Luana Reis , Esio B. Ramos , and Marcio S. Rocha
We have studied the interaction of the DNA molecule with the ligand 3,3'-Diaminobenzidine (DAB) by performing single molecule stretching experiments with optical tweezers and dynamic light scattering (DLS) on the DNA-DAB complexes. In the stretching experiments, the persistence and contour lengths of the complexes were measured as a function of DAB concentration, allowing one to infer the main binding mechanism and also to determine the physicochemical parameters of the interaction. In the DLS experiments, the effective size of the complexes, measured as the hydrodynamic radius, was monitored as a function of DAB concentration. We found a qualitative agreement between the results obtained from the two techniques by comparing the behaviors of the hydrodynamics radius and the radius of gyration, since this last one can be expressed as a function of the persistence and contour lengths.
DOI
Thursday, November 7, 2013
Memory effects for a trapped Brownian particle in viscoelastic shear flows
Romi Mankin, Katrin Laas, and Neeme Lumi
The long-time limit behavior of the positional distribution for an underdamped Brownian particle in a fluctuating harmonic potential well, which is simultaneously exposed to an oscillatory viscoelastic shear flow is investigated using the generalized Langevin equation with a power-law-type memory kernel. The influence of a fluctuating environment is modeled by a multiplicative white noise (fluctuations of the stiffness of the trapping potential) and by an additive internal fractional Gaussian noise. The exact expressions of the second-order moments of the fluctuating position for the Brownian particle in the shear plane have been calculated. Also, shear-induced cross correlation between particle fluctuations along orthogonal directions as well as the angular momentum are found. It is shown that interplay of shear flow, memory, and multiplicative noise can generate a variety of cooperation effects, such as energetic instability, multiresonance versus the shear frequency, and memory-induced anomalous diffusion in the direction of the shear flow. Particularly, two different critical memory exponents have been found, which mark dynamical transitions from a stationary regime to a subdiffusive (or superdiffusive) regime of the system. Similarities and differences between the behaviors of the models with oscillatory and nonoscillatory shear flow are also discussed.
DOI
The long-time limit behavior of the positional distribution for an underdamped Brownian particle in a fluctuating harmonic potential well, which is simultaneously exposed to an oscillatory viscoelastic shear flow is investigated using the generalized Langevin equation with a power-law-type memory kernel. The influence of a fluctuating environment is modeled by a multiplicative white noise (fluctuations of the stiffness of the trapping potential) and by an additive internal fractional Gaussian noise. The exact expressions of the second-order moments of the fluctuating position for the Brownian particle in the shear plane have been calculated. Also, shear-induced cross correlation between particle fluctuations along orthogonal directions as well as the angular momentum are found. It is shown that interplay of shear flow, memory, and multiplicative noise can generate a variety of cooperation effects, such as energetic instability, multiresonance versus the shear frequency, and memory-induced anomalous diffusion in the direction of the shear flow. Particularly, two different critical memory exponents have been found, which mark dynamical transitions from a stationary regime to a subdiffusive (or superdiffusive) regime of the system. Similarities and differences between the behaviors of the models with oscillatory and nonoscillatory shear flow are also discussed.
DOI
Determining the unique refractive index properties of solid polystyrene aerosol using broadband Mie scattering from optically trapped beads
Stephanie H. Jones, Martin D. King and Andrew D. Ward
A method is described to measure the refractive index dispersion with wavelength of optically trapped solid particles in air. Knowledge of the refraction properties of solid particles is critical for the study of aerosol; both in the laboratory and in the atmosphere for climate studies. Single micron-sized polystyrene beads were optically trapped in air using a vertically aligned counter-propagating configuration of focussed laser beams. Each bead was illuminated using white light from a broadband light emitting diode (LED) and elastic scattering within the bead was collected onto a spectrograph. The resulting Mie spectra were analysed to accurately determine polystyrene bead radii to ±0.4 nm and values of the refractive index to ±0.0005 over a wavelength range of 480–700 nm. We demonstrate that optical trapping combined with elastic scattering can be used to both accurately size polystyrene beads suspended in air and determine their wavelength dependent refractive index. The refractive index dispersions are in close agreement with reported values for polystyrene beads in aqueous dispersion. Our results also demonstrate a variation in the refractive index of polystyrene, from bead to bead, in a commercial sample. The measured variation highlights that care must be taken when using polystyrene beads as a calibration aerosol.
