Thymus-derived lymphocytes protect mammalian hosts against virus- or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric αβ, CD3ϵγ, CD3ϵδ, and CD3ζζ subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3ϵ lobe that they approach diagonally, adjacent to the lever-like Cβ FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3ϵ and CD3γ in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force (∼50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands.
Monday, November 9, 2009
The αβ T Cell Receptor Is an Anisotropic Mechanosensor
Sun Taek Kim, Koh Takeuchi, Zhen-Yu J. Sun, Maki Touma, Carlos E. Castro, Amr Fahmy, Matthew J. Lang, Gerhard Wagner and Ellis L. Reinherz
Thymus-derived lymphocytes protect mammalian hosts against virus- or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric αβ, CD3ϵγ, CD3ϵδ, and CD3ζζ subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3ϵ lobe that they approach diagonally, adjacent to the lever-like Cβ FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3ϵ and CD3γ in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force (∼50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands.
Thymus-derived lymphocytes protect mammalian hosts against virus- or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric αβ, CD3ϵγ, CD3ϵδ, and CD3ζζ subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3ϵ lobe that they approach diagonally, adjacent to the lever-like Cβ FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3ϵ and CD3γ in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force (∼50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands.
Monday, November 2, 2009
Measurement of axial and transverse trapping stiffness of optical tweezers in air using a radially polarized beam
Masaki Michihata, Terutake Hayashi, and Yasuhiro Takaya
The trapping efficiency and stiffness of optical tweezers using radial polarization are evaluated; the ray-tracing method and a proposed measurement method are used for numerical and experimental analyses, respectively. The maximum axial trapping efficiency with radial polarization is 1.84 times that with linear polarization, while the maximum transverse trapping efficiency decreases by 0.58 times. Further, the axial and transverse trapping efficiencies are found to be 1.19 times larger and 0.83 times smaller, respectively, than the values with linear polarization. From the experiments, the axial and transverse stiffness values are 1.2 times larger and 0.8 times smaller, respectively, with radial polarization. Hence, radial polarization enhances the axial trapping properties while reducing the transverse trapping properties.
The trapping efficiency and stiffness of optical tweezers using radial polarization are evaluated; the ray-tracing method and a proposed measurement method are used for numerical and experimental analyses, respectively. The maximum axial trapping efficiency with radial polarization is 1.84 times that with linear polarization, while the maximum transverse trapping efficiency decreases by 0.58 times. Further, the axial and transverse trapping efficiencies are found to be 1.19 times larger and 0.83 times smaller, respectively, than the values with linear polarization. From the experiments, the axial and transverse stiffness values are 1.2 times larger and 0.8 times smaller, respectively, with radial polarization. Hence, radial polarization enhances the axial trapping properties while reducing the transverse trapping properties.
Rotation Detection in Light-Driven Nanorotors
P. H. Jones, F. Palmisano, F. Bonaccorso, P. G. Gucciardi, G. Calogero, A. C. Ferrari and O. M. Marago
We analyze the rotational dynamics of light driven nanorotors, made of nanotube bundles and gold nanorods aggregates, with nonsymmetric shapes, trapped in optical tweezers. We identify two different regimes depending on dimensions and optical properties of the nanostructures. These correspond to alignment with either the laser propagation axis or the dominant polarization direction, or rotational motions caused by either unbalanced radiation pressure or polarization torque. By analyzing the motion correlations of the trapped nanostructures, we measure with high accuracy both the optical trapping parameters and the rotation frequency induced by the radiation pressure. Our results pave the way to improved all-optical detection, control over rotating nanomachines, and rotation detection in nano-optomechanics.
We analyze the rotational dynamics of light driven nanorotors, made of nanotube bundles and gold nanorods aggregates, with nonsymmetric shapes, trapped in optical tweezers. We identify two different regimes depending on dimensions and optical properties of the nanostructures. These correspond to alignment with either the laser propagation axis or the dominant polarization direction, or rotational motions caused by either unbalanced radiation pressure or polarization torque. By analyzing the motion correlations of the trapped nanostructures, we measure with high accuracy both the optical trapping parameters and the rotation frequency induced by the radiation pressure. Our results pave the way to improved all-optical detection, control over rotating nanomachines, and rotation detection in nano-optomechanics.
