 µW to 1.12 mW, the application of a saturation-modified FCS model yields very good fit to each data set and the fitted concentration of 0.64 nM is comparable to the 0.7 nM given by the inductively coupled plasma mass spectrometry measurement. The FCS assay appears to be an efficient method for the quantification of gold nanorods when correctly interpreted. However, even with the saturation considered in the fitting model, the fitted rotational and translational diffusion rates are getting faster as the power increases. This indicates that other effects such as photothermal effects may raise the local temperature, and thus increasing the rotational and translational diffusion rate.
DOI
Owing to the highly efficient two-photon fluorescence of gold nanorods and very short fluorescence lifetime compared with the rotational correlation time, the rotation and diffusion of a single gold nanorod can be easily observed by two-photon fluorescence correlation spectroscopy (TP-FCS). This property, along with the previous successful use as a contrast agent in two-photon fluorescence imaging, suggests a potential application in TP-FCS as well. Although the FCS measurement becomes highly efficient with gold nanorods as probes, the amplitude and temporal decay of the measured correlation functions depend critically on excitation power. Here, we investigate various photophysical processes of gold nanorods to determine the cause of such a sensitive power dependency. This understanding provides a basis for choosing appropriate FCS models to recover reasonable physical parameters. Although the correlation function amplitude G(0) is 32 times lower when the excitation power increases from 20 µW to 1.12 mW, the application of a saturation-modified FCS model yields very good fit to each data set and the fitted concentration of 0.64 nM is comparable to the 0.7 nM given by the inductively coupled plasma mass spectrometry measurement. The FCS assay appears to be an efficient method for the quantification of gold nanorods when correctly interpreted. However, even with the saturation considered in the fitting model, the fitted rotational and translational diffusion rates are getting faster as the power increases. This indicates that other effects such as photothermal effects may raise the local temperature, and thus increasing the rotational and translational diffusion rate.
DOI
Friday, June 14, 2013
Microscopic and macroscopic manipulation of gold nanorod and its hybrid nanostructures
Jiafang Li, Honglian Guo, and Zhi-Yuan Li
Gold nanorods (GNRs) have potential applications ranging from biomedical sciences and emerging nanophotonics. In this paper, we will review some of our recent studies on both microscopic and macroscopic manipulation of GNRs. Unique properties of GNR nanoparticles, such as efficient surface plasmon amplifications effects, are introduced. The stable trapping, transferring, positioning and patterning of GNRs with nonintrusive optical tweezers will be shown. Vector beams are further employed to improve the trapping performance. On the other hand, alignment of GNRs and their hybrid nanostructures will be described by using a film stretch method, which induces the anisotropic and enhanced absorptive nonlinearities from aligned GNRs. Realization and engineering of polarized emission from aligned hybrid GNRs will be further demonstrated, with relative excitation–emission efficiency significantly enhanced. Our works presented in this review show that optical tweezers possess great potential in microscopic manipulation of metal nanoparticles and macroscopic alignment of anisotropic nanoparticles could help the macroscopic samples to flexibly represent the plasmonic properties of single nanoparticles for fast, cheap, and high-yield applications.
DOI
Gold nanorods (GNRs) have potential applications ranging from biomedical sciences and emerging nanophotonics. In this paper, we will review some of our recent studies on both microscopic and macroscopic manipulation of GNRs. Unique properties of GNR nanoparticles, such as efficient surface plasmon amplifications effects, are introduced. The stable trapping, transferring, positioning and patterning of GNRs with nonintrusive optical tweezers will be shown. Vector beams are further employed to improve the trapping performance. On the other hand, alignment of GNRs and their hybrid nanostructures will be described by using a film stretch method, which induces the anisotropic and enhanced absorptive nonlinearities from aligned GNRs. Realization and engineering of polarized emission from aligned hybrid GNRs will be further demonstrated, with relative excitation–emission efficiency significantly enhanced. Our works presented in this review show that optical tweezers possess great potential in microscopic manipulation of metal nanoparticles and macroscopic alignment of anisotropic nanoparticles could help the macroscopic samples to flexibly represent the plasmonic properties of single nanoparticles for fast, cheap, and high-yield applications.
DOI
All-optical particle trap using orthogonally intersecting beams
K. D. Leake, A. R. Hawkins, and H. Schmidt
We analyze the properties of a dual-beam trap of orthogonally intersecting beams in the geometrical optics regime. We derive analytical expressions for the trapping location and stability criteria for trapping a microparticle with uncollimated Gaussian beams. An upper limit for the beam waist is found. Optical forces and particle trajectories are calculated numerically for the realistic case of a microparticle in intersecting liquid-core waveguides.
DOI
We analyze the properties of a dual-beam trap of orthogonally intersecting beams in the geometrical optics regime. We derive analytical expressions for the trapping location and stability criteria for trapping a microparticle with uncollimated Gaussian beams. An upper limit for the beam waist is found. Optical forces and particle trajectories are calculated numerically for the realistic case of a microparticle in intersecting liquid-core waveguides.
DOI
Cell manipulation tool with combined microwell array and optical tweezers for cell isolation and deposition
Xiaolin Wang, Xue Gou, Shuxun Chen, Xiao Yan and Dong Sun
Isolation from rare cells and deposition of sorted cells with high accuracy for further study are critical to a wide range of biomedical applications. In the current paper, we report an automated cell manipulation tool with combined optical tweezers and a uniquely designed microwell array, which functions for recognition, isolation, assembly, transportation and deposition of the interesting cells. The microwell array allows the passive hydrodynamic docking of cells, while offering the opportunity to inspect the interesting cell phenotypes with high spatio-temporal resolution based on the flexible image processing technique. In addition, dynamic and parallel cell manipulation in three dimensions can realize the target cell levitation from microwell and pattern assembly with multiple optical traps. Integrated with the programmed motorized stage, the optically levitated and assembled cells can be transported and deposited to the predefined microenvironment, so the tool can facilitate the integration of other on-chip functionalities for further study without removing these isolated cells from the chip. Experiments on human embryonic stem cells and yeast cells are performed to demonstrate the effectiveness of the proposed cell manipulation tool. Besides the application to cell isolation and deposition, three other biological applications with this tool are also presented.
DOI
Isolation from rare cells and deposition of sorted cells with high accuracy for further study are critical to a wide range of biomedical applications. In the current paper, we report an automated cell manipulation tool with combined optical tweezers and a uniquely designed microwell array, which functions for recognition, isolation, assembly, transportation and deposition of the interesting cells. The microwell array allows the passive hydrodynamic docking of cells, while offering the opportunity to inspect the interesting cell phenotypes with high spatio-temporal resolution based on the flexible image processing technique. In addition, dynamic and parallel cell manipulation in three dimensions can realize the target cell levitation from microwell and pattern assembly with multiple optical traps. Integrated with the programmed motorized stage, the optically levitated and assembled cells can be transported and deposited to the predefined microenvironment, so the tool can facilitate the integration of other on-chip functionalities for further study without removing these isolated cells from the chip. Experiments on human embryonic stem cells and yeast cells are performed to demonstrate the effectiveness of the proposed cell manipulation tool. Besides the application to cell isolation and deposition, three other biological applications with this tool are also presented.