DOI
A method is described to measure the refractive index dispersion with wavelength of optically trapped solid particles in air. Knowledge of the refraction properties of solid particles is critical for the study of aerosol; both in the laboratory and in the atmosphere for climate studies. Single micron-sized polystyrene beads were optically trapped in air using a vertically aligned counter-propagating configuration of focussed laser beams. Each bead was illuminated using white light from a broadband light emitting diode (LED) and elastic scattering within the bead was collected onto a spectrograph. The resulting Mie spectra were analysed to accurately determine polystyrene bead radii to ±0.4 nm and values of the refractive index to ±0.0005 over a wavelength range of 480–700 nm. We demonstrate that optical trapping combined with elastic scattering can be used to both accurately size polystyrene beads suspended in air and determine their wavelength dependent refractive index. The refractive index dispersions are in close agreement with reported values for polystyrene beads in aqueous dispersion. Our results also demonstrate a variation in the refractive index of polystyrene, from bead to bead, in a commercial sample. The measured variation highlights that care must be taken when using polystyrene beads as a calibration aerosol.
DOI
Monday, November 4, 2013
Optical manipulation of charged microparticles in polar fluids
Giuseppe Pesce, Vincenzo Lisbino, Giulia Rusciano, Antonio Sasso
In this study, we report a systematic study of the response of a charged microparticle confined in an optical trap and driven by electric fields. The particle is embedded in a polar fluid, hence, the role of ions and counterions forming a double layer around the electrodes and the particle surface itself has been taken into account. We analyze two different cases: (i) electrodes energized by a step-wise voltage (DC mode) and (ii) electrodes driven by a sinusoidal voltage (AC mode). The experimental outcomes are analyzed in terms of a model that combines the electric response of the electrolytic cell and the motion of the trapped particle. In particular, for the DC mode we analyze the transient particle motion and correlate it with the electric current flowing in the cell. For the AC mode, the stochastic and deterministic motion of the trapped particle is analyzed either in the frequency domain (power spectral density, PSD) or in the time domain (autocorrelation function). Moreover, we will show how these different approaches (DC and AC modes) allow us, assuming predictable the applied electric field (here generated by plane parallel electrodes), to provide accurate estimation (3%) of the net charge carried by the microparticle. Vice versa, we also demonstrate how, once predetermined the charge, the trapped particle acts as a sensitive probe to reveal locally electric fields generated by arbitrary electrode geometries (in this work, wire-tip geometry).
DOI
In this study, we report a systematic study of the response of a charged microparticle confined in an optical trap and driven by electric fields. The particle is embedded in a polar fluid, hence, the role of ions and counterions forming a double layer around the electrodes and the particle surface itself has been taken into account. We analyze two different cases: (i) electrodes energized by a step-wise voltage (DC mode) and (ii) electrodes driven by a sinusoidal voltage (AC mode). The experimental outcomes are analyzed in terms of a model that combines the electric response of the electrolytic cell and the motion of the trapped particle. In particular, for the DC mode we analyze the transient particle motion and correlate it with the electric current flowing in the cell. For the AC mode, the stochastic and deterministic motion of the trapped particle is analyzed either in the frequency domain (power spectral density, PSD) or in the time domain (autocorrelation function). Moreover, we will show how these different approaches (DC and AC modes) allow us, assuming predictable the applied electric field (here generated by plane parallel electrodes), to provide accurate estimation (3%) of the net charge carried by the microparticle. Vice versa, we also demonstrate how, once predetermined the charge, the trapped particle acts as a sensitive probe to reveal locally electric fields generated by arbitrary electrode geometries (in this work, wire-tip geometry).
