Thomas Andersen, Anders Kyrsting and Poul-Martin Bendix
We reveal that the gel to fluid phase transition causes spherical membrane vesicles to release a finite number of molecules in several consecutive and localized events. By locally melting Giant Unilamellar lipid Vesicles (GUVs), using an optically trapped gold nanoparticle (AuNP) as a local heat source, we establish a local phase transition on the spherical GUV membrane clearly visualized by a phase sensitive fluorescent marker. We measure transient permeation events through this transition zone visualized as de-quenching of calcein as it escapes the interior of the GUV. Since biological membranes share several features with melting membranes, like nanoscale domain formation and critical density fluctuations, similar passive membrane transport could potentially be abundant in living cells.
DOI
Monday, March 31, 2014
The Grab-and-Drop Protocol: A Novel Strategy for Membrane Protein Isolation and Reconstitution from Single Cells
Angelika Schrems, John Phillips, Duncan Robert Casey, Douglas Wylie, Miroslava Novakova, Uwe B Sleytr, David Klug, Mark A A Neil, Bernhard Schuster and Oscar Ces
We present a rapid and robust technique for the sampling of membrane-associated proteins from the surface of a single, live cell and their subsequent deposition onto a solid-supported lipid bilayer. As a proof of principle, this method has been used to extract green fluorescent protein (EGFP) labelled K-ras proteins located at the inner leaflet of the plasma membrane of colon carcinoma cells and to transfer them to an S-layer supported lipid bilayer system. The technique is non-destructive, meaning that both the cell and proteins are intact after the sampling operation, offering the potential for repeated measurements of the same cell of interest. This system provides the ideal tool for the investigation of cellular heterogeneity, as well as a platform for the investigation of rare cell types such as circulating tumour cells.
DOI
We present a rapid and robust technique for the sampling of membrane-associated proteins from the surface of a single, live cell and their subsequent deposition onto a solid-supported lipid bilayer. As a proof of principle, this method has been used to extract green fluorescent protein (EGFP) labelled K-ras proteins located at the inner leaflet of the plasma membrane of colon carcinoma cells and to transfer them to an S-layer supported lipid bilayer system. The technique is non-destructive, meaning that both the cell and proteins are intact after the sampling operation, offering the potential for repeated measurements of the same cell of interest. This system provides the ideal tool for the investigation of cellular heterogeneity, as well as a platform for the investigation of rare cell types such as circulating tumour cells.
DOI
Trapping of individual airborne absorbing particles using a counterflow nozzle and photophoretic trap for continuous sampling and analysis
Yong-Le Pan, Chuji Wang, Steven C. Hill, Mark Coleman, Leonid A. Beresnev and Joshua L. Santarpia
We describe an integrated opto-aerodynamic system and demonstrate that it enables us to trap absorbing airborne micron-size particles from air, hold them and then release them, and to repeat this sequence many times as would be appropriate for continuous sampling of particles from air. The key parts of the system are a conical photophoretic optical trap and a counter-flow coaxial-double-nozzle that concentrates and then slows particles for trapping. This technology should be useful for on-line applications that require monitoring (by single particle analyses) of a series of successively arriving particles (e.g., from the atmosphere or pharmaceutical or other production facilities) where the total sampling time may last from minutes to days, but where each particle must be held for a short time for measurements (e.g., Raman scattering).
DOI
We describe an integrated opto-aerodynamic system and demonstrate that it enables us to trap absorbing airborne micron-size particles from air, hold them and then release them, and to repeat this sequence many times as would be appropriate for continuous sampling of particles from air. The key parts of the system are a conical photophoretic optical trap and a counter-flow coaxial-double-nozzle that concentrates and then slows particles for trapping. This technology should be useful for on-line applications that require monitoring (by single particle analyses) of a series of successively arriving particles (e.g., from the atmosphere or pharmaceutical or other production facilities) where the total sampling time may last from minutes to days, but where each particle must be held for a short time for measurements (e.g., Raman scattering).
DOI
Diffraction Gratings for Chiral Molecules and Their Applications
Robert Peter Cameron, Alison Yao, and Stephen Barnett
We suggest the use of certain readily producible types of light to exert a force that points in opposite directions for the enantiomers of a chiral molecule and propose multiple devices based upon this novel manifestation of optical activity: in particular, our ‘discriminatory chiral diffraction grating’; a device that could be employed, for example, to measure the enantiomeric excess of a sample of chiral molecules simply and to high precision. Our work is relevant for many types of molecule and our proposed devices may be realisable using currently existing technology.
DOI
We suggest the use of certain readily producible types of light to exert a force that points in opposite directions for the enantiomers of a chiral molecule and propose multiple devices based upon this novel manifestation of optical activity: in particular, our ‘discriminatory chiral diffraction grating’; a device that could be employed, for example, to measure the enantiomeric excess of a sample of chiral molecules simply and to high precision. Our work is relevant for many types of molecule and our proposed devices may be realisable using currently existing technology.
DOI
Monday, March 24, 2014
Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends
Vladimir A. Volkov, Anatoly V. Zaytsev, Ekaterina L. Grishchuk
Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.
DOI
Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.
DOI
Photonic crystal waveguide cavity with waist design for efficient trapping and detection of nanoparticles
Pin-Tso Lin, Tsan-Wen Lu, and Po-Tsung Lee
For manipulating nanometric particles, we propose a photonic crystal waveguide cavity design with a waist structure to enhance resonance characteristic of the cavity. For trapping a polystyrene particle of 50 nm radius on the lateral side of the waist, the optical force can reach 2308 pN/W with 24.7% signal transmission. Threshold power of only 0.32 mW is required for stable trapping. The total length of the device is relatively short with only ten photonic crystal periods, and the trapping can occur precisely and only at the waist. The designed cavity can also provide particle detection and surrounding medium sensing using the transmission spectrum with narrow linewidth. The simulated figure of merit of 110.6 is relatively high compared with those obtained from most plasmonic structures for sensing application. We anticipate this design with features of compact, efficient, and versatile in functionality will be beneficial for developing lab-on-chip in the future.
DOI
For manipulating nanometric particles, we propose a photonic crystal waveguide cavity design with a waist structure to enhance resonance characteristic of the cavity. For trapping a polystyrene particle of 50 nm radius on the lateral side of the waist, the optical force can reach 2308 pN/W with 24.7% signal transmission. Threshold power of only 0.32 mW is required for stable trapping. The total length of the device is relatively short with only ten photonic crystal periods, and the trapping can occur precisely and only at the waist. The designed cavity can also provide particle detection and surrounding medium sensing using the transmission spectrum with narrow linewidth. The simulated figure of merit of 110.6 is relatively high compared with those obtained from most plasmonic structures for sensing application. We anticipate this design with features of compact, efficient, and versatile in functionality will be beneficial for developing lab-on-chip in the future.
DOI
Bond Elasticity Controls Molecular Recognition Specificity in Antibody–Antigen Binding
Anna Alemany, Nuria Sanvicens, Sara de Lorenzo, M.-Pilar Marco, and Felix Ritort
Force-spectroscopy experiments make it possible to characterize single ligand–receptor pairs. Here we measure the spectrum of bond strengths and flexibilities in antibody–antigen interactions using optical tweezers. We characterize the mechanical evolution of polyclonal antibodies generated under infection and the ability of a monoclonal antibody to cross-react against different antigens. Our results suggest that bond flexibility plays a major role in remodeling antibody–antigen bonds in order to improve recognition during the maturation of the humoral immune system.
DOI
Force-spectroscopy experiments make it possible to characterize single ligand–receptor pairs. Here we measure the spectrum of bond strengths and flexibilities in antibody–antigen interactions using optical tweezers. We characterize the mechanical evolution of polyclonal antibodies generated under infection and the ability of a monoclonal antibody to cross-react against different antigens. Our results suggest that bond flexibility plays a major role in remodeling antibody–antigen bonds in order to improve recognition during the maturation of the humoral immune system.
DOI
Moving average process underlying the holographic–optical–tweezers experiments
Jakub Ślęzak, Sławomir Drobczyński, Karina Weron, and Jan Masajada
We study the statistical properties of recordings that contain time-dependent positions of a bead trapped in optical tweezers. Analysis of such a time series indicates that the commonly accepted model, i.e., the autoregressive process of first-order, is not sufficient to fit the data. We show the presence of a first-order moving average part in the dynamical model of the system. We explain the origin of this part as an influence of the high-frequency CCD camera on the measurements. We show that this influence evidently depends on the applied exposure time. The proposed autoregressive moving average model appears to reflect perfectly all statistical features of the high-frequency recording data.