DOI
Thursday, June 13, 2013
Separation of spin angular momentum in space-variant linearly polarized beam
Hao Chen, Zhongliang Yu, Jingjing Hao, Zhaozhong Chen, Ji Xu, Jianping Ding, Hui-Tian Wang
We show that the spin angular momentum (SAM) flux in a space-variant linearly polarized beam can be separated in the focal plane. Such a beam carries only orbital angular momentum (OAM) and develops a net SAM flux upon focusing. The radial splitting of the SAM flux density is mediated by the phase vortex (or OAM) and can be controlled by the topological charge of the phase vortex. Optical trapping experiments verify the separation of the SAM flux density. The proposed approach enriches the manipulation of the angular momentum of light fields and inspires more designs of focus engineering, which would benefit optical micromanipulation of microscopic particles.
DOI
We show that the spin angular momentum (SAM) flux in a space-variant linearly polarized beam can be separated in the focal plane. Such a beam carries only orbital angular momentum (OAM) and develops a net SAM flux upon focusing. The radial splitting of the SAM flux density is mediated by the phase vortex (or OAM) and can be controlled by the topological charge of the phase vortex. Optical trapping experiments verify the separation of the SAM flux density. The proposed approach enriches the manipulation of the angular momentum of light fields and inspires more designs of focus engineering, which would benefit optical micromanipulation of microscopic particles.
DOI
Wednesday, June 12, 2013
Polarization and Droplet Size Effects in the Laser-Trapping-Induced Reconfiguration in Individual Nematic Liquid Crystal Microdroplets
Anwar Usman, Wei-Yi Chiang, Takayuki Uwada, and Hiroshi Masuhara
We experimentally demonstrate reordering throughout the inside of an individual bipolar nematic liquid-crystalline microdroplet optically trapped by a highly focused laser beam, when the laser powers are above a definite threshold. The threshold depends on the droplet size and laser polarization. A physical interpretation of the results is presented by considering the nonlocal orientations of the nematic liquid-crystal molecules in the droplets with the dimensions on the order of the focal spot diameter or larger. On the basis of the finite size approximation, we show that the dependence of threshold power on the droplet size is calculated to be in qualitative agreement with the experimental data.
DOI
We experimentally demonstrate reordering throughout the inside of an individual bipolar nematic liquid-crystalline microdroplet optically trapped by a highly focused laser beam, when the laser powers are above a definite threshold. The threshold depends on the droplet size and laser polarization. A physical interpretation of the results is presented by considering the nonlocal orientations of the nematic liquid-crystal molecules in the droplets with the dimensions on the order of the focal spot diameter or larger. On the basis of the finite size approximation, we show that the dependence of threshold power on the droplet size is calculated to be in qualitative agreement with the experimental data.
DOI
Cell manipulation in microfluidics
Hoyoung Yun, Kisoo Kim and Won Gu Lee
Recent advances in the lab-on-a-chip field in association with nano/microfluidics have been made for new applications and functionalities to the fields of molecular biology, genetic analysis and proteomics, enabling the expansion of the cell biology field. Specifically, microfluidics has provided promising tools for enhancing cell biological research, since it has the ability to precisely control the cellular environment, to easily mimic heterogeneous cellular environment by multiplexing, and to analyze sub-cellular information by high-contents screening assays at the single-cell level. Various cell manipulation techniques in microfluidics have been developed in accordance with specific objectives and applications. In this review, we examine the latest achievements of cell manipulation techniques in microfluidics by categorizing externally applied forces for manipulation: (i) optical, (ii) magnetic, (iii) electrical, (iv) mechanical and (v) other manipulations. We furthermore focus on history where the manipulation techniques originate and also discuss future perspectives with key examples where available.
DOI
Recent advances in the lab-on-a-chip field in association with nano/microfluidics have been made for new applications and functionalities to the fields of molecular biology, genetic analysis and proteomics, enabling the expansion of the cell biology field. Specifically, microfluidics has provided promising tools for enhancing cell biological research, since it has the ability to precisely control the cellular environment, to easily mimic heterogeneous cellular environment by multiplexing, and to analyze sub-cellular information by high-contents screening assays at the single-cell level. Various cell manipulation techniques in microfluidics have been developed in accordance with specific objectives and applications. In this review, we examine the latest achievements of cell manipulation techniques in microfluidics by categorizing externally applied forces for manipulation: (i) optical, (ii) magnetic, (iii) electrical, (iv) mechanical and (v) other manipulations. We furthermore focus on history where the manipulation techniques originate and also discuss future perspectives with key examples where available.
DOI
Tuesday, June 11, 2013
Radiation pressure on a biconcave human Red Blood Cell and the resulting deformation in a pair of parallel optical traps
Guan-Bo Liao, Yin-Quan Chen, Paul B. Bareil, Yunlong Sheng, Arthur Chiou, Ming-Shien Chang
We calculated the three-dimensional optical stress distribution and the resulting deformation on a biconcave human red blood cell (RBC) in a pair of parallel optical trap. We assumed a Gaussian intensity distribution with a spherical wavefront for each trapping beam and calculated the optical stress from the momentum transfer associated with the reflection and refraction of the incident photons at each interface. The RBC was modelled as a biconcave thin elastic membrane with uniform elasticity and a uniform thickness of 0.25 μm. The resulting cell deformation was determined from the optical stress distribution by finite element software, Comsol Structure Mechanics Module, with Young's modulus (E) as a fitting parameter in order to fit the theoretical results for cell elongation to our experimental data.
DOI
We calculated the three-dimensional optical stress distribution and the resulting deformation on a biconcave human red blood cell (RBC) in a pair of parallel optical trap. We assumed a Gaussian intensity distribution with a spherical wavefront for each trapping beam and calculated the optical stress from the momentum transfer associated with the reflection and refraction of the incident photons at each interface. The RBC was modelled as a biconcave thin elastic membrane with uniform elasticity and a uniform thickness of 0.25 μm. The resulting cell deformation was determined from the optical stress distribution by finite element software, Comsol Structure Mechanics Module, with Young's modulus (E) as a fitting parameter in order to fit the theoretical results for cell elongation to our experimental data.