Friday, October 30, 2009
Design of nanoslotted photonic crystal waveguide cavities for single nanoparticle trapping and detection
Shiyun Lin, Juejun Hu, Lionel Kimerling, and Kenneth Crozier
We design and numerically simulate an on-chip photonic device that integrates both optical manipulation and detection functionalities for a single nanoparticle or macromolecule. A unique combination of a photonic crystal waveguide cavity and a nanoslot structure leads to a ~1300 times enhancement of the optical gradient trapping force compared with a conventional waveguide trapping device. Numerical simulations indicate that the designed device is capable of stably trapping a single nanoparticle inside the nanoslot cavity, and thus provides an ideal platform for single particle detection and analysis using cavity-enhanced spectroscopic technologies.
We design and numerically simulate an on-chip photonic device that integrates both optical manipulation and detection functionalities for a single nanoparticle or macromolecule. A unique combination of a photonic crystal waveguide cavity and a nanoslot structure leads to a ~1300 times enhancement of the optical gradient trapping force compared with a conventional waveguide trapping device. Numerical simulations indicate that the designed device is capable of stably trapping a single nanoparticle inside the nanoslot cavity, and thus provides an ideal platform for single particle detection and analysis using cavity-enhanced spectroscopic technologies.
Wednesday, October 28, 2009
Trapping and Rotation of Nanowires Assisted by Surface Plasmons
Miao , X. Wilson, B. K. Cao , G. Pun , S. H. Lin, L. Y.
We report long-range trapping of vanadium dioxide (VO2) and vanadium oxyhydroxide (H2V3O8) nanowires at a distance as large as 50 mu m outside the laser spot using plasmonic tweezers and controlled rotation of the nanowires by combining trapping with microfluidic drag force. The plasmonic tweezers are built upon a self-assembled gold nanoparticle array platform. In addition to the long-range trapping and rotation capability, the required optical intensity for the plasmonic tweezers to initiate trapping is much lower than that required by conventional optical tweezers for similar nanowires. We also investigate possible mechanisms for the unique long-range trapping of nanowires through performing control experiments.
We report long-range trapping of vanadium dioxide (VO2) and vanadium oxyhydroxide (H2V3O8) nanowires at a distance as large as 50 mu m outside the laser spot using plasmonic tweezers and controlled rotation of the nanowires by combining trapping with microfluidic drag force. The plasmonic tweezers are built upon a self-assembled gold nanoparticle array platform. In addition to the long-range trapping and rotation capability, the required optical intensity for the plasmonic tweezers to initiate trapping is much lower than that required by conventional optical tweezers for similar nanowires. We also investigate possible mechanisms for the unique long-range trapping of nanowires through performing control experiments.
Tuesday, October 27, 2009
Characterization of Photoactivated Singlet Oxygen Damage in Single-Molecule Optical Trap Experiments
Markita P. Landry, Patrick M. McCall, Zhi Qi and Yann R. Chemla
Optical traps or “tweezers” use high-power, near-infrared laser beams to manipulate and apply forces to biological systems, ranging from individual molecules to cells. Although previous studies have established that optical tweezers induce photodamage in live cells, the effects of trap irradiation have yet to be examined in vitro, at the single-molecule level. In this study, we investigate trap-induced damage in a simple system consisting of DNA molecules tethered between optically trapped polystyrene microspheres. We show that exposure to the trapping light affects the lifetime of the tethers, the efficiency with which they can be formed, and their structure. Moreover, we establish that these irreversible effects are caused by oxidative damage from singlet oxygen. This reactive state of molecular oxygen is generated locally by the optical traps in the presence of a sensitizer, which we identify as the trapped polystyrene microspheres. Trap-induced oxidative damage can be reduced greatly by working under anaerobic conditions, using additives that quench singlet oxygen, or trapping microspheres lacking the sensitizers necessary for singlet state photoexcitation. Our findings are relevant to a broad range of trap-based single-molecule experiments—the most common biological application of optical tweezers—and may guide the development of more robust experimental protocols.
Optical traps or “tweezers” use high-power, near-infrared laser beams to manipulate and apply forces to biological systems, ranging from individual molecules to cells. Although previous studies have established that optical tweezers induce photodamage in live cells, the effects of trap irradiation have yet to be examined in vitro, at the single-molecule level. In this study, we investigate trap-induced damage in a simple system consisting of DNA molecules tethered between optically trapped polystyrene microspheres. We show that exposure to the trapping light affects the lifetime of the tethers, the efficiency with which they can be formed, and their structure. Moreover, we establish that these irreversible effects are caused by oxidative damage from singlet oxygen. This reactive state of molecular oxygen is generated locally by the optical traps in the presence of a sensitizer, which we identify as the trapped polystyrene microspheres. Trap-induced oxidative damage can be reduced greatly by working under anaerobic conditions, using additives that quench singlet oxygen, or trapping microspheres lacking the sensitizers necessary for singlet state photoexcitation. Our findings are relevant to a broad range of trap-based single-molecule experiments—the most common biological application of optical tweezers—and may guide the development of more robust experimental protocols.