DOI
We study the statistical properties of recordings that contain time-dependent positions of a bead trapped in optical tweezers. Analysis of such a time series indicates that the commonly accepted model, i.e., the autoregressive process of first-order, is not sufficient to fit the data. We show the presence of a first-order moving average part in the dynamical model of the system. We explain the origin of this part as an influence of the high-frequency CCD camera on the measurements. We show that this influence evidently depends on the applied exposure time. The proposed autoregressive moving average model appears to reflect perfectly all statistical features of the high-frequency recording data.
DOI
Friday, March 21, 2014
Recent developments in the field of bending rigidity measurements on membranes
Rumiana Dimova
This review gives a brief overview of experimental approaches used to assess the bending rigidity of membranes. Emphasis is placed on techniques based on the use of giant unilamellar vesicles. We summarize the effect on the bending rigidity of membranes as a function of membrane composition, presence of various inclusions in the bilayer and molecules and ions in the bathing solutions. Examples for the impact of temperature, cholesterol, some peptides and proteins, sugars and salts are provided and the literature data discussed critically. Future directions, open questions and possible developments in this research field are also included.
DOI
This review gives a brief overview of experimental approaches used to assess the bending rigidity of membranes. Emphasis is placed on techniques based on the use of giant unilamellar vesicles. We summarize the effect on the bending rigidity of membranes as a function of membrane composition, presence of various inclusions in the bilayer and molecules and ions in the bathing solutions. Examples for the impact of temperature, cholesterol, some peptides and proteins, sugars and salts are provided and the literature data discussed critically. Future directions, open questions and possible developments in this research field are also included.
DOI
Optical trapping and rotation of airborne absorbing particles with a single focused laser beam
Jinda Lin and Yong-qing Li
We measure the periodic circular motion of single absorbing aerosol particles that are optically trapped with a single focused Gaussian beam and rotate around the laser propagation direction. The scattered light from the trapped particle is observed to be directional and change periodically at 0.4–20 kHz. The instantaneous positions of the moving particle within a rotation period are measured by a high-speed imaging technique using a charge coupled device camera and a repetitively pulsed light-emitting diode illumination. The centripetal acceleration of the trapped particle as high as ∼20 times the gravitational acceleration is observed and is attributed to the photophoretic forces.
DOI
We measure the periodic circular motion of single absorbing aerosol particles that are optically trapped with a single focused Gaussian beam and rotate around the laser propagation direction. The scattered light from the trapped particle is observed to be directional and change periodically at 0.4–20 kHz. The instantaneous positions of the moving particle within a rotation period are measured by a high-speed imaging technique using a charge coupled device camera and a repetitively pulsed light-emitting diode illumination. The centripetal acceleration of the trapped particle as high as ∼20 times the gravitational acceleration is observed and is attributed to the photophoretic forces.
DOI
Thursday, March 20, 2014
Escherichia coli swimming is robust against variations in flagellar number
Patrick J Mears, Santosh Koirala, Chris V Rao, Ido Golding, Yann R Chemla
Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Although the network underlying this process has been studied extensively, we do not yet have an end-to-end understanding of chemotaxis. Specifically, how the rotational states of a cell’s flagella cooperatively determine whether the cell ‘runs’ or ‘tumbles’ remains poorly characterized. Here, we measure the swimming behavior of individual E. coli cells while simultaneously detecting the rotational states of each flagellum. We find that a simple mathematical expression relates the cell’s run/tumble bias to the number and average rotational state of its flagella. However, due to inter-flagellar correlations, an ‘effective number’ of flagella—smaller than the actual number—enters into this relation. Data from a chemotaxis mutant and stochastic modeling suggest that fluctuations of the regulator CheY-P are the source of flagellar correlations. A consequence of inter-flagellar correlations is that run/tumble behavior is only weakly dependent on number of flagella.
DOI
Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Although the network underlying this process has been studied extensively, we do not yet have an end-to-end understanding of chemotaxis. Specifically, how the rotational states of a cell’s flagella cooperatively determine whether the cell ‘runs’ or ‘tumbles’ remains poorly characterized. Here, we measure the swimming behavior of individual E. coli cells while simultaneously detecting the rotational states of each flagellum. We find that a simple mathematical expression relates the cell’s run/tumble bias to the number and average rotational state of its flagella. However, due to inter-flagellar correlations, an ‘effective number’ of flagella—smaller than the actual number—enters into this relation. Data from a chemotaxis mutant and stochastic modeling suggest that fluctuations of the regulator CheY-P are the source of flagellar correlations. A consequence of inter-flagellar correlations is that run/tumble behavior is only weakly dependent on number of flagella.
DOI
A quasi-continuum model for human erythrocyte membrane based on the higher order Cauchy–Born rule
Xiangyang Wang, Xu Guo, Zheng Su
A nanoscale quasi-continuum (QC) model for exploring the mechanical properties of human erythrocyte/red blood cell (RBC) membranes is presented in this paper. The so-called higher order Cauchy–Born rule (HCB rule) is utilized as the linkage between the deformation of the spectrin network/cytoskeleton and that of the corresponding equivalent continuum. By incorporating the second order deformation gradients into kinematic description, the resulting QC model can capture the curvature effect of nanoscale membranes accurately in a geometrically consistent way. Based on the proposed QC model, a variationally consistent meshless computational scheme is developed for simulating the finite deformation of human erythrocyte/RBCs. The obtained deformation and wrinkling patterns are in good agreement with those from the existing experiments.
DOI
A nanoscale quasi-continuum (QC) model for exploring the mechanical properties of human erythrocyte/red blood cell (RBC) membranes is presented in this paper. The so-called higher order Cauchy–Born rule (HCB rule) is utilized as the linkage between the deformation of the spectrin network/cytoskeleton and that of the corresponding equivalent continuum. By incorporating the second order deformation gradients into kinematic description, the resulting QC model can capture the curvature effect of nanoscale membranes accurately in a geometrically consistent way. Based on the proposed QC model, a variationally consistent meshless computational scheme is developed for simulating the finite deformation of human erythrocyte/RBCs. The obtained deformation and wrinkling patterns are in good agreement with those from the existing experiments.
DOI
Fabrication of an On-Chip Nanorobot Integrating Functional Nanomaterials for Single-Cell Punctures
Hayakawa, T.; Fukada, S.; Arai, F.
Cell manipulations and cell surgeries are key techniques in biotechnology today. Micro/nanorobots integrated on a microfluidic chip (on-chip robot) are a promising technology for cell manipulations and cell surgeries because of their operator skill independency and robustness for external environments. These features enable high-throughput cell manipulations and cell surgeries on a microfluidic chip. However, it is difficult to apply previous on-chip robots for small cells of order ≈ 10 bm μ m because the manipulation or surgery probes of those robots are a few micrometers in size. This size has been restricted by their fabrication, employing a standard mask lithography process. We fabricated on-chip robots of nanometer size by femtosecond laser exposure (nanorobots). The processing resolutions were 270 nm (linewidth) and 600 nm (thickness). Furthermore, our fabrication technique enabled the nanorobot to have a hybrid structure integrating functional nanomaterials (hybrid nanorobot). By integrating the various functional nanomaterials on the nanorobot, we can create a new function for the nanorobot. In this study, we fabricated a hybrid nanorobot with carbon nanotubes (CNTs) of high photothermal efficiency. We demonstrated a single-cell puncture with this nanorobot by irradiating the CNTs with an infrared laser and generating heat at that point. Additionally, we demonstrated an optical manipulation of the nanorobot that makes it possible to perform a cell puncture with high spatial flexibility and high positioning accuracy.