DOI
Kinesin-8 Is a Low-Force Motor Protein with a Weakly Bound Slip State
Anita Jannasch, Volker Bormuth, Marko Storch, Jonathon Howard, Erik Schäffer
During the cell cycle, kinesin-8s control the length of microtubules by interacting with their plus ends. To reach these ends, the motors have to be able to take many steps without dissociating. However, the underlying mechanism for this high processivity and how stepping is affected by force are unclear. Here, we tracked the motion of yeast (Kip3) and human (Kif18A) kinesin-8s with high precision under varying loads using optical tweezers. Surprisingly, both kinesin-8 motors were much weaker compared with other kinesins. Furthermore, we discovered a force-induced stick-slip motion: the motor frequently slipped, recovered from this state, and then resumed normal stepping motility without detaching from the microtubule. The low forces are consistent with kinesin-8s being regulators of microtubule dynamics rather than cargo transporters. The weakly bound slip state, reminiscent of a molecular safety leash, may be an adaptation for high processivity.
DOI
During the cell cycle, kinesin-8s control the length of microtubules by interacting with their plus ends. To reach these ends, the motors have to be able to take many steps without dissociating. However, the underlying mechanism for this high processivity and how stepping is affected by force are unclear. Here, we tracked the motion of yeast (Kip3) and human (Kif18A) kinesin-8s with high precision under varying loads using optical tweezers. Surprisingly, both kinesin-8 motors were much weaker compared with other kinesins. Furthermore, we discovered a force-induced stick-slip motion: the motor frequently slipped, recovered from this state, and then resumed normal stepping motility without detaching from the microtubule. The low forces are consistent with kinesin-8s being regulators of microtubule dynamics rather than cargo transporters. The weakly bound slip state, reminiscent of a molecular safety leash, may be an adaptation for high processivity.
DOI
Variation of Trapping Strength with Size and Number of Particles in a Single Trap
Jitendra Bhatt, Sachin Bhatt, Shaival Buch, Ravindra Pratap Singh, Saiyed Nisar Ali Jaaffrey
Optical tweezers use the radiation pressure to trap and manipulate the microscopic particles. Using various algorithms multiple traps are being formed which can trap a number of particles simultaneously. In contrast to multiple traps, many particles can be trapped at a single trap position. It is known that when two or more particles are trapped in a single trap they align themselves in axial direction and it appears as if only one particle is trapped. We present a study of the dependence of the optical trapping force on the number of particles in a single trap using equipartition method; the study was carried out for particles of different sizes. The trapping force was first found to increase then decrease with number of particles in trap for all particle sizes. We feel that our studies will be useful in applications of optical tweezers involving trapping of multiple particles in a single trap.
DOI
Optical tweezers use the radiation pressure to trap and manipulate the microscopic particles. Using various algorithms multiple traps are being formed which can trap a number of particles simultaneously. In contrast to multiple traps, many particles can be trapped at a single trap position. It is known that when two or more particles are trapped in a single trap they align themselves in axial direction and it appears as if only one particle is trapped. We present a study of the dependence of the optical trapping force on the number of particles in a single trap using equipartition method; the study was carried out for particles of different sizes. The trapping force was first found to increase then decrease with number of particles in trap for all particle sizes. We feel that our studies will be useful in applications of optical tweezers involving trapping of multiple particles in a single trap.
DOI
Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin
Horia Nicolae Roman, Nedjma B. Zitouni, Linda Kachmar, Gijs Ijpma, Lennart Hilbert, Oleg Matusovskiy, Andrea Benedetti, Apolinary Sobieszek, Anne-Marie Lauzon
Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. Funb from F-actin alone (0.12 ± 0.01pN; mean ± SE) was significantly increased in the presence of calponin (0.20 ± 0.02pN). This enhancement was lost when calponin was phosphorylated (0.12 ± 0.01pN). To further verify that this enhancement of Funb was due to cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25 mM (0.21 ± 0.02pN; mean ± SE) was significantly decreased at a [KCl] of 150 mM, (0.13 ± 0.01pN). This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.
Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. Funb from F-actin alone (0.12 ± 0.01pN; mean ± SE) was significantly increased in the presence of calponin (0.20 ± 0.02pN). This enhancement was lost when calponin was phosphorylated (0.12 ± 0.01pN). To further verify that this enhancement of Funb was due to cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25 mM (0.21 ± 0.02pN; mean ± SE) was significantly decreased at a [KCl] of 150 mM, (0.13 ± 0.01pN). This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.
Monday, June 10, 2013
Resonant propulsion of a microparticle by a surface wave
A. V. Maslov, V. N. Astratov, M. I. Bakunov
We investigate the electromagnetic force experienced by a microparticle supporting high-quality whispering gallery modes that are excited by a surface wave. Our theoretical approach is based on an analytical representation of the solution of the scattering problem with a subsequent numerical treatment. It accounts rigorously for the interaction of the microparticle with the waveguiding surface and allows us to establish the balances of electromagnetic power and momentum flow for the system. We show that the resonant excitation of the whispering gallery modes and suppression of the transmitted surface wave lead to an almost complete transformation of the momentum flow of the initial surface wave into the propelling force on the microparticle. The validation of the momentum balance justifies the definition of the momentum flow of the surface wave as the ratio of carried power and phase velocity. A simple approximate relation between the propelling force and the power of the transmitted surface wave is also introduced. The transverse force can be either attractive or repulsive depending on the particle-to-surface distance, particle size, and operating frequencies, and it can significantly exceed the value of the propelling force. A comparison with a microparticle excited by a plane wave is also included.
DOI
We investigate the electromagnetic force experienced by a microparticle supporting high-quality whispering gallery modes that are excited by a surface wave. Our theoretical approach is based on an analytical representation of the solution of the scattering problem with a subsequent numerical treatment. It accounts rigorously for the interaction of the microparticle with the waveguiding surface and allows us to establish the balances of electromagnetic power and momentum flow for the system. We show that the resonant excitation of the whispering gallery modes and suppression of the transmitted surface wave lead to an almost complete transformation of the momentum flow of the initial surface wave into the propelling force on the microparticle. The validation of the momentum balance justifies the definition of the momentum flow of the surface wave as the ratio of carried power and phase velocity. A simple approximate relation between the propelling force and the power of the transmitted surface wave is also introduced. The transverse force can be either attractive or repulsive depending on the particle-to-surface distance, particle size, and operating frequencies, and it can significantly exceed the value of the propelling force. A comparison with a microparticle excited by a plane wave is also included.
DOI
Bragg scattering and Brownian motion dynamics in optically induced crystals of submicron particles
R. E. Sapiro, B. N. Slama, and G. Raithel
A set of four confocal laser beams of 1064-nm wavelength is used to prepare optically induced crystals of submicron particles in aqueous solution. Thousands of polystyrene spheres of about 200 nm in diameter are trapped in three dimensions. Bragg scattering patterns obtained with a probe beam of 532-nm wavelength are in agreement with the calculated lattice structure and its polarization dependence. The decay and rise of the Bragg scattering intensity upon switching the lattice off and on reveal the Brownian motion dynamics of the particles in the periodic optical trapping potential. Experimental results agree well with results from trajectory simulations based on the Langevin equation. The results exhibit the interplay between Brownian motion and deterministic forces in an inhomogeneous (near-)periodic optical trapping potential.
