Joshua L. Rovey, Paul D. Friz, Changyu Hu, Matthew S. Glascock, and Xiaodong Yang
Plasmonic space propulsion uses solar light focused onto deep-subwavelength nanostructures to excite strong optical forces that accelerate and expel nanoparticle propellant. Simulations predict that light within the solar spectrum can excite asymmetric nanostructures to create plasmonic forces that will accelerate and expel nanoparticles. A peak force of 55 pN/W is predicted for a 50-nm-wide, 400-nm-long nanostructure that resonates at 500 nm. Results for a conceptual design of a plasmonic thruster that has 35 layers, 86 array columns, a multistage length of 5 mm, a 5-cm-diam light focusing lens, and uses 100 nm polystyrene nanoparticles expelled at a rate of 1×106 per second would have a thrust of 250 nN, specific impulse of 10 s, and minimum impulse bit of 50 pN⋅s.
DOI
Monday, August 31, 2015
A U⋅U Pair-to-U⋅C Pair Mutation-Induced RNA Native Structure Destabilisation and Stretching-Force-Induced RNA Misfolding
Zhensheng Zhong, Lai Huat Soh, Ming Hui Lim andProf. Gang Chen
Little is known about how a non-Watson–Crick pair affects the RNA folding dynamics. We studied the effects of a U⋅U-to-U⋅C pair mutation on the folding of a hairpin in human telomerase RNA. The ensemble thermal melting of the hairpins shows an on-pathway intermediate with the disruption of the internal loop structure containing the U⋅U/U⋅C pairs. By using optical tweezers, we applied a stretching force on the terminal ends of the hairpins to probe directly the non-nearest-neighbour effects upon the mutations. The single U⋅U to U⋅C mutations are observed to 1) lower the mechanical unfolding force by approximately 1 picoNewton (pN) per mutation without affecting the unfolding reaction transition-state position (thus suggesting that removing a single hydrogen bond affects the structural dynamics at least two base pairs away), 2) result in more frequent misfolding into a small hairpin at approximately 10 pN and 3) shift the folding reaction transition-state position towards the native hairpin structure and slightly increase the mechanical folding kinetics (thus suggesting that untrapping from the misfolded state is not the rate-limiting step).
DOI
Little is known about how a non-Watson–Crick pair affects the RNA folding dynamics. We studied the effects of a U⋅U-to-U⋅C pair mutation on the folding of a hairpin in human telomerase RNA. The ensemble thermal melting of the hairpins shows an on-pathway intermediate with the disruption of the internal loop structure containing the U⋅U/U⋅C pairs. By using optical tweezers, we applied a stretching force on the terminal ends of the hairpins to probe directly the non-nearest-neighbour effects upon the mutations. The single U⋅U to U⋅C mutations are observed to 1) lower the mechanical unfolding force by approximately 1 picoNewton (pN) per mutation without affecting the unfolding reaction transition-state position (thus suggesting that removing a single hydrogen bond affects the structural dynamics at least two base pairs away), 2) result in more frequent misfolding into a small hairpin at approximately 10 pN and 3) shift the folding reaction transition-state position towards the native hairpin structure and slightly increase the mechanical folding kinetics (thus suggesting that untrapping from the misfolded state is not the rate-limiting step).
DOI
Stimulus-responsive colloidal sensors with fast holographic readout
Chen Wang, Henrique W. Moyses and David G. Grier
Colloidal spheres synthesized from polymer gels swell by absorbing molecules from solution. The resulting change in size can be monitored with nanometer precision using holographic video microscopy. When the absorbate is chemically similar to the polymer matrix, swelling is driven primarily by the entropy of mixing, and is limited by the surface tension of the swelling sphere and by the elastic energy of the polymer matrix. We demonstrate through a combination of optical micromanipulation and holographic particle characterization that the degree of swelling of a single polymer bead can be used to measure the monomer concentration in situ with spatial resolution comparable to the size of the sphere.
DOI
Colloidal spheres synthesized from polymer gels swell by absorbing molecules from solution. The resulting change in size can be monitored with nanometer precision using holographic video microscopy. When the absorbate is chemically similar to the polymer matrix, swelling is driven primarily by the entropy of mixing, and is limited by the surface tension of the swelling sphere and by the elastic energy of the polymer matrix. We demonstrate through a combination of optical micromanipulation and holographic particle characterization that the degree of swelling of a single polymer bead can be used to measure the monomer concentration in situ with spatial resolution comparable to the size of the sphere.