DOI
Cell manipulations and cell surgeries are key techniques in biotechnology today. Micro/nanorobots integrated on a microfluidic chip (on-chip robot) are a promising technology for cell manipulations and cell surgeries because of their operator skill independency and robustness for external environments. These features enable high-throughput cell manipulations and cell surgeries on a microfluidic chip. However, it is difficult to apply previous on-chip robots for small cells of order ≈ 10 bm μ m because the manipulation or surgery probes of those robots are a few micrometers in size. This size has been restricted by their fabrication, employing a standard mask lithography process. We fabricated on-chip robots of nanometer size by femtosecond laser exposure (nanorobots). The processing resolutions were 270 nm (linewidth) and 600 nm (thickness). Furthermore, our fabrication technique enabled the nanorobot to have a hybrid structure integrating functional nanomaterials (hybrid nanorobot). By integrating the various functional nanomaterials on the nanorobot, we can create a new function for the nanorobot. In this study, we fabricated a hybrid nanorobot with carbon nanotubes (CNTs) of high photothermal efficiency. We demonstrated a single-cell puncture with this nanorobot by irradiating the CNTs with an infrared laser and generating heat at that point. Additionally, we demonstrated an optical manipulation of the nanorobot that makes it possible to perform a cell puncture with high spatial flexibility and high positioning accuracy.
DOI
Onset of Non-Continuum Effects in Microrheology of Entangled Polymer Solutions
Cole D. Chapman, Kent Lee, Dean Henze, Douglas E. Smith, and Rae M. Robertson-Anderson
Microrheology has emerged as a powerful approach for elucidating mechanical properties of soft materials and complex fluids, especially biomaterials. In this technique, embedded microspheres are used to determine viscoelastic properties via generalized Stokes–Einstein relations, which assume the material behaves as a homogeneous continuum on the length scale of the probe. However, this condition can be violated if macromolecular systems form characteristic length scales that are larger than the probe size. Here we report observations of the onset of this effect in DNA solutions. We use microspheres driven with optical tweezers to determine the frequency dependence of the linear elastic and viscous moduli and their dependence on probe radius and DNA length. For well-entangled DNA, we find that the threshold probe radius yielding continuum behavior is 3× the reptation tube diameter, consistent with recent theoretical predictions. Notably, this threshold is significantly larger than the mesh size of the polymer network, and larger than typical probe sizes used in microrheology studies.
DOI
Microrheology has emerged as a powerful approach for elucidating mechanical properties of soft materials and complex fluids, especially biomaterials. In this technique, embedded microspheres are used to determine viscoelastic properties via generalized Stokes–Einstein relations, which assume the material behaves as a homogeneous continuum on the length scale of the probe. However, this condition can be violated if macromolecular systems form characteristic length scales that are larger than the probe size. Here we report observations of the onset of this effect in DNA solutions. We use microspheres driven with optical tweezers to determine the frequency dependence of the linear elastic and viscous moduli and their dependence on probe radius and DNA length. For well-entangled DNA, we find that the threshold probe radius yielding continuum behavior is 3× the reptation tube diameter, consistent with recent theoretical predictions. Notably, this threshold is significantly larger than the mesh size of the polymer network, and larger than typical probe sizes used in microrheology studies.
DOI
Wednesday, March 19, 2014
The phosphatidylserine receptor TIM4 utilizes integrins as coreceptors to effect phagocytosis
Ronald S. Flannagan, Johnathan Canton, Wendy Furuya, Michael Glogauer, and Sergio Grinstein
T-cell Immunoglobulin Mucin protein 4 (TIM4), a phosphatidylserine (PtdSer)-binding receptor, mediates the phagocytosis of apoptotic cells. How TIM4 exerts its function is unclear and conflicting data have emerged. To define the mode of action of TIM4 we used two distinct but complementary approaches: (i) we compared bone marrow-derived macrophages from wild-type and TIM4−/− mice, and (ii) heterologously expressed TIM4 in epithelioid AD293 cells, which rendered them competent for engulfment of PtdSer-bearing targets. Using these systems, we demonstrate that rather than serving merely as a tether, as proposed earlier (Toda et al. 2012), TIM4 is an active participant in the phagocytic process. Furthermore, we find that TIM4 operates independently of lactadherin, which had been proposed to act as a bridging molecule. Interestingly, TIM4-driven phagocytosis is dependent on the activation of integrins and involves stimulation of Src-family kinases and FAK, as well as the localized accumulation of phosphatidylinositol 3,4,5-Trisphosphate. These mediators promote the recruitment of the nucleotide-exchange factor Vav3, which in turn activates small Rho-family GTPases. Gene silencing or ablation experiments demonstrated that RhoA, Rac1 and Rac2 act synergistically to drive the remodeling of actin that underlies phagocytosis. Single-particle detection experiments demonstrated that TIM4 and β1 integrins associate upon receptor clustering. These findings support a model where TIM4 engages integrins as coreceptors to evoke the signal transduction needed to internalize PtdSer-bearing targets such as apoptotic cells.
DOI
T-cell Immunoglobulin Mucin protein 4 (TIM4), a phosphatidylserine (PtdSer)-binding receptor, mediates the phagocytosis of apoptotic cells. How TIM4 exerts its function is unclear and conflicting data have emerged. To define the mode of action of TIM4 we used two distinct but complementary approaches: (i) we compared bone marrow-derived macrophages from wild-type and TIM4−/− mice, and (ii) heterologously expressed TIM4 in epithelioid AD293 cells, which rendered them competent for engulfment of PtdSer-bearing targets. Using these systems, we demonstrate that rather than serving merely as a tether, as proposed earlier (Toda et al. 2012), TIM4 is an active participant in the phagocytic process. Furthermore, we find that TIM4 operates independently of lactadherin, which had been proposed to act as a bridging molecule. Interestingly, TIM4-driven phagocytosis is dependent on the activation of integrins and involves stimulation of Src-family kinases and FAK, as well as the localized accumulation of phosphatidylinositol 3,4,5-Trisphosphate. These mediators promote the recruitment of the nucleotide-exchange factor Vav3, which in turn activates small Rho-family GTPases. Gene silencing or ablation experiments demonstrated that RhoA, Rac1 and Rac2 act synergistically to drive the remodeling of actin that underlies phagocytosis. Single-particle detection experiments demonstrated that TIM4 and β1 integrins associate upon receptor clustering. These findings support a model where TIM4 engages integrins as coreceptors to evoke the signal transduction needed to internalize PtdSer-bearing targets such as apoptotic cells.
DOI
The effect of boundary proximity on the response of individual ultrasound contrast agent microbubbles
Brandon L Helfield, Ben Y C Leung and David E Goertz
The effect of boundary proximity on ultrasound contrast agent microbubble emissions can play an important role in the context of both targeted microbubble imaging and contrast imaging of microvascular perfusion. In this study, individual microbubbles (n = 104) were insonicated as a function of distance from either a polystyrene membrane (OpticellTM) or a compliant agarose boundary up to offset distances of 1000 µm by use of an optical trapping setup. An 'acoustic spectroscopy' approach was employed, which entailed transmitting a sequence of tone bursts with centre frequencies ranging from 4 to 13.5 MHz to determine the frequency and amplitude of maximum radial response (fMR and AMR, respectively). For the OpticellTM case, microbubble response exhibited a distinctly oscillatory pattern with increasing offset distance, with an average maximal change in peak frequency and scattered pressure amplitude of 29.6% and 73.2%, respectively, as compared to their values adjacent to the boundary. For the agarose case, microbubbles exhibited an increase in fMR and a decrease in AMR with respect to their values in free space. Simulations indicate the oscillatory dependence on OpticellTM distance stems from wavelength-dependent interference phenomena. A recent analytical bubble-boundary model is in broad agreement with the relative AMR changes due to the more compliant agarose layer, however underestimates the change in relative fMR at the boundary.
DOI
The effect of boundary proximity on ultrasound contrast agent microbubble emissions can play an important role in the context of both targeted microbubble imaging and contrast imaging of microvascular perfusion. In this study, individual microbubbles (n = 104) were insonicated as a function of distance from either a polystyrene membrane (OpticellTM) or a compliant agarose boundary up to offset distances of 1000 µm by use of an optical trapping setup. An 'acoustic spectroscopy' approach was employed, which entailed transmitting a sequence of tone bursts with centre frequencies ranging from 4 to 13.5 MHz to determine the frequency and amplitude of maximum radial response (fMR and AMR, respectively). For the OpticellTM case, microbubble response exhibited a distinctly oscillatory pattern with increasing offset distance, with an average maximal change in peak frequency and scattered pressure amplitude of 29.6% and 73.2%, respectively, as compared to their values adjacent to the boundary. For the agarose case, microbubbles exhibited an increase in fMR and a decrease in AMR with respect to their values in free space. Simulations indicate the oscillatory dependence on OpticellTM distance stems from wavelength-dependent interference phenomena. A recent analytical bubble-boundary model is in broad agreement with the relative AMR changes due to the more compliant agarose layer, however underestimates the change in relative fMR at the boundary.