DOI
Optical vault: A reconfigurable bottle beam based on conical refraction of light
A. Turpin, V. Shvedov, C. Hnatovsky, Yu. V. Loiko, J. Mompart, and W. Krolikowski
We employ conical refraction of light in a biaxial crystal to create an optical bottle for photophoretic trapping and manipulation of particles in gaseous media. We show that by only varying the polarization state of the input light beam the optical bottle can be opened and closed in order to load and unload particles in a highly controllable manner.
DOI
We employ conical refraction of light in a biaxial crystal to create an optical bottle for photophoretic trapping and manipulation of particles in gaseous media. We show that by only varying the polarization state of the input light beam the optical bottle can be opened and closed in order to load and unload particles in a highly controllable manner.
DOI
Selective Optical Assembly of Highly Uniform Nanoparticles by Doughnut-Shaped Beams
Syoji Ito, Hiroaki Yamauchi, Mamoru Tamura, Shimpei Hidaka, Hironori Hattori, Taichi Hamada, Keisuke Nishida, Shiho Tokonami, Tamitake Itoh, Hiroshi Miyasaka & Takuya Iida
A highly efficient natural light-harvesting antenna has a ring-like structure consisting of dye molecules whose absorption band changes through selective evolutionary processes driven by external stimuli, i.e., sunlight depending on its territory and thermal fluctuations. Inspired by this fact, here, we experimentally and theoretically demonstrate the selective assembling of ring-like arrangements of many silver nanorods with particular shapes and orientations onto a substrate by the light-induced force of doughnut beams with different colours (wavelengths) and polarizations in conjunction with thermal fluctuations at room temperature. Furthermore, the majority of nanorods are electromagnetically coupled to form a prominent red-shifted collective mode of localized surface plasmons resonant with the wavelength of the irradiated light, where a spectral broadening also appears for the efficient broadband optical response. The discovered principle is a promising route for "bio-inspired selective optical assembly" of various nanomaterials that can be used in the wide field of nanotechnology.
DOI
A highly efficient natural light-harvesting antenna has a ring-like structure consisting of dye molecules whose absorption band changes through selective evolutionary processes driven by external stimuli, i.e., sunlight depending on its territory and thermal fluctuations. Inspired by this fact, here, we experimentally and theoretically demonstrate the selective assembling of ring-like arrangements of many silver nanorods with particular shapes and orientations onto a substrate by the light-induced force of doughnut beams with different colours (wavelengths) and polarizations in conjunction with thermal fluctuations at room temperature. Furthermore, the majority of nanorods are electromagnetically coupled to form a prominent red-shifted collective mode of localized surface plasmons resonant with the wavelength of the irradiated light, where a spectral broadening also appears for the efficient broadband optical response. The discovered principle is a promising route for "bio-inspired selective optical assembly" of various nanomaterials that can be used in the wide field of nanotechnology.
DOI
The ClpXP Protease Unfolds Substrates Using a Constant Rate of Pulling but Different Gears
Maya Sen, Rodrigo A. Maillard, Kristofor Nyquist, Piere Rodriguez-Aliaga, Steve Presse Andreas Martin, and Carlos Bustamante
ATP-dependent proteases are vital to maintain cellular protein homeostasis. Here, we study the mechanisms of force generation and intersubunit coordination in the ClpXP protease from E. coli to understand how these machines couple ATP hydrolysis to mechanical protein unfolding. Single-molecule analyses reveal that phosphate release is the force generating step in the ATP-hydrolysis cycle and that ClpXP translocates substrate polypeptides in bursts resulting from highly coordinated conformational changes in two to four ATPase subunits. ClpXP must use its maximum successive firing capacity of four subunits to unfold stable substrates like GFP. The average dwell duration between individual bursts of translocation is constant, regardless of the number of translocating subunits, implying that ClpXP operates with constant ‘‘rpm’’ but uses different ‘‘gears.’’