DOI
Computational study of radiation torque on arbitrary shaped particles with MLFMA
Minglin Yang, Kuan Fang Ren, Theodor Petkov, Bernard Pouligny, Jean-Christophe Loudet, and Xinqing Sheng
The surface integral equation (SIE) method is used for the computational study of radiation torque on arbitrarily shaped homogeneous particles. The Multilevel Fast Multipole Algorithm (MLFMA) is employed to reduce memory requirements and improve the capability of SIE. The resultant matrix equations are solved iteratively to obtain equivalent electric and magnetic currents. Then, radiation torque is computed using the vector flux of the pseudotensor over a spherical surface tightly enclosing the particle. We use, therefore, the analytical electromagnetic field expression for incident waves in the near region, instead of the far-field approximation. This avoids the error which may be caused when describing the incident beam. The numerical results of three kinds of non-spherical particles are presented to illustrate the validity and capability of the developed method. It is shown that our method can be applied to predict, in the rigorous sense, the torque from a beam of any shape on a particle of complex configuration with a size parameter as large as 650. The radiation torques on large ellipsoids are exemplified to show the performance of the method and to study the influence that different aspect ratios have on the results. Then, the code is used for the calculation of radiation torque on objects of complex shape including a biconcave cell-like particle and a motor with a non-smooth surface.
DOI
The surface integral equation (SIE) method is used for the computational study of radiation torque on arbitrarily shaped homogeneous particles. The Multilevel Fast Multipole Algorithm (MLFMA) is employed to reduce memory requirements and improve the capability of SIE. The resultant matrix equations are solved iteratively to obtain equivalent electric and magnetic currents. Then, radiation torque is computed using the vector flux of the pseudotensor over a spherical surface tightly enclosing the particle. We use, therefore, the analytical electromagnetic field expression for incident waves in the near region, instead of the far-field approximation. This avoids the error which may be caused when describing the incident beam. The numerical results of three kinds of non-spherical particles are presented to illustrate the validity and capability of the developed method. It is shown that our method can be applied to predict, in the rigorous sense, the torque from a beam of any shape on a particle of complex configuration with a size parameter as large as 650. The radiation torques on large ellipsoids are exemplified to show the performance of the method and to study the influence that different aspect ratios have on the results. Then, the code is used for the calculation of radiation torque on objects of complex shape including a biconcave cell-like particle and a motor with a non-smooth surface.
DOI
Friday, August 28, 2015
Application of axial symmetric phase plate and circular diffraction waveplate in optical tweezers
V. K. Abrahamyan
In development of optical tweezers can be used diffractive optical elements, as laser beam steering systems, as well the phase plates, which allow modulating the intensity distribution of the laser beam for manipulating trapping forces. The system, consisting of an axial symmetric phase retarder–glass substrate coated by axially symmetric oriented liquid crystal (LC) polymer, and a circular diffraction wave plate–glass substrate, coated by LC polymer with polarization patterned orientation, is considered. Diffracted beams are obtained at the output of the system in ±1 order, the intensity distribution in which is determined by the state of light polarization at the system input. The possibility of use this system for trapping, scrolling and moving the particles of micro- and nanosizes by modification of the shape and intensity of the beams at the system output is considered.
In development of optical tweezers can be used diffractive optical elements, as laser beam steering systems, as well the phase plates, which allow modulating the intensity distribution of the laser beam for manipulating trapping forces. The system, consisting of an axial symmetric phase retarder–glass substrate coated by axially symmetric oriented liquid crystal (LC) polymer, and a circular diffraction wave plate–glass substrate, coated by LC polymer with polarization patterned orientation, is considered. Diffracted beams are obtained at the output of the system in ±1 order, the intensity distribution in which is determined by the state of light polarization at the system input. The possibility of use this system for trapping, scrolling and moving the particles of micro- and nanosizes by modification of the shape and intensity of the beams at the system output is considered.
Optically Trapped Surface-Enhanced Raman Probes Prepared by Silver Photoreduction to 3D Microstructures
Gaszton Vizsnyiczai, Tamás Lestyán, Jaroslava Joniova, Badri L. Aekbote, Alena Strejčková, Pál Ormos, Pavol Miskovsky, Lóránd Kelemen, and Gregor Bánó
3D microstructures partially covered by silver nanoparticles have been developed and tested for surface-enhanced Raman spectroscopy (SERS) in combination with optical tweezers. The microstructures made by two-photon polymerization of SU-8 photoresist were manipulated in a dual beam optical trap. The active area of the structures was covered by a SERS-active silver layer using chemically assisted photoreduction from silver nitrate solutions. Silver layers of different grain size distributions were created by changing the photoreduction parameters and characterized by scanning electron microscopy. The structures were tested by measuring the SERS spectra of emodin and hypericin.