DOI
Catch and release: how do kinetochores hook the right microtubules during mitosis?
Krishna K. Sarangapani, Charles L. Asbury
Sport fishermen keep tension on their lines to prevent hooked fish from releasing. A molecular version of this angler's trick, operating at kinetochores, ensures accuracy during mitosis: the mitotic spindle attaches randomly to chromosomes and then correctly bioriented attachments are stabilized due to the tension exerted on them by opposing microtubules. Incorrect attachments, which lack tension, are unstable and release quickly, allowing another chance for biorientation. Stabilization of molecular interactions by tension also occurs in other physiological contexts, such as cell adhesion, motility, hemostasis, and tissue morphogenesis. Here, we review models for the stabilization of kinetochore attachments with an eye toward emerging models for other force-activated systems. Although attention in the mitosis field has focused mainly on one kinase-based mechanism, multiple mechanisms may act together to stabilize properly bioriented kinetochores and some principles governing other tension-sensitive systems may also apply to kinetochores.
DOI
Sport fishermen keep tension on their lines to prevent hooked fish from releasing. A molecular version of this angler's trick, operating at kinetochores, ensures accuracy during mitosis: the mitotic spindle attaches randomly to chromosomes and then correctly bioriented attachments are stabilized due to the tension exerted on them by opposing microtubules. Incorrect attachments, which lack tension, are unstable and release quickly, allowing another chance for biorientation. Stabilization of molecular interactions by tension also occurs in other physiological contexts, such as cell adhesion, motility, hemostasis, and tissue morphogenesis. Here, we review models for the stabilization of kinetochore attachments with an eye toward emerging models for other force-activated systems. Although attention in the mitosis field has focused mainly on one kinase-based mechanism, multiple mechanisms may act together to stabilize properly bioriented kinetochores and some principles governing other tension-sensitive systems may also apply to kinetochores.
DOI
Application of flat-top focus to 2D trapping of large particles
Hao Chen and K. C. Toussaint, Jr.
The 2D optical trapping ability of larger-than average-particles is compared for three different beam types: a flat-top, a Gaussian beam, and a donut shaped beam. Optical force-displacement curves are calculated in four different size regimes of particle diameters (1.5-20 μm). We find that the trapping efficiency increases linearly with ratio of particle diameter to wavelength for all three beams. As the ratio reaches a specific threshold value, the flat-top focus exhibits the largest trapping efficiency compared to the other two beam types. We experimentally demonstrate that flat-top focusing provides the largest transverse trapping efficiency for particles as large as 20 μm in diameter for our given experimental conditions. The overall trend in our experimental results follows that observed in our simulation model. The results from this study could facilitate light manipulation of large particles.
DOI
The 2D optical trapping ability of larger-than average-particles is compared for three different beam types: a flat-top, a Gaussian beam, and a donut shaped beam. Optical force-displacement curves are calculated in four different size regimes of particle diameters (1.5-20 μm). We find that the trapping efficiency increases linearly with ratio of particle diameter to wavelength for all three beams. As the ratio reaches a specific threshold value, the flat-top focus exhibits the largest trapping efficiency compared to the other two beam types. We experimentally demonstrate that flat-top focusing provides the largest transverse trapping efficiency for particles as large as 20 μm in diameter for our given experimental conditions. The overall trend in our experimental results follows that observed in our simulation model. The results from this study could facilitate light manipulation of large particles.
DOI
Study for optical manipulation of a surfactant-covered droplet using lattice Boltzmann method
Se Bin Choi, Sasidhar Kondaraju and Joon Sang Lee
In this study, we simulated deformation and surfactant distribution on the interface of a surfactant-covered droplet using optical tweezers as an external source. Two optical forces attracted a single droplet from the center to both sides. This resulted in an elliptical shape deformation. The droplet deformation was characterized as the change of the magnitudes of surface tension and optical force. In this process, a non-linear relationship among deformation, surface tension, and optical forces was observed. The change in the local surfactant concentration resulting from the application of optical forces was also analyzed and compared with the concentration of surfactants subjected to an extensional flow. Under the optical force influence, the surfactant molecules were concentrated at the droplet equator, which is totally opposite to the surfactants behavior under extensional flow, where the molecules were concentrated at the poles. Lastly, the quasi-equilibrium surfactant distribution was obtained by combining the effects of the optical forces with the extensional flow. All simulations were executed by the lattice Boltzmann method which is a powerful tool for solving micro-scale problems.
DOI
In this study, we simulated deformation and surfactant distribution on the interface of a surfactant-covered droplet using optical tweezers as an external source. Two optical forces attracted a single droplet from the center to both sides. This resulted in an elliptical shape deformation. The droplet deformation was characterized as the change of the magnitudes of surface tension and optical force. In this process, a non-linear relationship among deformation, surface tension, and optical forces was observed. The change in the local surfactant concentration resulting from the application of optical forces was also analyzed and compared with the concentration of surfactants subjected to an extensional flow. Under the optical force influence, the surfactant molecules were concentrated at the droplet equator, which is totally opposite to the surfactants behavior under extensional flow, where the molecules were concentrated at the poles. Lastly, the quasi-equilibrium surfactant distribution was obtained by combining the effects of the optical forces with the extensional flow. All simulations were executed by the lattice Boltzmann method which is a powerful tool for solving micro-scale problems.
DOI
Long term Raman spectral study of power-dependent photodamage in red blood cell
Marcos A. S. de Oliveira, Zachary J. Smith, Florian Knorr, Renato E. de Araujo and Sebastian Wachsmann-Hogiu
We monitored time-dependent changes in the Raman spectra of optically trapped red blood cells. By fitting the Raman peaks of individual spectra over time, high-precision time evolutions of peak positions and intensities were obtained. These changes are dependent on the trapping laser power. Characteristic times for these changes were determined for each laser power by fitting the time courses with multi-exponential curves. Raman spectral dynamics showed significant and irreversible changes as a function of trapping duration that we attribute to a combination of photodamage of hemoglobin at short times followed by diffusion of hemoglobin out of the cell at longer times.
DOI
We monitored time-dependent changes in the Raman spectra of optically trapped red blood cells. By fitting the Raman peaks of individual spectra over time, high-precision time evolutions of peak positions and intensities were obtained. These changes are dependent on the trapping laser power. Characteristic times for these changes were determined for each laser power by fitting the time courses with multi-exponential curves. Raman spectral dynamics showed significant and irreversible changes as a function of trapping duration that we attribute to a combination of photodamage of hemoglobin at short times followed by diffusion of hemoglobin out of the cell at longer times.
DOI
Monday, March 17, 2014
Absolute Quantification of Protein Copy Number using a Single-Molecule-Sensitive Microarray
Edward Burgin, Ali Salehi-Reyhani, Michael Barclay, Aidan Thomas Brown, Joseph Kaplinsky, Miroslava Novakova, Mark A A Neil, Oscar Ces, Keith R Willison and David Klug
We report the use of a microfluidic microarray incorporating single molecule detection for the absolute quantification of protein copy number in solution. In this paper we demonstrate protocols which enable calibration free detection for two protein detection assays. An EGFP protein assay has a limit of detection of <30 EGFP proteins in a microfluidic analysis chamber (limited by non-specific background binding), with a measured limit of linearity of approximately 6×106 molecules of analyte in the analysis chamber and a dynamic range of >5 orders of magnitude in protein concentration. An antibody sandwich assay was used to detect unlabelled human tumour suppressor protein p53 with a limit of detection of approximately 21 p53 proteins and a dynamic range of >3 orders of magnitude. We show that these protocols can be used to determine the absolute protein copy number at the single cell level in two human cancer cell lines.
DOI
We report the use of a microfluidic microarray incorporating single molecule detection for the absolute quantification of protein copy number in solution. In this paper we demonstrate protocols which enable calibration free detection for two protein detection assays. An EGFP protein assay has a limit of detection of <30 EGFP proteins in a microfluidic analysis chamber (limited by non-specific background binding), with a measured limit of linearity of approximately 6×106 molecules of analyte in the analysis chamber and a dynamic range of >5 orders of magnitude in protein concentration. An antibody sandwich assay was used to detect unlabelled human tumour suppressor protein p53 with a limit of detection of approximately 21 p53 proteins and a dynamic range of >3 orders of magnitude. We show that these protocols can be used to determine the absolute protein copy number at the single cell level in two human cancer cell lines.
