Through evolution, nature has produced exquisite nanometric structures, with features unrealized in the most advanced man-made devices. Type IV pili (Tfp) represent such a structure: 6-nm-wide retractable filamentous appendages found in many bacteria, including human pathogens. Whereas the structure of Neisseria gonorrhoeae Tfp has been defined by conventional structural techniques, it remains difficult to explain the wide spectrum of functions associated with Tfp. Here we uncover a previously undescribed force-induced quaternary structure of the N. gonorrhoeae Tfp. By using a combination of optical and magnetic tweezers, atomic force microscopy, and molecular combing to apply forces on purified Tfp, we demonstrate that Tfp subjected to approximately 100 pN of force will transition into a new conformation. The new structure is roughly 3 times longer and 40% narrower than the original structure. Upon release of the force, the Tfp fiber regains its original form, indicating a reversible transition. Equally important, we show that the force-induced conformation exposes hidden epitopes previously buried in the Tfp fiber. We postulate that this transition provides a means for N. gonorrhoeae to maintain attachment to its host while withstanding intermittent forces encountered in the environment. Our findings demonstrate the need to reassess our understanding of Tfp dynamics and functions. They could also explain the structural diversity of other helical polymers while presenting a unique mechanism for polymer elongation and exemplifying the extreme structural plasticity of biological polymers.
Thursday, August 12, 2010
Force-dependent polymorphism in type IV pili reveals hidden epitopes
Nicolas Biais, Dustin L. Higashi, Jasna Brujić, Magdalene So, and Michael P. Sheetz
Through evolution, nature has produced exquisite nanometric structures, with features unrealized in the most advanced man-made devices. Type IV pili (Tfp) represent such a structure: 6-nm-wide retractable filamentous appendages found in many bacteria, including human pathogens. Whereas the structure of Neisseria gonorrhoeae Tfp has been defined by conventional structural techniques, it remains difficult to explain the wide spectrum of functions associated with Tfp. Here we uncover a previously undescribed force-induced quaternary structure of the N. gonorrhoeae Tfp. By using a combination of optical and magnetic tweezers, atomic force microscopy, and molecular combing to apply forces on purified Tfp, we demonstrate that Tfp subjected to approximately 100 pN of force will transition into a new conformation. The new structure is roughly 3 times longer and 40% narrower than the original structure. Upon release of the force, the Tfp fiber regains its original form, indicating a reversible transition. Equally important, we show that the force-induced conformation exposes hidden epitopes previously buried in the Tfp fiber. We postulate that this transition provides a means for N. gonorrhoeae to maintain attachment to its host while withstanding intermittent forces encountered in the environment. Our findings demonstrate the need to reassess our understanding of Tfp dynamics and functions. They could also explain the structural diversity of other helical polymers while presenting a unique mechanism for polymer elongation and exemplifying the extreme structural plasticity of biological polymers.
Through evolution, nature has produced exquisite nanometric structures, with features unrealized in the most advanced man-made devices. Type IV pili (Tfp) represent such a structure: 6-nm-wide retractable filamentous appendages found in many bacteria, including human pathogens. Whereas the structure of Neisseria gonorrhoeae Tfp has been defined by conventional structural techniques, it remains difficult to explain the wide spectrum of functions associated with Tfp. Here we uncover a previously undescribed force-induced quaternary structure of the N. gonorrhoeae Tfp. By using a combination of optical and magnetic tweezers, atomic force microscopy, and molecular combing to apply forces on purified Tfp, we demonstrate that Tfp subjected to approximately 100 pN of force will transition into a new conformation. The new structure is roughly 3 times longer and 40% narrower than the original structure. Upon release of the force, the Tfp fiber regains its original form, indicating a reversible transition. Equally important, we show that the force-induced conformation exposes hidden epitopes previously buried in the Tfp fiber. We postulate that this transition provides a means for N. gonorrhoeae to maintain attachment to its host while withstanding intermittent forces encountered in the environment. Our findings demonstrate the need to reassess our understanding of Tfp dynamics and functions. They could also explain the structural diversity of other helical polymers while presenting a unique mechanism for polymer elongation and exemplifying the extreme structural plasticity of biological polymers.