DOI
A set of four confocal laser beams of 1064-nm wavelength is used to prepare optically induced crystals of submicron particles in aqueous solution. Thousands of polystyrene spheres of about 200 nm in diameter are trapped in three dimensions. Bragg scattering patterns obtained with a probe beam of 532-nm wavelength are in agreement with the calculated lattice structure and its polarization dependence. The decay and rise of the Bragg scattering intensity upon switching the lattice off and on reveal the Brownian motion dynamics of the particles in the periodic optical trapping potential. Experimental results agree well with results from trajectory simulations based on the Langevin equation. The results exhibit the interplay between Brownian motion and deterministic forces in an inhomogeneous (near-)periodic optical trapping potential.
DOI
The Essential Role of Stacking Adenines in a Two-Base-Pair RNA Kissing Complex
William Stephenson, Papa Nii Asare-Okai, Alan A. Chen, Sean Keller, Rachel Santiago, Scott A. Tenenbaum, Angel E. Garcia, Daniele Fabris, and Pan T. X. Li
In minimal RNA kissing complexes formed between hairpins with cognate GACG tetraloops, the two tertiary GC pairs are likely stabilized by the stacking of 5′-unpaired adenines at each end of the short helix. To test this hypothesis, we mutated the flanking adenines to various nucleosides and examined their effects on the kissing interaction. Electrospray ionization mass spectrometry was used to detect kissing dimers in a multiequilibria mixture, whereas optical tweezers were applied to monitor the (un)folding trajectories of single RNA molecules. The experimental findings were rationalized by molecular dynamics simulations. Together, the results showed that the stacked adenines are indispensable for the tertiary interaction. By shielding the tertiary base pairs from solvent and reducing their fraying, the stacked adenines made terminal pairs act more like interior base pairs. The purine double-ring of adenine was essential for effective stacking, whereas additional functional groups modulated the stabilizing effects through varying hydrophobic and electrostatic forces. Furthermore, formation of the kissing complex was dominated by base pairing, whereas its dissociation was significantly influenced by the flanking bases. Together, these findings indicate that unpaired flanking nucleotides play essential roles in the formation of otherwise unstable two-base-pair RNA tertiary interactions.
DOI
In minimal RNA kissing complexes formed between hairpins with cognate GACG tetraloops, the two tertiary GC pairs are likely stabilized by the stacking of 5′-unpaired adenines at each end of the short helix. To test this hypothesis, we mutated the flanking adenines to various nucleosides and examined their effects on the kissing interaction. Electrospray ionization mass spectrometry was used to detect kissing dimers in a multiequilibria mixture, whereas optical tweezers were applied to monitor the (un)folding trajectories of single RNA molecules. The experimental findings were rationalized by molecular dynamics simulations. Together, the results showed that the stacked adenines are indispensable for the tertiary interaction. By shielding the tertiary base pairs from solvent and reducing their fraying, the stacked adenines made terminal pairs act more like interior base pairs. The purine double-ring of adenine was essential for effective stacking, whereas additional functional groups modulated the stabilizing effects through varying hydrophobic and electrostatic forces. Furthermore, formation of the kissing complex was dominated by base pairing, whereas its dissociation was significantly influenced by the flanking bases. Together, these findings indicate that unpaired flanking nucleotides play essential roles in the formation of otherwise unstable two-base-pair RNA tertiary interactions.
DOI
Theory of microdroplet and microbubble deformation by Gaussian laser beam
Simen Å. Ellingsen
The theory for linear deformations of fluid microparticles in a laser beam of Gaussian profile is presented, when the beam focus is at the particle center as in optical trapping. Three different fluid systems are considered: water microdroplet in air, air microbubble in water, and a special oil-emulsion in water system used in experiments with optical deformation of fluid interfaces. We compare interface deformations of the three systems when illuminated by wide (compared to particle radius) and narrow laser beams and analyze differences. Deformations of droplets are radically different from bubbles under otherwise identical conditions, due to the opposite lensing effect (converging and diverging, respectively) of the two; a droplet is deformed far more than a bubble, cetera paribus. Optical contrast is found to be of great importance to the shape obtained when comparing the relatively low-contrast oil-emulsion system to that of water droplets. We finally analyze the dynamics of particle motion when the laser beam is turned on, and compare a static beam to the case of a short pulse. The very different surface tension coefficient implies a very different time scale for dynamics: microseconds for the water–air interface and tens of milliseconds for the oil-emulsion. Surface oscillations of a water microdroplet are found always to be underdamped, while those of the oil-emulsion are overdamped; deformations of a microbubble can be either, depending on physical parameters.
DOI
The theory for linear deformations of fluid microparticles in a laser beam of Gaussian profile is presented, when the beam focus is at the particle center as in optical trapping. Three different fluid systems are considered: water microdroplet in air, air microbubble in water, and a special oil-emulsion in water system used in experiments with optical deformation of fluid interfaces. We compare interface deformations of the three systems when illuminated by wide (compared to particle radius) and narrow laser beams and analyze differences. Deformations of droplets are radically different from bubbles under otherwise identical conditions, due to the opposite lensing effect (converging and diverging, respectively) of the two; a droplet is deformed far more than a bubble, cetera paribus. Optical contrast is found to be of great importance to the shape obtained when comparing the relatively low-contrast oil-emulsion system to that of water droplets. We finally analyze the dynamics of particle motion when the laser beam is turned on, and compare a static beam to the case of a short pulse. The very different surface tension coefficient implies a very different time scale for dynamics: microseconds for the water–air interface and tens of milliseconds for the oil-emulsion. Surface oscillations of a water microdroplet are found always to be underdamped, while those of the oil-emulsion are overdamped; deformations of a microbubble can be either, depending on physical parameters.
DOI
The photonic wheel - demonstration of a state of light with purely transverse angular momentum
P. Banzer, M. Neugebauer, A. Aiello, C. Marquardt, N. Lindlein, T. Bauer, G. Leuchs
In classical mechanics, a system may possess angular momentum which can be either transverse (e.g. in a spinning wheel) or longitudinal(e.g. for a spiraling seed falling from a tree) with respect to the direction of motion. However, for light, a typical massless wave system,the situation is less versatile. Photons are well-known to exhibit intrinsic angular momentum which is longitudinal only: the spin angular momentum defining the polarization and the orbital angular momentum associated with a spiraling phase front. Here we show that it is possible to generate a novel state of the light field that contains purely transverse angular momentum, the analogue of a spinning mechanical wheel. We realize this state by tight focusing of a polarization tailored light beam and measure it using an optical nano-probing technique. Such a novel state of the light field can find applications in optical tweezers and spanners where it allows for additional rotational degree of freedom not achievable in single-beam configurations so far.