DOI
Optical clearing at cellular level
Matti Kinnunen ; Alexander V. Bykov ; Juho Tuorila ; Tomi Haapalainen ; Artashes V. Karmenyan ; Valery V. Tuchin
Strong light scattering in tissues and blood reduces the usability of many optical techniques. By reducing scattering, optical clearing enables deeper light penetration and improves resolution in several optical imaging applications. We demonstrate the usage of optical tweezers and elastic light scattering to study optical clearing [one of the major mechanisms—matching of refractive indices (RIs)] at the single particle and cell level. We used polystyrene spheres and human red blood cells (RBCs) as samples and glycerol or glucose water solutions as clearing agents. Optical tweezers kept single microspheres and RBCs in place during the measurement of light scattering patterns. The results show that optical clearing reduces the scattering cross section and increases g. Glucose also decreased light scattering from a RBC. Optical clearing affected the anisotropy factor g of 23.25-μm polystyrene spheres, increasing it by 0.5% for an RI change of 2.2% (20% glycerol) and 0.3% for an RI change of 1.1% (13% glucose).
DOI
Strong light scattering in tissues and blood reduces the usability of many optical techniques. By reducing scattering, optical clearing enables deeper light penetration and improves resolution in several optical imaging applications. We demonstrate the usage of optical tweezers and elastic light scattering to study optical clearing [one of the major mechanisms—matching of refractive indices (RIs)] at the single particle and cell level. We used polystyrene spheres and human red blood cells (RBCs) as samples and glycerol or glucose water solutions as clearing agents. Optical tweezers kept single microspheres and RBCs in place during the measurement of light scattering patterns. The results show that optical clearing reduces the scattering cross section and increases g. Glucose also decreased light scattering from a RBC. Optical clearing affected the anisotropy factor g of 23.25-μm polystyrene spheres, increasing it by 0.5% for an RI change of 2.2% (20% glycerol) and 0.3% for an RI change of 1.1% (13% glucose).
DOI
A Novel Temperature Sensor Based on Optical Trapping Technology
Yu Zhang, Peibo Liang, Zhihai Liu, Jiaojie Lei, Jun Yang, and Libo Yuan
We propose and fabricate a novel temperature sensor based on the optical trapping technology. The temperature sensing cell is constructed by putting a “test-micro-particle” enclosed in a space built by a quartz capillary tube and two opposite-inserted optical fibers. In order to make the temperature sensor have the ability of auto-ready and easy-reset, we design and fabricate the special concavities in the ends of two fibers. This ability of auto-ready and easy-reset makes the sensor convenient to be applied in industrial fields for long-term-use. These properties provide a new probably development direction in sensing research fields for the optical tweezers technology, and solve the optical tweezers measurement repeatability problems.
DOI
We propose and fabricate a novel temperature sensor based on the optical trapping technology. The temperature sensing cell is constructed by putting a “test-micro-particle” enclosed in a space built by a quartz capillary tube and two opposite-inserted optical fibers. In order to make the temperature sensor have the ability of auto-ready and easy-reset, we design and fabricate the special concavities in the ends of two fibers. This ability of auto-ready and easy-reset makes the sensor convenient to be applied in industrial fields for long-term-use. These properties provide a new probably development direction in sensing research fields for the optical tweezers technology, and solve the optical tweezers measurement repeatability problems.
DOI
Saturday, March 15, 2014
Optical manipulation of single molecules in the living cell
Kamilla Norregaard, Liselotte Jauffred, Kirstine Berg-Sørensen and Lene Broeng Oddershede
Optical tweezers are the only nano-tool capable of manipulating and performing force-measurements on individual molecules and organelles within the living cell without performing a destructive penetration through the cell wall and without the need of inserting a non-endogenous probe. Here, we describe how optical tweezers are used to manipulate individual molecules and perform accurate force and distance measurements within the complex cytoplasm of the living cell. Optical tweezers can grab individual molecules or organelles, if their optical contrast to the medium is large enough, as is the case, e.g., for lipid granules or chromosomes. However, often the molecule of interest is specifically attached to a handle manipulated by the optical trap. The most commonly used handles, their insertion into the cytoplasm, and the relevant micro-rheology of the cell are here discussed and we also review recent and exciting results achieved through optical force manipulation of individual molecules in vivo.
DOI
Optical tweezers are the only nano-tool capable of manipulating and performing force-measurements on individual molecules and organelles within the living cell without performing a destructive penetration through the cell wall and without the need of inserting a non-endogenous probe. Here, we describe how optical tweezers are used to manipulate individual molecules and perform accurate force and distance measurements within the complex cytoplasm of the living cell. Optical tweezers can grab individual molecules or organelles, if their optical contrast to the medium is large enough, as is the case, e.g., for lipid granules or chromosomes. However, often the molecule of interest is specifically attached to a handle manipulated by the optical trap. The most commonly used handles, their insertion into the cytoplasm, and the relevant micro-rheology of the cell are here discussed and we also review recent and exciting results achieved through optical force manipulation of individual molecules in vivo.
DOI
Engineered Tumor Cell Apoptosis Monitoring Method Based on Dynamic Laser Tweezers
Yuquan Zhang, Xiaojing Wu, Changjun Min, Siwei Zhu, Xiaodong Yuan, and Paul Urbach
Monitoring the cells’ apoptosis progression could provide a valuable insight into the
temporal events that initiate cell death as well as the potential for rescue of apoptotic cells. In
this paper, we engineered a novel and robust method for monitoring apoptosis of tumor
cellsbased on dynamic laser tweezers, using A549 and HeLa cell line as typical samples. The entire experiment can be completed in a few hours withsmall amount of fluid sample, presenting great advantages of celerity, micro-scaled measurement, and label-free explorations without perturbing experimental conditions in combination with other probes. Validity and stability of this method are verified experimentally in terms of physical parameters of the system. The proposed technique hasgreat potential in improving cancer treatment by monitoring the objective efficacy of tumor cell killing.
DOI
Monitoring the cells’ apoptosis progression could provide a valuable insight into the
temporal events that initiate cell death as well as the potential for rescue of apoptotic cells. In
this paper, we engineered a novel and robust method for monitoring apoptosis of tumor
cellsbased on dynamic laser tweezers, using A549 and HeLa cell line as typical samples. The entire experiment can be completed in a few hours withsmall amount of fluid sample, presenting great advantages of celerity, micro-scaled measurement, and label-free explorations without perturbing experimental conditions in combination with other probes. Validity and stability of this method are verified experimentally in terms of physical parameters of the system. The proposed technique hasgreat potential in improving cancer treatment by monitoring the objective efficacy of tumor cell killing.
DOI
Superadiabatic optical forces on a dipole: exactly solvable model for a vortex field
M V Berry and Pragya Shukla
The forces exerted by light on a small particle are modified by the particle's motion, giving a series of superadiabatic corrections to the lowest-order approximation in which the motion is neglected. The correction forces can be calculated recursively for an electric dipole modelled as a damped oscillator. In lowest order, there is, as is known, a non-potential though non-dissipative 'curl force', in addition to the familiar gradient force. In the next order, there are forces of geometric magnetism and friction, related to the geometric phase 2-form and the metric of the driving field. For the paraxial field of an optical vortex, the hierarchy of superadiabatic forces can be calculated explicitly, revealing a four-sheeted Riemann surface on which fast and slow dynamics are connected. This leads to an exact 'slow manifold', on which the dipole is driven without oscillations by the same forces as in the first two adiabatic orders, but with frequency-renormalized strengths.
DOI
The forces exerted by light on a small particle are modified by the particle's motion, giving a series of superadiabatic corrections to the lowest-order approximation in which the motion is neglected. The correction forces can be calculated recursively for an electric dipole modelled as a damped oscillator. In lowest order, there is, as is known, a non-potential though non-dissipative 'curl force', in addition to the familiar gradient force. In the next order, there are forces of geometric magnetism and friction, related to the geometric phase 2-form and the metric of the driving field. For the paraxial field of an optical vortex, the hierarchy of superadiabatic forces can be calculated explicitly, revealing a four-sheeted Riemann surface on which fast and slow dynamics are connected. This leads to an exact 'slow manifold', on which the dipole is driven without oscillations by the same forces as in the first two adiabatic orders, but with frequency-renormalized strengths.