DOI
ATP-dependent proteases are vital to maintain cellular protein homeostasis. Here, we study the mechanisms of force generation and intersubunit coordination in the ClpXP protease from E. coli to understand how these machines couple ATP hydrolysis to mechanical protein unfolding. Single-molecule analyses reveal that phosphate release is the force generating step in the ATP-hydrolysis cycle and that ClpXP translocates substrate polypeptides in bursts resulting from highly coordinated conformational changes in two to four ATPase subunits. ClpXP must use its maximum successive firing capacity of four subunits to unfold stable substrates like GFP. The average dwell duration between individual bursts of translocation is constant, regardless of the number of translocating subunits, implying that ClpXP operates with constant ‘‘rpm’’ but uses different ‘‘gears.’’
DOI
Following the Mechanisms of Bacteriostatic versus Bactericidal Action Using Raman Spectroscopy
Silvie Bernatová, Ota Samek, Zdeněk Pilát, Mojmír Šerý, Jan Ježek, Petr Jákl, Martin Šiler, Vladislav Krzyžánek, Pavel Zemánek, Veronika Holá, Milada Dvořáčková and Filip Růžička
Antibiotics cure infections by influencing bacterial growth or viability. Antibiotics can be divided to two groups on the basis of their effect on microbial cells through two main mechanisms, which are either bactericidal or bacteriostatic. Bactericidal antibiotics kill the bacteria and bacteriostatic antibiotics suppress the growth of bacteria (keep them in the stationary phase of growth). One of many factors to predict a favorable clinical outcome of the potential action of antimicrobial chemicals may be provided using in vitro bactericidal/bacteriostatic data (e.g., minimum inhibitory concentrations—MICs). Consequently, MICs are used in clinical situations mainly to confirm resistance, and to determine the in vitro activities of new antimicrobials. We report on the combination of data obtained from MICs with information on microorganisms’ “fingerprint” (e.g., DNA/RNA, and proteins) provided by Raman spectroscopy. Thus, we could follow mechanisms of the bacteriostatic versus bactericidal action simply by detecting the Raman bands corresponding to DNA. The Raman spectra of Staphylococcus epidermidis treated with clindamycin (a bacteriostatic agent) indeed show little effect on DNA which is in contrast with the action of ciprofloxacin (a bactericidal agent), where the Raman spectra show a decrease in strength of the signal assigned to DNA, suggesting DNA fragmentation.
DOI
Antibiotics cure infections by influencing bacterial growth or viability. Antibiotics can be divided to two groups on the basis of their effect on microbial cells through two main mechanisms, which are either bactericidal or bacteriostatic. Bactericidal antibiotics kill the bacteria and bacteriostatic antibiotics suppress the growth of bacteria (keep them in the stationary phase of growth). One of many factors to predict a favorable clinical outcome of the potential action of antimicrobial chemicals may be provided using in vitro bactericidal/bacteriostatic data (e.g., minimum inhibitory concentrations—MICs). Consequently, MICs are used in clinical situations mainly to confirm resistance, and to determine the in vitro activities of new antimicrobials. We report on the combination of data obtained from MICs with information on microorganisms’ “fingerprint” (e.g., DNA/RNA, and proteins) provided by Raman spectroscopy. Thus, we could follow mechanisms of the bacteriostatic versus bactericidal action simply by detecting the Raman bands corresponding to DNA. The Raman spectra of Staphylococcus epidermidis treated with clindamycin (a bacteriostatic agent) indeed show little effect on DNA which is in contrast with the action of ciprofloxacin (a bactericidal agent), where the Raman spectra show a decrease in strength of the signal assigned to DNA, suggesting DNA fragmentation.