Monday, October 26, 2009
Probing the dynamic differential stiffness of dsDNA interacting with RecA in the enthalpic regime
Chia-Hui Lien, Ming-Tzo Wei, Te- Yu Tseng, Chien-Der Lee, Chung Wang, Ting-Fang Wang, H. Daniel Ou-Yang, and Arthur Chiou
RecA plays a central role in homologous recombination of DNA. When RecA combines with dsDNA to form RecA-dsDNA nucleofilament, it unwinds dsDNA and changes its structure. The unwinding length extension of a DNA segment interacting with RecA has been studied by various techniques, but the dynamic differential stiffness of dsDNA conjugating with RecA has not been well characterized. We applied oscillatory optical tweezers to measure the differential stiffness of dsDNA molecules, interacting with RecA, as a function of time at a constant stretching force of 33.6pN. The values of the differential stiffness of DNA (for stretching force in the range of 20.0pN to 33.6pN) measured by oscillatory optical tweezers, both before and after its interaction with RecA, are consistent with those measured by stationary optical tweezers. In the dynamic measurement, we have shown that the association (or binding) rate increases with higher concentration of RecA; besides, we have also monitored in real-time the dissociation of RecA from the stretched RecA-dsDNA filament as ATPγS was washed off from the sample chamber. Finally, we verified that RecA (I26C), a form of RecA mutant, does not affect the differential stiffness of the stretched DNA sample. It implies that mutant RecA (I26C) does not bind to the DNA, which is consistent with the result obtained by conventional biochemical approach.
RecA plays a central role in homologous recombination of DNA. When RecA combines with dsDNA to form RecA-dsDNA nucleofilament, it unwinds dsDNA and changes its structure. The unwinding length extension of a DNA segment interacting with RecA has been studied by various techniques, but the dynamic differential stiffness of dsDNA conjugating with RecA has not been well characterized. We applied oscillatory optical tweezers to measure the differential stiffness of dsDNA molecules, interacting with RecA, as a function of time at a constant stretching force of 33.6pN. The values of the differential stiffness of DNA (for stretching force in the range of 20.0pN to 33.6pN) measured by oscillatory optical tweezers, both before and after its interaction with RecA, are consistent with those measured by stationary optical tweezers. In the dynamic measurement, we have shown that the association (or binding) rate increases with higher concentration of RecA; besides, we have also monitored in real-time the dissociation of RecA from the stretched RecA-dsDNA filament as ATPγS was washed off from the sample chamber. Finally, we verified that RecA (I26C), a form of RecA mutant, does not affect the differential stiffness of the stretched DNA sample. It implies that mutant RecA (I26C) does not bind to the DNA, which is consistent with the result obtained by conventional biochemical approach.
Friday, October 23, 2009
Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging
Joost van Mameren, Peter Gross, Geraldine Farge, Pleuni Hooijman, Mauro Modesti, Maria Falkenberg, Gijs J. L. Wuite and Erwin J. G. Peterman
Single-molecule manipulation studies have revealed that double-stranded DNA undergoes a structural transition when subjected to tension. At forces that depend on the attachment geometry of the DNA (65 pN or 110 pN), it elongates ≈1.7-fold and its elastic properties change dramatically. The nature of this overstretched DNA has been under debate. In one model, the DNA cooperatively unwinds, while base pairing remains intact. In a competing model, the hydrogen bonds between base pairs break and two single DNA strands are formed, comparable to thermal DNA melting. Here, we resolve the structural basis of DNA overstretching using a combination of fluorescence microscopy, optical tweezers, and microfluidics. In DNA molecules undergoing the transition, we visualize double- and single-stranded segments using specific fluorescent labels. Our data directly demonstrate that overstretching comprises a gradual conversion from double-stranded to single-stranded DNA, irrespective of the attachment geometry. We found that these conversions favorably initiate from nicks or free DNA ends. These discontinuities in the phosphodiester backbone serve as energetically favorable nucleation points for melting. When both DNA strands are intact and no nicks or free ends are present, the overstretching force increases from 65 to 110 pN and melting initiates throughout the molecule, comparable to thermal melting. These results provide unique insights in the thermodynamics of DNA and DNA-protein interactions.