DOI
5D-Tracking of a nanorod in a focused laser beam - a theoretical concept
Markus Grießhammer and Alexander Rohrbach
Back-focal plane (BFP) interferometry is a very fast and precise method to track the 3D position of a sphere within a focused laser beam using a simple quadrant photo diode (QPD). Here we present a concept of how to track and recover the 5D state of a cylindrical nanorod (3D position and 2 tilt angles) in a laser focus by analyzing the interference of unscattered light and light scattered at the cylinder. The analytical theoretical approach is based on Rayleigh-Gans scattering together with a local field approximation for an infinitely thin cylinder. The approximated BFP intensities compare well with those from a more rigorous numerical approach. It turns out that a displacement of the cylinder results in a modulation of the BFP intensity pattern, whereas a tilt of the cylinder results in a shift of this pattern. We therefore propose the concept of a local QPD in the BFP of a detection lens, where the QPD center is shifted by the angular coordinates of the cylinder tilt.
DOI
Back-focal plane (BFP) interferometry is a very fast and precise method to track the 3D position of a sphere within a focused laser beam using a simple quadrant photo diode (QPD). Here we present a concept of how to track and recover the 5D state of a cylindrical nanorod (3D position and 2 tilt angles) in a laser focus by analyzing the interference of unscattered light and light scattered at the cylinder. The analytical theoretical approach is based on Rayleigh-Gans scattering together with a local field approximation for an infinitely thin cylinder. The approximated BFP intensities compare well with those from a more rigorous numerical approach. It turns out that a displacement of the cylinder results in a modulation of the BFP intensity pattern, whereas a tilt of the cylinder results in a shift of this pattern. We therefore propose the concept of a local QPD in the BFP of a detection lens, where the QPD center is shifted by the angular coordinates of the cylinder tilt.
DOI
Lateral optical force on chiral particles near a surface
S. B. Wang & C. T. Chan
Light can exert radiation pressure on any object it encounters and that resulting optical force can be used to manipulate particles. It is commonly assumed that light should move a particle forward and indeed an incident plane wave with a photon momentum ħk can only push any particle, independent of its properties, in the direction of k. Here we demonstrate, using full-wave simulations, that an anomalous lateral force can be induced in a direction perpendicular to that of the incident photon momentum if a chiral particle is placed above a substrate that does not break any left–right symmetry. Analytical theory shows that the lateral force emerges from the coupling between structural chirality (the handedness of the chiral particle) and the light reflected from the substrate surface. Such coupling induces a sideway force that pushes chiral particles with opposite handedness in opposite directions.
DOI
Light can exert radiation pressure on any object it encounters and that resulting optical force can be used to manipulate particles. It is commonly assumed that light should move a particle forward and indeed an incident plane wave with a photon momentum ħk can only push any particle, independent of its properties, in the direction of k. Here we demonstrate, using full-wave simulations, that an anomalous lateral force can be induced in a direction perpendicular to that of the incident photon momentum if a chiral particle is placed above a substrate that does not break any left–right symmetry. Analytical theory shows that the lateral force emerges from the coupling between structural chirality (the handedness of the chiral particle) and the light reflected from the substrate surface. Such coupling induces a sideway force that pushes chiral particles with opposite handedness in opposite directions.
DOI
Monolithic integration of DUV-induced waveguides into plastic microfluidic chip for optical manipulation
M. Khoury, C. Vannahme, K.T. Sørensen, A. Kristensen, K. Berg-Sørensen
A monolithic polymer optofluidic chip for manipulation of microbeads in flow is demonstrated. On this chip, polymer waveguides induced by Deep UV lithography are integrated with microfluidic channels. The optical propagation losses of the waveguides are measured to be 0.66±0.130.66±0.13 dB/mm at a wavelength of λλ = 808 nm. An optimized bead tracking algorithm is implemented, allowing for determination of the optical forces acting on the particles. The algorithm features a spatio-temporal mapping of coordinates for uniting partial trajectories, without increased processing time. With an external laser power of 250 mW, a maximum scattering force of 0.84 pN is achieved for 5 μm diameter polystyrene beads in water.
DOI
A monolithic polymer optofluidic chip for manipulation of microbeads in flow is demonstrated. On this chip, polymer waveguides induced by Deep UV lithography are integrated with microfluidic channels. The optical propagation losses of the waveguides are measured to be 0.66±0.130.66±0.13 dB/mm at a wavelength of λλ = 808 nm. An optimized bead tracking algorithm is implemented, allowing for determination of the optical forces acting on the particles. The algorithm features a spatio-temporal mapping of coordinates for uniting partial trajectories, without increased processing time. With an external laser power of 250 mW, a maximum scattering force of 0.84 pN is achieved for 5 μm diameter polystyrene beads in water.
DOI
Wednesday, March 12, 2014
Yoctoliter Thermometry for Single-Molecule Investigations: A Generic Bead-on-a-Tip Temperature-Control Module
Deepak Koirala, Jibin Abraham Punnoose, Prakash Shrestha, Prof. Hanbin Mao
A new temperature-jump (T-jump) strategy avoids photo-damage of individual molecules by focusing a low-intensity laser on a black microparticle at the tip of a capillary. The black particle produces an efficient photothermal effect that enables a wide selection of lasers with powers in the milliwatt range to achieve a T-jump of 65 °C within milliseconds. To measure the temperature in situ in single-molecule experiments, the temperature-dependent mechanical unfolding of a single DNA hairpin molecule was monitored by optical tweezers within a yoctoliter volume. Using this bead-on-a-tip module and the robust single-molecule thermometer, full thermodynamic landscapes for the unfolding of this DNA hairpin were retrieved. These approaches are likely to provide powerful tools for the microanalytical investigation of dynamic processes with a combination of T-jump and single-molecule techniques.
DOI
A new temperature-jump (T-jump) strategy avoids photo-damage of individual molecules by focusing a low-intensity laser on a black microparticle at the tip of a capillary. The black particle produces an efficient photothermal effect that enables a wide selection of lasers with powers in the milliwatt range to achieve a T-jump of 65 °C within milliseconds. To measure the temperature in situ in single-molecule experiments, the temperature-dependent mechanical unfolding of a single DNA hairpin molecule was monitored by optical tweezers within a yoctoliter volume. Using this bead-on-a-tip module and the robust single-molecule thermometer, full thermodynamic landscapes for the unfolding of this DNA hairpin were retrieved. These approaches are likely to provide powerful tools for the microanalytical investigation of dynamic processes with a combination of T-jump and single-molecule techniques.
DOI
Seminal Plasma Initiates a Neisseria gonorrhoeae Transmission State
Mark T. Anderson, Lena Dewenter, Berenike Maier, H. Steven Seifert
Niche-restricted pathogens are evolutionarily linked with the specific biological fluids that are encountered during infection. Neisseria gonorrhoeae causes the genital infection gonorrhea and is exposed to seminal fluid during sexual transmission. Treatment of N. gonorrhoeae with seminal plasma or purified semen proteins lactoferrin, serum albumin, and prostate-specific antigen each facilitated type IV pilus-mediated twitching motility of the bacterium. Motility in the presence of seminal plasma was characterized by high velocity and low directional persistence. In addition, infection of epithelial cells with N. gonorrhoeae in the presence of seminal plasma resulted in enhanced microcolony formation. Close association of multiple pili in the form of bundles was also disrupted after seminal plasma treatment leading to an increase in the number of single pilus filaments on the bacterial surface. Thus, exposure of N. gonorrhoeae to seminal plasma is proposed to alter bacterial motility and aggregation characteristics to influence the processes of transmission and colonization.