DOI
In classical mechanics, a system may possess angular momentum which can be either transverse (e.g. in a spinning wheel) or longitudinal(e.g. for a spiraling seed falling from a tree) with respect to the direction of motion. However, for light, a typical massless wave system,the situation is less versatile. Photons are well-known to exhibit intrinsic angular momentum which is longitudinal only: the spin angular momentum defining the polarization and the orbital angular momentum associated with a spiraling phase front. Here we show that it is possible to generate a novel state of the light field that contains purely transverse angular momentum, the analogue of a spinning mechanical wheel. We realize this state by tight focusing of a polarization tailored light beam and measure it using an optical nano-probing technique. Such a novel state of the light field can find applications in optical tweezers and spanners where it allows for additional rotational degree of freedom not achievable in single-beam configurations so far.
DOI
Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers
Marijn T. J. van Loenhout, Iwijn De Vlaminck, Benedetta Flebus, Johan F. den Blanken, Ludovit P. Zweifel, Koen M. Hooning, Jacob W. J. Kerssemakers, Cees Dekker
The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33±5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.
DOI
The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33±5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.
DOI
Saturday, June 8, 2013
Kinetics and non-exponential binding of DNA-coated colloids
William Benjamin Rogers, Talid Sinno and John C. Crocker
Transient bridges of DNA have been used to direct the self-assembly of colloidal particles into interesting soft materials, but the particle binding kinetics are often slow or anomalous. Using line optical tweezers, we quantify the dynamics of two DNA-coated microspheres as a function of DNA density and strength of the DNA-induced pair interaction potential. At high DNA density, the binding kinetics is limited by the rate of microsphere diffusion and displays the expected dependence on the interaction potential energy. At low DNA density, the particle binding kinetics is set by single molecular binding events and exhibits bound times having a non-exponential distribution, suggesting that individual DNA bridges may also have intrinsic non-exponential kinetics. A dynamic model that includes such dispersion in the lifetimes of molecular bridges reproduces our observations, while an alternative model based on fluctuations in DNA density does not.
DOI
Transient bridges of DNA have been used to direct the self-assembly of colloidal particles into interesting soft materials, but the particle binding kinetics are often slow or anomalous. Using line optical tweezers, we quantify the dynamics of two DNA-coated microspheres as a function of DNA density and strength of the DNA-induced pair interaction potential. At high DNA density, the binding kinetics is limited by the rate of microsphere diffusion and displays the expected dependence on the interaction potential energy. At low DNA density, the particle binding kinetics is set by single molecular binding events and exhibits bound times having a non-exponential distribution, suggesting that individual DNA bridges may also have intrinsic non-exponential kinetics. A dynamic model that includes such dispersion in the lifetimes of molecular bridges reproduces our observations, while an alternative model based on fluctuations in DNA density does not.
DOI
Transport of particles by a thermally induced gradient of the order parameter in nematic liquid crystals
M. Škarabot, Ž. Lokar, and I. Muševič
We demonstrate manipulation and transport of microparticles and even fluorescent molecules by the thermally induced gradient of the order parameter in the nematic liquid crystal. We use IR light absorption of the tightly focused beam of laser tweezers to heat locally a thin layer of the nematic liquid crystal by several degrees. This creates a spatial gradient of temperature of the nematic liquid crystal over separations of several tens of micrometers. We show that a dipolar colloidal particle is attracted into the hot spot of the laser tweezers. The depth of the trapping potential scales linearly with particle radius, indicating that the trapping mechanism is due to elastic self-energy of the distorted nematic liquid crystal around the particle and softening of the elasticity with increased temperature of the liquid crystal. We also demonstrate that this thermal trapping mechanism is efficient down to the nanoscale, as fluorescent molecules are also transported into hotter regions of the liquid crystal. This effect is absent in the isotropic phase, which calls into question particle transport due to the Soret effect.
DOI
We demonstrate manipulation and transport of microparticles and even fluorescent molecules by the thermally induced gradient of the order parameter in the nematic liquid crystal. We use IR light absorption of the tightly focused beam of laser tweezers to heat locally a thin layer of the nematic liquid crystal by several degrees. This creates a spatial gradient of temperature of the nematic liquid crystal over separations of several tens of micrometers. We show that a dipolar colloidal particle is attracted into the hot spot of the laser tweezers. The depth of the trapping potential scales linearly with particle radius, indicating that the trapping mechanism is due to elastic self-energy of the distorted nematic liquid crystal around the particle and softening of the elasticity with increased temperature of the liquid crystal. We also demonstrate that this thermal trapping mechanism is efficient down to the nanoscale, as fluorescent molecules are also transported into hotter regions of the liquid crystal. This effect is absent in the isotropic phase, which calls into question particle transport due to the Soret effect.
DOI
Trapping and delivery of Escherichia coli in a microfluidic channel using an optical nanofiber
Hongbao Xin, Chang Cheng and Baojun Li
Stable trapping and direct delivery of bacteria are very important in studying their nanoscopic biochemical processes. Here, we report stable optical trapping and direct delivery of Escherichia coli in a microfluidic channel. By placing an optical nanofiber (NF, diameter: 600 nm) in the channel and launching a laser beam at 980 nm wavelength with an optical power over 25 mW into the NF, E. coli were stably trapped by the NF. Trapping stability was tested in flowing suspensions and delivery efficiency of the trapped E. coli was demonstrated. Experimental results were interpreted by numerical simulations and theoretical calculations.
DOI
Stable trapping and direct delivery of bacteria are very important in studying their nanoscopic biochemical processes. Here, we report stable optical trapping and direct delivery of Escherichia coli in a microfluidic channel. By placing an optical nanofiber (NF, diameter: 600 nm) in the channel and launching a laser beam at 980 nm wavelength with an optical power over 25 mW into the NF, E. coli were stably trapped by the NF. Trapping stability was tested in flowing suspensions and delivery efficiency of the trapped E. coli was demonstrated. Experimental results were interpreted by numerical simulations and theoretical calculations.
DOI
Thursday, June 6, 2013
Copper ion-exchanged channel waveguides optimization for optical trapping
K.N. Khor, A.H. Reshak, M.M. Shahimin, S.A.Z. Murad
Optical trapping of particles has become a powerful non-mechanical and non-destructive technique for precise particle positioning. The manipulation of particles in the evanescent field of a channel waveguide potentially allows for sorting and trapping of several particles and cells simultaneously. Channel waveguide designs can be further optimized to increase evanescent field prior to the fabrication process. This is crucial in order to make sure that the surface intensity is sufficient for optical trapping. Simulation configurations are explained in detail with specific simulation flow. Discussion on parameters optimization; physical geometry, optical polarization and wavelength is included in this paper. The effect of physical, optical parameters and beam spot size on evanescent field has been thoroughly discussed. These studies will continue toward the development of a novel copper ion-exchanged waveguide as a method of particle sorting, with biological cell propulsion studies presently underway.