DOI
3D microstructures partially covered by silver nanoparticles have been developed and tested for surface-enhanced Raman spectroscopy (SERS) in combination with optical tweezers. The microstructures made by two-photon polymerization of SU-8 photoresist were manipulated in a dual beam optical trap. The active area of the structures was covered by a SERS-active silver layer using chemically assisted photoreduction from silver nitrate solutions. Silver layers of different grain size distributions were created by changing the photoreduction parameters and characterized by scanning electron microscopy. The structures were tested by measuring the SERS spectra of emodin and hypericin.
DOI
Thursday, August 27, 2015
Tracking of colloids close to contact
Chi Zhang, Georges Brügger, and Frank Scheffold
The precise tracking of micron sized colloidal particles - held in the vicinity of each other using optical tweezers - is an elegant way to gain information about the particle-particle pair interaction potential. The accuracy of the method, however, relies strongly on the tracking precision. Particularly the elimination of systematic errors in the position detection due to overlapping particle diffraction patterns remains a great challenge. Here we propose a template based particle finding algorithm that circumvents these problems by tracking only a fraction of the particle image that is insignificantly affected by nearby colloids. Under realistic experimental conditions we show that our algorithm significantly reduces systematic errors compared to standard tracking methods. Moreover our approach should in principle be applicable to almost arbitrary shaped particles as the template can be adapted to any geometry.
DOI
The precise tracking of micron sized colloidal particles - held in the vicinity of each other using optical tweezers - is an elegant way to gain information about the particle-particle pair interaction potential. The accuracy of the method, however, relies strongly on the tracking precision. Particularly the elimination of systematic errors in the position detection due to overlapping particle diffraction patterns remains a great challenge. Here we propose a template based particle finding algorithm that circumvents these problems by tracking only a fraction of the particle image that is insignificantly affected by nearby colloids. Under realistic experimental conditions we show that our algorithm significantly reduces systematic errors compared to standard tracking methods. Moreover our approach should in principle be applicable to almost arbitrary shaped particles as the template can be adapted to any geometry.
DOI
Structural features of the αβTCR mechanotransduction apparatus that promote pMHC discrimination
Kristine N. Brazin, Robert J. Mallis, Dibyendu K. Das, Yinnian Feng, Wonmuk Hwang, Jia-huai Wang, Gerhard Wagner, Matthew J. Lang and Ellis L. Reinherz
The αβTCR was recently revealed to function as a mechanoreceptor. That is, it leverages mechanical energy generated during immune surveillance and at the immunological synapse to drive biochemical signaling following ligation by a specific foreign peptide-MHC complex (pMHC). Here we review the structural features that optimize this transmembrane receptor for mechanotransduction. Specialized adaptations include: 1) the CβFG loop region positioned between Vβ and Cβ domains that allosterically gates both dynamic TCR-pMHC bond formation and lifetime; 2) the rigid super β-sheet amalgams of heterodimeric CD3εγ as well as CD3εδ ectodomain components of the αβTCR complex; 3) the αβTCR subunit connecting peptides (CP) linking the extracellular and transmembrane (TM) segments, particularly the oxidized CxxC motif in each CD3 heterodimeric subunit that facilitates force transfer through the TM segments and surrounding lipid, impacting cytoplasmic tail conformation; and 4) quaternary changes in the αβTCR complex that accompany pMHC ligation under load. How bioforces foster specific αβTCR-based pMHC discrimination and why dynamic bond formation is a primary basis for kinetic proofreading are discussed. We suggest that the details of the molecular rearrangements of individual αβTCR subunit components can be analyzed utilizing a combination of structural biology, single molecule FRET, optical tweezers and nanobiology, guided by insightful atomistic molecular dynamic studies. Finally, we review very recent data showing that the preTCR complex employs a similar mechanobiology to that of the αβTCR to interact with self-pMHC ligands, impacting early thymic repertoire selection prior to the CD4+CD8+ double positive thymocyte stage of development.
DOI
The αβTCR was recently revealed to function as a mechanoreceptor. That is, it leverages mechanical energy generated during immune surveillance and at the immunological synapse to drive biochemical signaling following ligation by a specific foreign peptide-MHC complex (pMHC). Here we review the structural features that optimize this transmembrane receptor for mechanotransduction. Specialized adaptations include: 1) the CβFG loop region positioned between Vβ and Cβ domains that allosterically gates both dynamic TCR-pMHC bond formation and lifetime; 2) the rigid super β-sheet amalgams of heterodimeric CD3εγ as well as CD3εδ ectodomain components of the αβTCR complex; 3) the αβTCR subunit connecting peptides (CP) linking the extracellular and transmembrane (TM) segments, particularly the oxidized CxxC motif in each CD3 heterodimeric subunit that facilitates force transfer through the TM segments and surrounding lipid, impacting cytoplasmic tail conformation; and 4) quaternary changes in the αβTCR complex that accompany pMHC ligation under load. How bioforces foster specific αβTCR-based pMHC discrimination and why dynamic bond formation is a primary basis for kinetic proofreading are discussed. We suggest that the details of the molecular rearrangements of individual αβTCR subunit components can be analyzed utilizing a combination of structural biology, single molecule FRET, optical tweezers and nanobiology, guided by insightful atomistic molecular dynamic studies. Finally, we review very recent data showing that the preTCR complex employs a similar mechanobiology to that of the αβTCR to interact with self-pMHC ligands, impacting early thymic repertoire selection prior to the CD4+CD8+ double positive thymocyte stage of development.