DOI
Niche-restricted pathogens are evolutionarily linked with the specific biological fluids that are encountered during infection. Neisseria gonorrhoeae causes the genital infection gonorrhea and is exposed to seminal fluid during sexual transmission. Treatment of N. gonorrhoeae with seminal plasma or purified semen proteins lactoferrin, serum albumin, and prostate-specific antigen each facilitated type IV pilus-mediated twitching motility of the bacterium. Motility in the presence of seminal plasma was characterized by high velocity and low directional persistence. In addition, infection of epithelial cells with N. gonorrhoeae in the presence of seminal plasma resulted in enhanced microcolony formation. Close association of multiple pili in the form of bundles was also disrupted after seminal plasma treatment leading to an increase in the number of single pilus filaments on the bacterial surface. Thus, exposure of N. gonorrhoeae to seminal plasma is proposed to alter bacterial motility and aggregation characteristics to influence the processes of transmission and colonization.
DOI
Direct optical monitoring of flow generated by bacterial flagellar rotation
Silke R. Kirchner, Spas Nedev, Sol Carretero-Palacios, Andreas Mader, Madeleine Opitz, Theobald Lohmüller and Jochen Feldmann
We report on a highly sensitive approach to measure and quantify the time dependent changes of the flow generated by the flagella bundle rotation of single bacterial cells. This is achieved by observing the interactions between a silica particle and a bacterium, which are both trapped next to each other in a dual beam optical tweezer. In this configuration, the particle serves as a sensitive detector where the fast-Fourier analysis of the particle trajectory renders, it possible to access information about changes of bacterial activity.
DOI
We report on a highly sensitive approach to measure and quantify the time dependent changes of the flow generated by the flagella bundle rotation of single bacterial cells. This is achieved by observing the interactions between a silica particle and a bacterium, which are both trapped next to each other in a dual beam optical tweezer. In this configuration, the particle serves as a sensitive detector where the fast-Fourier analysis of the particle trajectory renders, it possible to access information about changes of bacterial activity.
DOI
Accurate position tracking of optically trapped live cells
Niall McAlinden, David G. Glass, Owain R. Millington, and Amanda J. Wright
Optical trapping is a powerful tool in Life Science research and is becoming common place in many microscopy laboratories and facilities. There is a growing need to directly trap the cells of interest rather than introduce beads to the sample that can affect the fundamental biological functions of the sample and impact on the very properties the user wishes to observe and measure. However, instabilities while tracking large inhomogeneous objects, such as cells, can make tracking position, calibrating trap strength and making reliable measurements challenging. These instabilities often manifest themselves as cell roll or re-orientation and can occur as a result of viscous drag forces and thermal convection, as well as spontaneously due to Brownian forces. In this paper we discuss and mathematically model the cause of this roll and present several experimental approaches for tackling these issues, including using a novel beam profile consisting of three closely spaced traps and tracking a trapped object by analysing fluorescence images. The approaches presented here trap T cells which form part of the adaptive immune response system, but in principle can be applied to a wide range of samples where the size and inhomogeneous nature of the trapped object can hinder particle tracking experiments.
DOI
Optical trapping is a powerful tool in Life Science research and is becoming common place in many microscopy laboratories and facilities. There is a growing need to directly trap the cells of interest rather than introduce beads to the sample that can affect the fundamental biological functions of the sample and impact on the very properties the user wishes to observe and measure. However, instabilities while tracking large inhomogeneous objects, such as cells, can make tracking position, calibrating trap strength and making reliable measurements challenging. These instabilities often manifest themselves as cell roll or re-orientation and can occur as a result of viscous drag forces and thermal convection, as well as spontaneously due to Brownian forces. In this paper we discuss and mathematically model the cause of this roll and present several experimental approaches for tackling these issues, including using a novel beam profile consisting of three closely spaced traps and tracking a trapped object by analysing fluorescence images. The approaches presented here trap T cells which form part of the adaptive immune response system, but in principle can be applied to a wide range of samples where the size and inhomogeneous nature of the trapped object can hinder particle tracking experiments.
DOI
Monday, March 10, 2014
Three-dimensional manipulation with scanning near-field optical nanotweezers
J. Berthelot, S. S. Aćimović, M. L. Juan, M. P. Kreuzer, J. Renger & R. Quidant
Recent advances in nanotechnologies have prompted the need for tools to accurately and non-invasively manipulate individual nano-objects1. Among the possible strategies, optical forces have been predicted to provide researchers with nano-optical tweezers capable of trapping a specimen and moving it in three dimensions2, 3, 4. In practice, however, the combination of weak optical forces and photothermal issues has thus far prevented their experimental realization. Here, we demonstrate the first three-dimensional optical manipulation of single 50 nm dielectric objects with near-field nanotweezers. The nano-optical trap is built by engineering a bowtie plasmonic aperture at the extremity of a tapered metal-coated optical fibre. Both the trapping operation and monitoring are performed through the optical fibre, making these nanotweezers totally autonomous and free of bulky optical elements. The achieved trapping performances allow for the trapped specimen to be moved over tens of micrometres over a period of several minutes with very low in-trap intensities. This non-invasive approach is foreseen to open new horizons in nanosciences by offering an unprecedented level of control of nanosized objects, including heat-sensitive biospecimens.
Recent advances in nanotechnologies have prompted the need for tools to accurately and non-invasively manipulate individual nano-objects1. Among the possible strategies, optical forces have been predicted to provide researchers with nano-optical tweezers capable of trapping a specimen and moving it in three dimensions2, 3, 4. In practice, however, the combination of weak optical forces and photothermal issues has thus far prevented their experimental realization. Here, we demonstrate the first three-dimensional optical manipulation of single 50 nm dielectric objects with near-field nanotweezers. The nano-optical trap is built by engineering a bowtie plasmonic aperture at the extremity of a tapered metal-coated optical fibre. Both the trapping operation and monitoring are performed through the optical fibre, making these nanotweezers totally autonomous and free of bulky optical elements. The achieved trapping performances allow for the trapped specimen to be moved over tens of micrometres over a period of several minutes with very low in-trap intensities. This non-invasive approach is foreseen to open new horizons in nanosciences by offering an unprecedented level of control of nanosized objects, including heat-sensitive biospecimens.
Rotational Dynamics of Optically Trapped Human Spermatozoa
Elavarasan Subramani, Himanish Basu, Shyam Thangaraju, Sucheta Dandekar, Deepak Mathur, and Koel Chaudhury
Introduction. Optical trapping is a laser-based method for probing the physiological and mechanical properties of cells in a noninvasive manner. As sperm motility is an important criterion for assessing the male fertility potential, this technique is used to study sperm cell motility behavior and rotational dynamics. Methods and Patients. An integrated optical system with near-infrared laser beam has been used to analyze rotational dynamics of live sperm cells from oligozoospermic and asthenozoospermic cases and compared with controls. Results. The linear, translational motion of the sperm is converted into rotational motion on being optically trapped, without causing any adverse effect on spermatozoa. The rotational speed of sperm cells from infertile men is observed to be significantly less as compared to controls. Conclusions. Distinguishing normal and abnormal sperm cells on the basis of beat frequency above 5.6 Hz may be an important step in modern reproductive biology to sort and select good quality spermatozoa. The application of laser-assisted technique in biology has the potential to be a valuable tool for assessment of sperm fertilization capacity for improving assisted reproductive technology.
DOI
Introduction. Optical trapping is a laser-based method for probing the physiological and mechanical properties of cells in a noninvasive manner. As sperm motility is an important criterion for assessing the male fertility potential, this technique is used to study sperm cell motility behavior and rotational dynamics. Methods and Patients. An integrated optical system with near-infrared laser beam has been used to analyze rotational dynamics of live sperm cells from oligozoospermic and asthenozoospermic cases and compared with controls. Results. The linear, translational motion of the sperm is converted into rotational motion on being optically trapped, without causing any adverse effect on spermatozoa. The rotational speed of sperm cells from infertile men is observed to be significantly less as compared to controls. Conclusions. Distinguishing normal and abnormal sperm cells on the basis of beat frequency above 5.6 Hz may be an important step in modern reproductive biology to sort and select good quality spermatozoa. The application of laser-assisted technique in biology has the potential to be a valuable tool for assessment of sperm fertilization capacity for improving assisted reproductive technology.