DOI
Optical trapping of particles has become a powerful non-mechanical and non-destructive technique for precise particle positioning. The manipulation of particles in the evanescent field of a channel waveguide potentially allows for sorting and trapping of several particles and cells simultaneously. Channel waveguide designs can be further optimized to increase evanescent field prior to the fabrication process. This is crucial in order to make sure that the surface intensity is sufficient for optical trapping. Simulation configurations are explained in detail with specific simulation flow. Discussion on parameters optimization; physical geometry, optical polarization and wavelength is included in this paper. The effect of physical, optical parameters and beam spot size on evanescent field has been thoroughly discussed. These studies will continue toward the development of a novel copper ion-exchanged waveguide as a method of particle sorting, with biological cell propulsion studies presently underway.
DOI
Mechanical unfolding of long human telomeric RNA (TERRA)
J. Ricardo Arias-Gonzalez, Miguel Garavís, Rebeca Bocanegra, Elías Herrero-Galán, Alfredo Villasante and Carlos Gonzalez
We report the first single molecule investigation of TERRA molecules. By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplexes blocks, which unfold at lower forces than their DNA counterparts.
DOI
We report the first single molecule investigation of TERRA molecules. By using optical-tweezers and other biophysical techniques, we have found that long RNA constructions of up to 25 GGGUUA repeats form higher order structures comprised of single parallel G-quadruplexes blocks, which unfold at lower forces than their DNA counterparts.
DOI
Particle Manipulation Based on Optically Controlled Free Surface Hydrodynamics
Subramanyan Namboodiri Varanakkottu‡, Dr. Sajan Daniel George, Dr. Tobias Baier, Prof. Steffen Hardt, Martina Ewald, Prof. Markus Biesalski
Microparticle manipulation: The photoisomerization of surfactants adsorbed at a gas–liquid interface drives a Marangoni flow that can be used for the trapping and manipulation of small particles. By switching the laser wavelength, a flow either into or away from the focal spot can be induced. The picture shows a microparticle trapped in the focal region by the inflow.
DOI
Microparticle manipulation: The photoisomerization of surfactants adsorbed at a gas–liquid interface drives a Marangoni flow that can be used for the trapping and manipulation of small particles. By switching the laser wavelength, a flow either into or away from the focal spot can be induced. The picture shows a microparticle trapped in the focal region by the inflow.
DOI
Wednesday, June 5, 2013
The bacterial type IV pilus system – a tunable molecular motor
Berenike Maier
Bacteria have evolved surface-exposed polymers called pili with remarkable physical properties. This highlight describes recent advances in the biophysics of type IV pilus systems. They are strong molecular motors whose velocity and force are tunable by external inputs. Coordination of multiple pili for mediating pilus-driven surface motility depends on cell shape, surface interaction, and cooperation with other motors. Rational design of surfaces can control pilus-mediated surface movement and potentially biofilm architecture.
DOI
Bacteria have evolved surface-exposed polymers called pili with remarkable physical properties. This highlight describes recent advances in the biophysics of type IV pilus systems. They are strong molecular motors whose velocity and force are tunable by external inputs. Coordination of multiple pili for mediating pilus-driven surface motility depends on cell shape, surface interaction, and cooperation with other motors. Rational design of surfaces can control pilus-mediated surface movement and potentially biofilm architecture.
DOI
Topology of nematic liquid crystal colloids confined to two dimensions
Uroš Tkalec and Igor Muševič
We present a brief overview of recent development in the field of nematic colloids with an emphasis on the topology of colloidal structures and recently discovered topologically nontrivial defect configurations. Nematic colloids are complex soft-matter systems, in which the topology of defects, induced by colloidal inclusions, can be controlled and varied locally by laser tweezers and other external fields. We discuss the role of elasticity driven colloidal interactions and demonstrate the importance of precise optical manipulation of topological defects for a targeted design of entangled structures. We conclude that the interplay between particle and defect topologies in liquid crystals provides an exciting journey to the burgeoning area of applied topology and opens several new directions in advanced engineering of soft materials.
DOI
We present a brief overview of recent development in the field of nematic colloids with an emphasis on the topology of colloidal structures and recently discovered topologically nontrivial defect configurations. Nematic colloids are complex soft-matter systems, in which the topology of defects, induced by colloidal inclusions, can be controlled and varied locally by laser tweezers and other external fields. We discuss the role of elasticity driven colloidal interactions and demonstrate the importance of precise optical manipulation of topological defects for a targeted design of entangled structures. We conclude that the interplay between particle and defect topologies in liquid crystals provides an exciting journey to the burgeoning area of applied topology and opens several new directions in advanced engineering of soft materials.
DOI
Single particle detection, manipulation and analysis with resonant optical trapping in photonic crystals
Nicolas Descharmes, Ulagalandha Perumal Dharanipathy, Zhaolu Diao, Mario Tonin and Romuald Houdré
We demonstrate a resonant optical trapping mechanism based on two-dimensional hollow photonic crystal cavities. This approach benefits simultaneously from the resonant nature and unprecedented field overlap with the trapped specimen. The photonic crystal structures are implemented in a 30 mm x 12 mm optofluidic chip consisting of a patterned silicon substrate and an ultrathin microfluidic membrane for particle injection and control. Firstly, we demonstrate permanent trapping of single 250 and 500 nm-sized particles with sub-mW powers. Secondly, the particle induces a large resonance shift of the cavity mode amounting up to several linewidths. This shift is exploited to detect the presence of a particle within the trap and to retrieve information on the trapped particle. The individual addressability of multiple cavities on a single photonic crystal device is also demonstrated.
DOI
We demonstrate a resonant optical trapping mechanism based on two-dimensional hollow photonic crystal cavities. This approach benefits simultaneously from the resonant nature and unprecedented field overlap with the trapped specimen. The photonic crystal structures are implemented in a 30 mm x 12 mm optofluidic chip consisting of a patterned silicon substrate and an ultrathin microfluidic membrane for particle injection and control. Firstly, we demonstrate permanent trapping of single 250 and 500 nm-sized particles with sub-mW powers. Secondly, the particle induces a large resonance shift of the cavity mode amounting up to several linewidths. This shift is exploited to detect the presence of a particle within the trap and to retrieve information on the trapped particle. The individual addressability of multiple cavities on a single photonic crystal device is also demonstrated.