Hummer and Szabo-like Potential of Mean Force Estimator for Bidirectional Nonequilibrium Pulling Experiments/Simulations
Paolo Nicolini, Piero Procacci and Riccardo Chelli
In the framework of single-molecule pulling experiments, the system is typically driven out of equilibrium by a time-dependent external potential V(t) acting on a collective coordinate such that the total Hamiltonian is the sum of V(t) and the Hamiltonian in the absence of external perturbation. Nonequilibrium work theorems such as Jarzynski equality and Crooks fluctuation theorem have been devised to recover free energy differences between states of this extended system. However, one is often more interested in the potential of mean force of the unperturbed Hamiltonian, i.e., in the effective potential dictating the equilibrium distribution of the collective coordinate in the absence of the external potential. In this respect, Hummer and Szabo proposed an algorithm to estimate the desired free energy differences when pulling experiments are performed in only one direction of the process (Proc. Natl. Acad. Sci. USA 2001, 98, 3658). In this paper, we present a potential of mean force estimator of the unperturbed system that exploits the work measured in both forward and backward directions of the process. The method is based on the reweighting technique of Hummer and Szabo and on the Bennett acceptance ratio. Using Brownian-dynamics simulations on a double-well free energy surface, we show that the estimator works satisfactorily in any pulling situation, from nearly equilibrium to strongly dissipative regimes. The method is also applied to the unfolding/refolding process of decaalanine, a system vastly used to illustrate and to test nonequilibrium methodologies. A thorough comparative analysis with another bidirectional potential of mean force estimator (Minh, D. D. L.; Adib, A. B. Phys. Rev. Lett. 2008, 100, 180602) is also presented. The proposed approach is well-suited to recover free energy profiles from single-molecule bidirectional-pulling experiments such as those performed by optical tweezers or atomic force microscopes.
DOI
In the framework of single-molecule pulling experiments, the system is typically driven out of equilibrium by a time-dependent external potential V(t) acting on a collective coordinate such that the total Hamiltonian is the sum of V(t) and the Hamiltonian in the absence of external perturbation. Nonequilibrium work theorems such as Jarzynski equality and Crooks fluctuation theorem have been devised to recover free energy differences between states of this extended system. However, one is often more interested in the potential of mean force of the unperturbed Hamiltonian, i.e., in the effective potential dictating the equilibrium distribution of the collective coordinate in the absence of the external potential. In this respect, Hummer and Szabo proposed an algorithm to estimate the desired free energy differences when pulling experiments are performed in only one direction of the process (Proc. Natl. Acad. Sci. USA 2001, 98, 3658). In this paper, we present a potential of mean force estimator of the unperturbed system that exploits the work measured in both forward and backward directions of the process. The method is based on the reweighting technique of Hummer and Szabo and on the Bennett acceptance ratio. Using Brownian-dynamics simulations on a double-well free energy surface, we show that the estimator works satisfactorily in any pulling situation, from nearly equilibrium to strongly dissipative regimes. The method is also applied to the unfolding/refolding process of decaalanine, a system vastly used to illustrate and to test nonequilibrium methodologies. A thorough comparative analysis with another bidirectional potential of mean force estimator (Minh, D. D. L.; Adib, A. B. Phys. Rev. Lett. 2008, 100, 180602) is also presented. The proposed approach is well-suited to recover free energy profiles from single-molecule bidirectional-pulling experiments such as those performed by optical tweezers or atomic force microscopes.
DOI
Single-step replication of a highly integrated PDMS optofluidic analysis system
Martin Amberg, Sebastian Stoebenau, and Stefan Sinzinger
Micromilling is a promising technology for the fabrication of surface profiles with optical quality. We present a highly integrated optofluidic system made of polydimethylsiloxane (PDMS). The system is replicated in a single-step process from a micromilled polymethyl methacrylate master mold. It already includes the reservoirs, the channel system, as well as the optical interconnect surfaces for high numerical aperture objectives. We demonstrate the potential of this approach by laser-based three-dimensional optical manipulation within the replicated system. To our knowledge, this is the first time that a PDMS membrane is used as a well-defined channel wall for an optical trapping setup.