DOI
Nano-opto-mechanical effects in plasmonic waveguides
Alexander S. Shalin, Pavel Ginzburg, Pavel A. Belov, Yuri S. Kivshar, Anatoly V. Zayats
In order to achieve interaction between light beams, a mediating material object is required. Nonlinear materials are commonly used for this purpose. Here a new approach to control light with light, based on a nano-opto-mechanical system integrated in a plasmonic waveguide is proposed. Optomechanics of a free-floating resonant nanoparticle in a subwavelength plasmonic V-groove waveguide is studied. It is shown that nanoparticle auto-oscillations in the waveguide induced by a control light result in the periodic modulation of a transmitted plasmonic signal. The modulation depth of 10% per single nanoparticle of 25 nm diameter with the clock frequencies of tens of MHz and the record low energy-per-bit energies of 10−18 J is observed. The frequency of auto-oscillations depends on the intensity of the continuous control light. The efficient modulation and deep-subwavelength dimensions make this nano-optomechanical system of significant interest for opto-electronic and opto-fluidic technologies.
DOI
In order to achieve interaction between light beams, a mediating material object is required. Nonlinear materials are commonly used for this purpose. Here a new approach to control light with light, based on a nano-opto-mechanical system integrated in a plasmonic waveguide is proposed. Optomechanics of a free-floating resonant nanoparticle in a subwavelength plasmonic V-groove waveguide is studied. It is shown that nanoparticle auto-oscillations in the waveguide induced by a control light result in the periodic modulation of a transmitted plasmonic signal. The modulation depth of 10% per single nanoparticle of 25 nm diameter with the clock frequencies of tens of MHz and the record low energy-per-bit energies of 10−18 J is observed. The frequency of auto-oscillations depends on the intensity of the continuous control light. The efficient modulation and deep-subwavelength dimensions make this nano-optomechanical system of significant interest for opto-electronic and opto-fluidic technologies.
DOI
Improved Laser Manipulation for On-chip Fabricated Microstructures Based on Solution Replacement and Its Application in Single Cell Analysis
Tao Yue, Masahiro Nakajima, Masaru Takeuchi and Toshio Fukuda
In this paper, we present the fabrication and assembly of microstructures inside a microfluidic device based on a photocrosslinkable resin and optical tweezers. We also report a method of solution replacement inside the microfluidic channel in order to improve the manipulation performance and apply the assembled microstructures for single cell cultivation. By the illumination of patterned ultraviolet (UV) through a microscope, microstructures of arbitrary shape were fabricated by the photocrosslinkable resin inside a microfluidic channel. Based on the microfluidic channel with both glass and polydimethylsiloxane (PDMS) surfaces, immovable and movable microstructures were fabricated and manipulated. The microstructures were fabricated at the desired places and manipulated by the optical tweezers. A rotational microstructure including a microgear and a rotation axis was assembled and rotated in demonstrating this technique. The improved laser manipulation of microstructures was achieved based on the on-chip solution replacement method. The manipulation speed of the microstructures increased when the viscosity of the solvent decreased. The movement efficiency of the fabricated microstructures inside the lower viscosity solvent was evaluated and compared with those microstructures inside the former high viscosity solvent. A novel cell cage was fabricated and the cultivation of a single yeast cell (w303) was demonstrated in the cell cage, inside the microfluidic device.
DOI
In this paper, we present the fabrication and assembly of microstructures inside a microfluidic device based on a photocrosslinkable resin and optical tweezers. We also report a method of solution replacement inside the microfluidic channel in order to improve the manipulation performance and apply the assembled microstructures for single cell cultivation. By the illumination of patterned ultraviolet (UV) through a microscope, microstructures of arbitrary shape were fabricated by the photocrosslinkable resin inside a microfluidic channel. Based on the microfluidic channel with both glass and polydimethylsiloxane (PDMS) surfaces, immovable and movable microstructures were fabricated and manipulated. The microstructures were fabricated at the desired places and manipulated by the optical tweezers. A rotational microstructure including a microgear and a rotation axis was assembled and rotated in demonstrating this technique. The improved laser manipulation of microstructures was achieved based on the on-chip solution replacement method. The manipulation speed of the microstructures increased when the viscosity of the solvent decreased. The movement efficiency of the fabricated microstructures inside the lower viscosity solvent was evaluated and compared with those microstructures inside the former high viscosity solvent. A novel cell cage was fabricated and the cultivation of a single yeast cell (w303) was demonstrated in the cell cage, inside the microfluidic device.
DOI
Wednesday, March 5, 2014
Unraveling secrets of telomeres: One molecule at a time
Jiangguo Lin, Parminder Kaur, Preston Countryman, Patricia L. Opresko, Hong Wang
Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure–function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins.
DOI
Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure–function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins.
DOI
Impact of heating on passive and active biomechanics of suspended cells
C. J. Chan, G. Whyte, L. Boyde, G. Salbreux and J. Guck
A cell is a complex material whose mechanical properties are essential for its normal functions. Heating can have a dramatic effect on these mechanical properties, similar to its impact on the dynamics of artificial polymer networks. We investigated such mechanical changes by the use of a microfluidic optical stretcher, which allowed us to probe cell mechanics when the cells were subjected to different heating conditions at different time scales. We find that HL60/S4 myeloid precursor cells become mechanically more compliant and fluid-like when subjected to either a sudden laser-induced temperature increase or prolonged exposure to higher ambient temperature. Above a critical temperature of 52 ± 1°C, we observed active cell contraction, which was strongly correlated with calcium influx through temperature-sensitive transient receptor potential vanilloid 2 (TRPV2) ion channels, followed by a subsequent expansion in cell volume. The change from passive to active cellular response can be effectively described by a mechanical model incorporating both active stress and viscoelastic components. Our work highlights the role of TRPV2 in regulating the thermomechanical response of cells. It also offers insights into how cortical tension and osmotic pressure govern cell mechanics and regulate cell-shape changes in response to heat and mechanical stress.
DOI
A cell is a complex material whose mechanical properties are essential for its normal functions. Heating can have a dramatic effect on these mechanical properties, similar to its impact on the dynamics of artificial polymer networks. We investigated such mechanical changes by the use of a microfluidic optical stretcher, which allowed us to probe cell mechanics when the cells were subjected to different heating conditions at different time scales. We find that HL60/S4 myeloid precursor cells become mechanically more compliant and fluid-like when subjected to either a sudden laser-induced temperature increase or prolonged exposure to higher ambient temperature. Above a critical temperature of 52 ± 1°C, we observed active cell contraction, which was strongly correlated with calcium influx through temperature-sensitive transient receptor potential vanilloid 2 (TRPV2) ion channels, followed by a subsequent expansion in cell volume. The change from passive to active cellular response can be effectively described by a mechanical model incorporating both active stress and viscoelastic components. Our work highlights the role of TRPV2 in regulating the thermomechanical response of cells. It also offers insights into how cortical tension and osmotic pressure govern cell mechanics and regulate cell-shape changes in response to heat and mechanical stress.
DOI
Torque Measurement at the Single-Molecule Level
Scott Forth, Maxim Y. Sheinin, James Inman, and Michelle D. Wang
Methods for exerting and measuring forces on single molecules have revolutionized the study of the physics of biology. However, it is often the case that biological processes involve rotation or torque generation, and these parameters have been more difficult to access experimentally. Recent advances in the single-molecule field have led to the development of techniques that add the capability of torque measurement. By combining force, displacement, torque, and rotational data, a more comprehensive description of the mechanics of a biomolecule can be achieved. In this review, we highlight a number of biological processes for which torque plays a key mechanical role. We describe the various techniques that have been developed to directly probe the torque experienced by a single molecule, and detail a variety of measurements made to date using these new technologies. We conclude by discussing a number of open questions and propose systems of study that would be well suited for analysis with torsional measurement techniques.
DOI
Methods for exerting and measuring forces on single molecules have revolutionized the study of the physics of biology. However, it is often the case that biological processes involve rotation or torque generation, and these parameters have been more difficult to access experimentally. Recent advances in the single-molecule field have led to the development of techniques that add the capability of torque measurement. By combining force, displacement, torque, and rotational data, a more comprehensive description of the mechanics of a biomolecule can be achieved. In this review, we highlight a number of biological processes for which torque plays a key mechanical role. We describe the various techniques that have been developed to directly probe the torque experienced by a single molecule, and detail a variety of measurements made to date using these new technologies. We conclude by discussing a number of open questions and propose systems of study that would be well suited for analysis with torsional measurement techniques.
DOI
Two steps forward, one step back: Determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers
Maria Spies
XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5′–3′ polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interactions within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein–DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).
DOI
XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5′–3′ polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interactions within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein–DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).
DOI
Subscribe to:
Posts (Atom)