DOI
Ultrafast folding kinetics and cooperativity of villin headpiece in single-molecule force spectroscopy
Gabriel Žoldák, Johannes Stigler, Benjamin Pelz, Hongbin Li, and Matthias Rief
In this study we expand the accessible dynamic range of single-molecule force spectroscopy by optical tweezers to the microsecond range by fast sampling. We are able to investigate a single molecule for up to 15 min and with 300-kHz bandwidth as the protein undergoes tens of millions of folding/unfolding transitions. Using equilibrium analysis and autocorrelation analysis of the time traces, the full energetics as well as real-time kinetics of the ultrafast folding of villin headpiece 35 and a stable asparagine 68 alanine/lysine 70 methionine variant can be measured directly. We also performed Brownian dynamics simulations of the response of the bead-DNA system to protein-folding fluctuations. All key features of the force-dependent deflection fluctuations could be reproduced: SD, skewness, and autocorrelation function. Our measurements reveal a difference in folding pathway and cooperativity between wild-type and stable variant of headpiece 35. Autocorrelation force spectroscopy pushes the time resolution of single-molecule force spectroscopy to ∼10 µs thus approaching the timescales accessible for all atom molecular dynamics simulations.
DOI
In this study we expand the accessible dynamic range of single-molecule force spectroscopy by optical tweezers to the microsecond range by fast sampling. We are able to investigate a single molecule for up to 15 min and with 300-kHz bandwidth as the protein undergoes tens of millions of folding/unfolding transitions. Using equilibrium analysis and autocorrelation analysis of the time traces, the full energetics as well as real-time kinetics of the ultrafast folding of villin headpiece 35 and a stable asparagine 68 alanine/lysine 70 methionine variant can be measured directly. We also performed Brownian dynamics simulations of the response of the bead-DNA system to protein-folding fluctuations. All key features of the force-dependent deflection fluctuations could be reproduced: SD, skewness, and autocorrelation function. Our measurements reveal a difference in folding pathway and cooperativity between wild-type and stable variant of headpiece 35. Autocorrelation force spectroscopy pushes the time resolution of single-molecule force spectroscopy to ∼10 µs thus approaching the timescales accessible for all atom molecular dynamics simulations.
DOI
Hydrodynamic and subdiffusive motion of tracers in a viscoelastic medium
Denis S. Grebenkov, Mahsa Vahabi, Elena Bertseva, László Forró, and Sylvia Jeney
We investigate the diffusive motion of micron-sized spherical tracers in a viscoelastic actin filament network over the time span of 8 orders of magnitude using optical-tweezers single-particle tracking. The hydrodynamic interactions of a tracer with the surrounding fluid are shown to dominate at microsecond time scales, while subdiffusive scaling due to viscoelastic properties of the medium emerges at millisecond time scales. The transition between these two regimes is analyzed in the frame of a minimal phenomenological model which combines the Basset force and the generalized Stokes force. The resulting Langevin equation accounts for various dynamical features of the thermal motion of endogenous or exogenous tracers in viscoelastic media such as inertial and hydrodynamic effects at short times, subdiffusive scaling at intermediate times, and eventual optical trapping at long times. Simple analytical formulas for the mean-square displacement and velocity autocorrelation function are derived.
DOI
We investigate the diffusive motion of micron-sized spherical tracers in a viscoelastic actin filament network over the time span of 8 orders of magnitude using optical-tweezers single-particle tracking. The hydrodynamic interactions of a tracer with the surrounding fluid are shown to dominate at microsecond time scales, while subdiffusive scaling due to viscoelastic properties of the medium emerges at millisecond time scales. The transition between these two regimes is analyzed in the frame of a minimal phenomenological model which combines the Basset force and the generalized Stokes force. The resulting Langevin equation accounts for various dynamical features of the thermal motion of endogenous or exogenous tracers in viscoelastic media such as inertial and hydrodynamic effects at short times, subdiffusive scaling at intermediate times, and eventual optical trapping at long times. Simple analytical formulas for the mean-square displacement and velocity autocorrelation function are derived.
DOI
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