DOI
Micromilling is a promising technology for the fabrication of surface profiles with optical quality. We present a highly integrated optofluidic system made of polydimethylsiloxane (PDMS). The system is replicated in a single-step process from a micromilled polymethyl methacrylate master mold. It already includes the reservoirs, the channel system, as well as the optical interconnect surfaces for high numerical aperture objectives. We demonstrate the potential of this approach by laser-based three-dimensional optical manipulation within the replicated system. To our knowledge, this is the first time that a PDMS membrane is used as a well-defined channel wall for an optical trapping setup.
DOI
Tuesday, August 10, 2010
Dynamic axial stabilization of counter-propagating beam-traps with feedback control
Sandeep Tauro, Andrew Bañas, Darwin Palima, and Jesper Glückstad
Optical trapping in a counter-propagating (CP) beam-geometry provides unique advantages in terms of working distance, aberration requirements and intensity hotspots. However, its axial performance is governed by the wave propagation of the opposing beams, which can limit the practical geometries. Here we propose a dynamic method for controlling axial forces to overcome this constraint. The technique uses computer-vision object tracking of the axial position, in conjunction with software-based feedback, for dynamically stabilizing the axial forces. We present proof-of-concept experiments showing real-time rapid repositioning coupled with a strongly enhanced axial trapping for a plurality of particles of varying sizes. We also demonstrate the technique’s adaptability for real-time reconfigurable feedback-trapping of a dynamically growing structure that mimics a continuously dividing cell colony. Advanced implementation of this feedback-driven approach can help make CP-trapping resistant to a host of perturbations such as laser fluctuations, mechanical vibrations and other distortions emphasizing its experimental versatility.
DOI
Optical trapping in a counter-propagating (CP) beam-geometry provides unique advantages in terms of working distance, aberration requirements and intensity hotspots. However, its axial performance is governed by the wave propagation of the opposing beams, which can limit the practical geometries. Here we propose a dynamic method for controlling axial forces to overcome this constraint. The technique uses computer-vision object tracking of the axial position, in conjunction with software-based feedback, for dynamically stabilizing the axial forces. We present proof-of-concept experiments showing real-time rapid repositioning coupled with a strongly enhanced axial trapping for a plurality of particles of varying sizes. We also demonstrate the technique’s adaptability for real-time reconfigurable feedback-trapping of a dynamically growing structure that mimics a continuously dividing cell colony. Advanced implementation of this feedback-driven approach can help make CP-trapping resistant to a host of perturbations such as laser fluctuations, mechanical vibrations and other distortions emphasizing its experimental versatility.
DOI
Optical Manipulation with Planar Silicon Microring Resonators
Shiyun Lin, Ethan Schonbrun and Kenneth Crozier
We demonstrate optically trapping of microparticles on silicon microring resonators. Once trapped on a microring, a particle can be confined in an optical potential with a depth of 25kBT over the entire microring’s circumference. The particles are propelled around the microring at hundreds of micrometers per second, producing periodic revolutions at a few hertz. We anticipate that the increased force and highly accurate positioning obtainable with this system will lead to various nanomanipulation applications.
DOI
We demonstrate optically trapping of microparticles on silicon microring resonators. Once trapped on a microring, a particle can be confined in an optical potential with a depth of 25kBT over the entire microring’s circumference. The particles are propelled around the microring at hundreds of micrometers per second, producing periodic revolutions at a few hertz. We anticipate that the increased force and highly accurate positioning obtainable with this system will lead to various nanomanipulation applications.