Single-molecule manipulation studies have revealed that double-stranded DNA undergoes a structural transition when subjected to tension. At forces that depend on the attachment geometry of the DNA (65 pN or 110 pN), it elongates ≈1.7-fold and its elastic properties change dramatically. The nature of this overstretched DNA has been under debate. In one model, the DNA cooperatively unwinds, while base pairing remains intact. In a competing model, the hydrogen bonds between base pairs break and two single DNA strands are formed, comparable to thermal DNA melting. Here, we resolve the structural basis of DNA overstretching using a combination of fluorescence microscopy, optical tweezers, and microfluidics. In DNA molecules undergoing the transition, we visualize double- and single-stranded segments using specific fluorescent labels. Our data directly demonstrate that overstretching comprises a gradual conversion from double-stranded to single-stranded DNA, irrespective of the attachment geometry. We found that these conversions favorably initiate from nicks or free DNA ends. These discontinuities in the phosphodiester backbone serve as energetically favorable nucleation points for melting. When both DNA strands are intact and no nicks or free ends are present, the overstretching force increases from 65 to 110 pN and melting initiates throughout the molecule, comparable to thermal melting. These results provide unique insights in the thermodynamics of DNA and DNA-protein interactions.
Optically driven micropump with a twin spiral microrotor
Shoji Maruo, Akira Takaura, and Yohei Saito
An optically driven micropump that employs viscous drag exerted on a spinning microrotor with left- and right-handed spiral blades on its rotational axis has been developed using two-photon microfabrication. It was demonstrated that the twin spiral microrotor provides a higher rotation speed than a single spiral microrotor. The rotation speed reached 560 rpm at a laser power of 500 mW. The twin spiral microrotor was also applied to a viscous micropump with a U-shaped microchannel. To pump fluid, the twin spiral microrotor located at the corner of the U-shaped microchannel was rotated by focusing a laser beam. The flow field inside the U-shaped microchannel was analyzed using the finite element method (FEM) based on the Navier-Stokes equation to optimize the shape of the microchannel. It was confirmed that the rotation of the twin spiral microrotor generated a unidirectional laminar flow. Finally, a tandem micropump using two twin spiral microrotors was driven by a dual optical trapping system using a spatial light modulation technique.
An optically driven micropump that employs viscous drag exerted on a spinning microrotor with left- and right-handed spiral blades on its rotational axis has been developed using two-photon microfabrication. It was demonstrated that the twin spiral microrotor provides a higher rotation speed than a single spiral microrotor. The rotation speed reached 560 rpm at a laser power of 500 mW. The twin spiral microrotor was also applied to a viscous micropump with a U-shaped microchannel. To pump fluid, the twin spiral microrotor located at the corner of the U-shaped microchannel was rotated by focusing a laser beam. The flow field inside the U-shaped microchannel was analyzed using the finite element method (FEM) based on the Navier-Stokes equation to optimize the shape of the microchannel. It was confirmed that the rotation of the twin spiral microrotor generated a unidirectional laminar flow. Finally, a tandem micropump using two twin spiral microrotors was driven by a dual optical trapping system using a spatial light modulation technique.
Laser-induced self-assembly of silver nanoparticles via plasmonic interactions
Yoshito Tanaka, Hiroyuki Yoshikawa, Tamitake Itoh, and Mitsuru Ishikawa
We report laser induced self-assembly of silver nanoparticles via plasmonic interactions. By focusing a near-infrared laser in silver nanoparticle suspension, nanoparticle assembly is formed as a result of optical trapping. The shape of Rayleigh scattering spectra of the nanoassembly strongly depends on the polarization of the laser beam. Particularly, a linearly polarized laser induces the formation of arrayed structure along the laser polarization, that shows a sharp plasmon resonance band and harnesses excellent plasmonic properties applicable for nonlinear surface enhanced spectroscopy.
We report laser induced self-assembly of silver nanoparticles via plasmonic interactions. By focusing a near-infrared laser in silver nanoparticle suspension, nanoparticle assembly is formed as a result of optical trapping. The shape of Rayleigh scattering spectra of the nanoassembly strongly depends on the polarization of the laser beam. Particularly, a linearly polarized laser induces the formation of arrayed structure along the laser polarization, that shows a sharp plasmon resonance band and harnesses excellent plasmonic properties applicable for nonlinear surface enhanced spectroscopy.
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