DOI
Electrostatic Binding and Hydrophobic Collapse of Peptide–Nucleic Acid Aggregates Quantified Using Force Spectroscopy
Joan Camunas-Soler, Silvia Frutos, Cristiano V. Bizarro, Sara de Lorenzo, Maria Eugenia Fuentes-Perez, Roland Ramsch, Susana Vilchez, Conxita Solans, Fernando Moreno-Herrero, Fernando Albericio, Ramón Eritja, Ernest Giralt, Sukhendu B. Dev , and Felix Ritort
Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g., Alzheimer’s and Parkinson’s diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide that contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers, and lengths of DNA segments sequestered within the KF–DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces and discuss how the bending rigidity of the nucleic acid affects this process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant.
DOI
Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g., Alzheimer’s and Parkinson’s diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide that contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers, and lengths of DNA segments sequestered within the KF–DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces and discuss how the bending rigidity of the nucleic acid affects this process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant.
DOI
Traffic of Secondary Metabolites to Cell Surface in the Red Alga Laurencia dendroidea Depends on a Two-Step Transport by the Cytoskeleton
Vanessa M. Reis, Louisi S. Oliveira, Raoni M. F. Passos, Nathan B. Viana, Cláudia Mermelstein, Celso Sant'Anna, Renato C. Pereira, Wladimir C. Paradas, Fabiano L. Thompson, Gilberto M. Amado-Filho, Leonardo T. Salgado
In Laurencia dendroidea, halogenated secondary metabolites are primarily located in the vacuole named the corps en cerise (CC). For chemical defence at the surface level, these metabolites are intracellularly mobilised through vesicle transport from the CC to the cell periphery for posterior exocytosis of these chemicals. The cell structures involved in this specific vesicle traffic as well as the cellular structures related to the positioning and anchoring of the CC within the cell are not well known. Here, we aimed to investigate the role of cytoskeletal elements in both processes. Cellular and molecular assays were conducted to i) determine the ultrastructural apparatus involved in the vesicle traffic, ii) localise cytoskeletal filaments, iii) evaluate the role of different cytoskeletal filaments in the vesicle transport, iv) identify the cytoskeletal filaments responsible for the positioning and anchoring of the CC, and v) identify the transcripts related to cytoskeletal activity and vesicle transport. Our results show that microfilaments are found within the connections linking the CC to the cell periphery, playing an essential role in the vesicle traffic at these connections, which means a first step of the secondary metabolites transport to the cell surface. After that, the microtubules work in the positioning of the vesicles along the cell periphery towards specific regions where exocytosis takes place, which corresponds to the second step of the secondary metabolites transport to the cell surface. In addition, microtubules are involved in anchoring and positioning the CC to the cell periphery. Transcriptomic analysis revealed the expression of genes coding for actin filaments, microtubules, motor proteins and cytoskeletal accessory proteins. Genes related to vesicle traffic, exocytosis and membrane recycling were also identified. Our findings show, for the first time, that actin microfilaments and microtubules play an underlying cellular role in the chemical defence of red algae.
DOI
In Laurencia dendroidea, halogenated secondary metabolites are primarily located in the vacuole named the corps en cerise (CC). For chemical defence at the surface level, these metabolites are intracellularly mobilised through vesicle transport from the CC to the cell periphery for posterior exocytosis of these chemicals. The cell structures involved in this specific vesicle traffic as well as the cellular structures related to the positioning and anchoring of the CC within the cell are not well known. Here, we aimed to investigate the role of cytoskeletal elements in both processes. Cellular and molecular assays were conducted to i) determine the ultrastructural apparatus involved in the vesicle traffic, ii) localise cytoskeletal filaments, iii) evaluate the role of different cytoskeletal filaments in the vesicle transport, iv) identify the cytoskeletal filaments responsible for the positioning and anchoring of the CC, and v) identify the transcripts related to cytoskeletal activity and vesicle transport. Our results show that microfilaments are found within the connections linking the CC to the cell periphery, playing an essential role in the vesicle traffic at these connections, which means a first step of the secondary metabolites transport to the cell surface. After that, the microtubules work in the positioning of the vesicles along the cell periphery towards specific regions where exocytosis takes place, which corresponds to the second step of the secondary metabolites transport to the cell surface. In addition, microtubules are involved in anchoring and positioning the CC to the cell periphery. Transcriptomic analysis revealed the expression of genes coding for actin filaments, microtubules, motor proteins and cytoskeletal accessory proteins. Genes related to vesicle traffic, exocytosis and membrane recycling were also identified. Our findings show, for the first time, that actin microfilaments and microtubules play an underlying cellular role in the chemical defence of red algae.
DOI
Two dimensional interferometric optical trapping of multiple particles and Escherichia coli bacterial cells using a lensed multicore fiber
Ashleigh L. Barron, Ajoy K. Kar, Thomas J. Aspray, Andrew J. Waddie, Mohammad R. Taghizadeh, and Henry T. Bookey
Two dimensional interferometric trapping of multiple microspheres and Escherichia coli has been demonstrated using a multicore fiber lensed with an electric arc fusion splicer. Light was coupled evenly into all four cores using a diffractive optical element. The visibility of the fringes and also the appearance of the lattice can be altered by rotating a half wave-plate. As a result the particles can be manipulated from one dimensional trapping to two dimensional trapping or a variety of different two dimensional arrangements. The ability to align bacterial populations has potential application for quorum sensing, floc and biofilm and, metabolic co-operation studies.
DOI
Two dimensional interferometric trapping of multiple microspheres and Escherichia coli has been demonstrated using a multicore fiber lensed with an electric arc fusion splicer. Light was coupled evenly into all four cores using a diffractive optical element. The visibility of the fringes and also the appearance of the lattice can be altered by rotating a half wave-plate. As a result the particles can be manipulated from one dimensional trapping to two dimensional trapping or a variety of different two dimensional arrangements. The ability to align bacterial populations has potential application for quorum sensing, floc and biofilm and, metabolic co-operation studies.
DOI
Monday, June 3, 2013
Laser patterning for the study of MSC cardiogenic differentiation at the single-cell level
Zhen Ma, Qiuying Liu, Huaxiao Yang, Raymond B Runyan, Carol A Eisenberg, Meifeng Xu, Thomas K Borg, Roger Markwald, Yifei Wang and Bruce Z Gao
Mesenchymal stem cells (MSCs) have been cited as contributors to heart repair through cardiogenic differentiation and multiple cellular interactions, including the paracrine effect, cell fusion, and mechanical and electrical couplings. Due to heart–muscle complexity, progress in the development of knowledge concerning the role of MSCs in cardiac repair is heavily based on MSC–cardiomyocyte coculture. In conventional coculture systems, however, the in vivo cardiac muscle structure, in which rod-shaped cells are connected end-to-end, is not sustained; instead, irregularly shaped cells spread randomly, resulting in randomly distributed cell junctions. Consequently, contact-mediated cell–cell interactions (e.g., the electrical triggering signal and the mechanical contraction wave that propagate through MSC–cardiomyocyte junctions) occur randomly. Thus, the data generated on the beneficial effects of MSCs may be irrelevant to in vivo biological processes. In this study, we explored whether cardiomyocyte alignment, the most important phenotype, is relevant to stem cell cardiogenic differentiation. Here, we report (i) the construction of a laser-patterned, biochip-based, stem cell–cardiomyocyte coculture model with controlled cell alignment; and (ii) single-cell-level data on stem cell cardiogenic differentiation under in vivo-like cardiomyocyte alignment conditions.