DOI
Monday, August 9, 2010
Simulation of single-molecule trapping in a nanochannel
William Neil Robinson and Lloyd M. Davis
The detection and trapping of single fluorescent molecules in solution within a nanochannel is studied using numerical simulations. As optical forces are insufficient for trapping molecules much smaller than the optical wavelength, a means for sensing a molecule's position along the nanochannel and adjusting electrokinetic motion to compensate diffusion is assessed. Fluorescence excitation is provided by two adjacently focused laser beams containing temporally interleaved laser pulses. Photon detection is time-gated, and the displacement of the molecule from the middle of the two foci alters the count rates collected in the two detection channels. An algorithm for feedback control of the electrokinetic motion in response to the timing of photons, to reposition the molecule back toward the middle for trapping and to rapidly reload the trap after a molecule photobleaches or escapes, is evaluated. While accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware, the algorithmis shown to be effective for trapping fast-diffusing single-chromophore molecules within a micron-sized confocal region. Studies show that there is an optimum laser power for which loss of molecules from the trap due to either photobleaching or shot-noise fluctuations isminimized.
DOI
The detection and trapping of single fluorescent molecules in solution within a nanochannel is studied using numerical simulations. As optical forces are insufficient for trapping molecules much smaller than the optical wavelength, a means for sensing a molecule's position along the nanochannel and adjusting electrokinetic motion to compensate diffusion is assessed. Fluorescence excitation is provided by two adjacently focused laser beams containing temporally interleaved laser pulses. Photon detection is time-gated, and the displacement of the molecule from the middle of the two foci alters the count rates collected in the two detection channels. An algorithm for feedback control of the electrokinetic motion in response to the timing of photons, to reposition the molecule back toward the middle for trapping and to rapidly reload the trap after a molecule photobleaches or escapes, is evaluated. While accommodating the limited electrokinetic speed and the finite latency of feedback imposed by experimental hardware, the algorithmis shown to be effective for trapping fast-diffusing single-chromophore molecules within a micron-sized confocal region. Studies show that there is an optimum laser power for which loss of molecules from the trap due to either photobleaching or shot-noise fluctuations isminimized.
DOI
Phase contrast optical tweezers
Ali Mahmoudi and S. Nader S. Reihani
In this paper, for the first time, we report on systematic theoretical and experimental investigation of Phase Contrast Optical Tweezers (PCOT) which could be an indispensable tool for micromanipulation of the transparent micro and nano objects such as biological tissues and vesicles. The quadrant photodiode detection scheme and the power-spectrum calibration method is shown to be valid for this case. We have shown that the phase objective with new designed phase plates can provide nearly aberration-free condition at a desired depth. This could be a valuable advantage for simultaneous in-depth micro-manipulations and visualization of the sample.
DOI
In this paper, for the first time, we report on systematic theoretical and experimental investigation of Phase Contrast Optical Tweezers (PCOT) which could be an indispensable tool for micromanipulation of the transparent micro and nano objects such as biological tissues and vesicles. The quadrant photodiode detection scheme and the power-spectrum calibration method is shown to be valid for this case. We have shown that the phase objective with new designed phase plates can provide nearly aberration-free condition at a desired depth. This could be a valuable advantage for simultaneous in-depth micro-manipulations and visualization of the sample.
DOI
Electrochemical Characterization of a Single Electricity-Producing Bacterial Cell of Shewanella by Using Optical Tweezers
Huan Liu Dr., Greg J. Newton, Ryuhei Nakamura Dr., Kazuhito Hashimoto Prof. Dr., Shuji Nakanishi Dr.
Members of genus Shewanella are Gram-negative bacteria that can utilize solid-state metal oxide as a terminal electron acceptor for respiration. The direct electrical connection between a single cell and a microelectrode is characterized by an optical tweezers technique.
DOI
Members of genus Shewanella are Gram-negative bacteria that can utilize solid-state metal oxide as a terminal electron acceptor for respiration. The direct electrical connection between a single cell and a microelectrode is characterized by an optical tweezers technique.
DOI
A clustered speckle approach to optical trapping
J.P. Staforelli, J.M. Brito, E. Vera, P. Solano and A. Lencina
An in situ study of the clustered speckle 3D structure using an optical tweezer setup is presented. Clustered speckles appear when a coherently illuminated diffuser is imaged through a pupil mask with several apertures, properly distributed over a closed path, which is placed before the objective lens of a standard optical trapping system. Thus, light volumes are reduced several times when compared with standard speckles, being even smaller than the focus volume of a Gaussian beam commonly used to trap. Moreover, clustered speckles have odd statistical properties which differentiated it from standard speckles. Then, geometrically ordered multiple trapping arrays with statistical random distribution of intensities can be created with this technique. This fact could enable different studies concerning optical binding or new developments in coherent matter wave transport where Optical Trapping has been proven with standard speckles. In this work, a qualitative analysis of clustered speckles in an optical tweezer setup relative to the number of apertures in the mask and their size is carried on. Besides, in the Rayleigh regime, a general quantitative method to characterize the trapping capability of an optical field is proposed. Then, it is applied to clustered speckles. As a result, a relation between aperture size and the maximum size of the particles that could be trapped is found. This fact opens the possibility of engineering the statistic of the trapped particles by properly selecting the pupil mask.
DOI
An in situ study of the clustered speckle 3D structure using an optical tweezer setup is presented. Clustered speckles appear when a coherently illuminated diffuser is imaged through a pupil mask with several apertures, properly distributed over a closed path, which is placed before the objective lens of a standard optical trapping system. Thus, light volumes are reduced several times when compared with standard speckles, being even smaller than the focus volume of a Gaussian beam commonly used to trap. Moreover, clustered speckles have odd statistical properties which differentiated it from standard speckles. Then, geometrically ordered multiple trapping arrays with statistical random distribution of intensities can be created with this technique. This fact could enable different studies concerning optical binding or new developments in coherent matter wave transport where Optical Trapping has been proven with standard speckles. In this work, a qualitative analysis of clustered speckles in an optical tweezer setup relative to the number of apertures in the mask and their size is carried on. Besides, in the Rayleigh regime, a general quantitative method to characterize the trapping capability of an optical field is proposed. Then, it is applied to clustered speckles. As a result, a relation between aperture size and the maximum size of the particles that could be trapped is found. This fact opens the possibility of engineering the statistic of the trapped particles by properly selecting the pupil mask.
DOI
Friday, August 6, 2010
Linear diode laser bar optical stretchers for cell deformation
Ihab Sraj, David W. M. Marr, and Charles D. Eggleton
To investigate the use of linear diode laser bars to optically stretch cells and measure their mechanical properties, we present numerical simulations using the immersed boundary method (IBM) coupled with classic ray optics. Cells are considered as three-dimensional (3D) spherical elastic capsules immersed in a fluid subjected to both optical and hydrodynamic forces in a periodic domain. We simulate cell deformation induced by both single and dual diode laser bar configurations and show that a single diode laser bar induces significant stretching but also induces cell translation of speed < 10 µm/sec for applied 6.6 mW/µm power in unconfined systems. The dual diode laser bar configuration, however, can be used to both stretch and optically trap cells at a fixed position. The net cell deformation was found to be a function of the total laser power and not the power distribution between single or dual diode laser bar configurations.
DOI
To investigate the use of linear diode laser bars to optically stretch cells and measure their mechanical properties, we present numerical simulations using the immersed boundary method (IBM) coupled with classic ray optics. Cells are considered as three-dimensional (3D) spherical elastic capsules immersed in a fluid subjected to both optical and hydrodynamic forces in a periodic domain. We simulate cell deformation induced by both single and dual diode laser bar configurations and show that a single diode laser bar induces significant stretching but also induces cell translation of speed < 10 µm/sec for applied 6.6 mW/µm power in unconfined systems. The dual diode laser bar configuration, however, can be used to both stretch and optically trap cells at a fixed position. The net cell deformation was found to be a function of the total laser power and not the power distribution between single or dual diode laser bar configurations.
DOI
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