DOI
Mesenchymal stem cells (MSCs) have been cited as contributors to heart repair through cardiogenic differentiation and multiple cellular interactions, including the paracrine effect, cell fusion, and mechanical and electrical couplings. Due to heart–muscle complexity, progress in the development of knowledge concerning the role of MSCs in cardiac repair is heavily based on MSC–cardiomyocyte coculture. In conventional coculture systems, however, the in vivo cardiac muscle structure, in which rod-shaped cells are connected end-to-end, is not sustained; instead, irregularly shaped cells spread randomly, resulting in randomly distributed cell junctions. Consequently, contact-mediated cell–cell interactions (e.g., the electrical triggering signal and the mechanical contraction wave that propagate through MSC–cardiomyocyte junctions) occur randomly. Thus, the data generated on the beneficial effects of MSCs may be irrelevant to in vivo biological processes. In this study, we explored whether cardiomyocyte alignment, the most important phenotype, is relevant to stem cell cardiogenic differentiation. Here, we report (i) the construction of a laser-patterned, biochip-based, stem cell–cardiomyocyte coculture model with controlled cell alignment; and (ii) single-cell-level data on stem cell cardiogenic differentiation under in vivo-like cardiomyocyte alignment conditions.
DOI
Modeling of micropipette aspiration and optical tweezers stretching of erythrocytes with or without Malaria parasite
Guyue Jiao, Ruojing ZhangThe erythrocytes play an important role in the human body. The healthy erythrocytes can undergo extremely large deformation while passing through small capillaries. Their infection by Malaria Plasmodium falcipurum (P.f.) will lead to capillary blockage and blood flow obstruction. Many experimental and computational methods have been applied to study the increase in stickiness and decrease in deformability of the Malaria (P.f.) infected erythrocytes. The novelty of this paper lies in the establishment of an multi-component model for investigating mechanical properties of Malaria (P.f.) infected erythrocytes, especially of their enclosed parasites. Finite element method was applied to simulate the erythrocytes’ deformation in micropipette aspiration and optical tweezers stretching using the computational software ABAQUS. The comparisons between simulations and experiments were able to quantitatively conclude the effects of stiffness and stickiness of the parasitophorous vacuole membrane on the cells’ deformation, which could not be obtained from experiments directly.
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DNA Interaction with Hoechst 33258: Stretching Experiments Decouple the Different Binding Modes
Eduardo F. Silva , Esio B. Ramos , and Marcio S. Rocha
By performing single molecule stretching experiments with optical tweezers, we have studied the DNA interaction with the ligand Hoechst 33258. The mechanical properties of the complexes formed as a function of ligand concentration were directly determined from these measurements by fitting the force $\times$ extension curve to the WormLike Chain model of semiflexible polymers. In addition, the physico-chemical parameters of the interaction were extracted from the persistence length data by using a previously developed two-sites quenched disorder statistical model, allowing the determination of the binding isotherm. Such approach has allowed us to decouple the two different binding modes present in this system. In particular, it was found that the binding isotherm consists of two Hill-type processes, one non-cooperative and the other strongly cooperative. Finally, DNA condensation due to the interaction with the ligand was also verified and characterized here by analyzing the apparent contour length of the complexes.
DOI
By performing single molecule stretching experiments with optical tweezers, we have studied the DNA interaction with the ligand Hoechst 33258. The mechanical properties of the complexes formed as a function of ligand concentration were directly determined from these measurements by fitting the force $\times$ extension curve to the WormLike Chain model of semiflexible polymers. In addition, the physico-chemical parameters of the interaction were extracted from the persistence length data by using a previously developed two-sites quenched disorder statistical model, allowing the determination of the binding isotherm. Such approach has allowed us to decouple the two different binding modes present in this system. In particular, it was found that the binding isotherm consists of two Hill-type processes, one non-cooperative and the other strongly cooperative. Finally, DNA condensation due to the interaction with the ligand was also verified and characterized here by analyzing the apparent contour length of the complexes.
DOI
Optical manipulation of complex molecular systems by high density green photons: experimental and theoretical evidence
Sorin Comorosan, Silviu Polosan, Irinel Popescu, Ioan Stamatin, Elena Ionescu, Sorin Avramescu, Liviu Cristian Cune, Marian Apostol
The recent revolution in modern optical techniques revealed that light interaction with matter generates a force, known as optical force, which produces material properties known in physics as optical matter. The basic technique of the domain uses forces exerted by a strongly focused beam of light to trap small objects and subsequently to manipulate their local structures. The purpose of this paper is to develop an alternative approach, using irradiations with high-density-green-photons, which induce electric dipoles by polarization effects. The materials used for the experiments were long carbon chains which represent the framework of biological macromolecules. The physical techniques used to reveal the locally induced molecular arrangements were: dynamic viscosity, zeta potential, chemiluminescence, liquid chromatography; mass spectrometry, and Raman and infrared spectroscopy. The principal result of our experiments was the detection of different molecular arrangements within the mixture of alkane chains, generated by our optical manipulations. This induced “optical matter” displayed two material properties: antioxidant effects and large molecular aggregation effects. In order to bring the experimental results in relation with theory, we developed a physical model and the interacting force between polarizable bodies was computed. By numerical calculations stable structures for N = 6 and N = 8 particles were obtained.
DOI
The recent revolution in modern optical techniques revealed that light interaction with matter generates a force, known as optical force, which produces material properties known in physics as optical matter. The basic technique of the domain uses forces exerted by a strongly focused beam of light to trap small objects and subsequently to manipulate their local structures. The purpose of this paper is to develop an alternative approach, using irradiations with high-density-green-photons, which induce electric dipoles by polarization effects. The materials used for the experiments were long carbon chains which represent the framework of biological macromolecules. The physical techniques used to reveal the locally induced molecular arrangements were: dynamic viscosity, zeta potential, chemiluminescence, liquid chromatography; mass spectrometry, and Raman and infrared spectroscopy. The principal result of our experiments was the detection of different molecular arrangements within the mixture of alkane chains, generated by our optical manipulations. This induced “optical matter” displayed two material properties: antioxidant effects and large molecular aggregation effects. In order to bring the experimental results in relation with theory, we developed a physical model and the interacting force between polarizable bodies was computed. By numerical calculations stable structures for N = 6 and N = 8 particles were obtained.
DOI
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