DOI
Controlled modulation of laser beam and dynamic patterning of colloidal particles using optical tweezers
Brijesh Kumar Singh, Dalip Singh Mehta, Ranjeet Kumar & Paramasivam Senthilkumaran
We present controlled generation of complex-structured beam profiles using diffractive optical element and demonstrate multiple dynamic trapping of colloidal particles. The phase element is programmed to generate various tailored optical fields having structures, similar to that of number three, spiral, and circle but in a tractable manner. Thus, the generated spatially tailored optical fields are confined to focal volume in optical tweezers. This enabled real-time trapping of multiple microscopic objects whereby its transverse organization was controlled in a dynamic manner from one structure to another with the help of spatial light modulator. Such a controlled beam shaping finds potential applications in biophotonics, super resolution imaging, and measurement of biophysical parameters, cell sorting, and micro-manipulation of colloidal particles.
DOI
We present controlled generation of complex-structured beam profiles using diffractive optical element and demonstrate multiple dynamic trapping of colloidal particles. The phase element is programmed to generate various tailored optical fields having structures, similar to that of number three, spiral, and circle but in a tractable manner. Thus, the generated spatially tailored optical fields are confined to focal volume in optical tweezers. This enabled real-time trapping of multiple microscopic objects whereby its transverse organization was controlled in a dynamic manner from one structure to another with the help of spatial light modulator. Such a controlled beam shaping finds potential applications in biophotonics, super resolution imaging, and measurement of biophysical parameters, cell sorting, and micro-manipulation of colloidal particles.
DOI
A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology
Marcel Ander, Sivaraman Subramaniam, Karim Fahmy, A. Francis Stewart, Erik Schäffer
Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]), such as eukaryotic RAD52 and λ phage Redβ. These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. However, it is unclear how SSAPs detect homology and catalyze annealing. Using single-molecule experiments, we provide evidence that homology is recognized by Redβ monomers that weakly hold single DNA strands together. Once annealing begins, dimerization of Redβ clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. The clamp is characterized by a structural change of Redβ and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, noncovalent, DNA–protein interaction.
DOI
Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]), such as eukaryotic RAD52 and λ phage Redβ. These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. However, it is unclear how SSAPs detect homology and catalyze annealing. Using single-molecule experiments, we provide evidence that homology is recognized by Redβ monomers that weakly hold single DNA strands together. Once annealing begins, dimerization of Redβ clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. The clamp is characterized by a structural change of Redβ and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, noncovalent, DNA–protein interaction.
DOI
Thursday, August 20, 2015
Rotation of large asymmetrical absorbing objects by Laguerre–Gauss beams
Catherine M. Herne, Kristina M. Capuzzi, Emily Sobel, and Ryan T. Kropas
In this Letter, we show the manipulation and rotation of opaque graphite through adhesion with optically trapped polystyrene spheres. The absorbing graphite is rotated by the orbital angular momentum transfer from a Laguerre–Gauss laser mode and is trapped due to the presence of refracting spheres. This technique is effective for trapping and rotating absorbing objects of all sizes, including those larger than the laser mode.
DOI
In this Letter, we show the manipulation and rotation of opaque graphite through adhesion with optically trapped polystyrene spheres. The absorbing graphite is rotated by the orbital angular momentum transfer from a Laguerre–Gauss laser mode and is trapped due to the presence of refracting spheres. This technique is effective for trapping and rotating absorbing objects of all sizes, including those larger than the laser mode.
DOI
Evanescent wave optical binding forces on spherical microparticles
Xiang Han and Philip H. Jones
In this Letter, we demonstrate stable optical binding of spherical microparticles in counter-propagating evanescent optical fields formed by total reflection at a dielectric interface. The microspheres are observed to form one-dimensional chains oriented parallel to the direction of propagation of the beams. We characterize the strength of the optical binding interaction by measuring the extent of Brownian position fluctuations of the optically bound microspheres and relating this to a binding spring constant acting between adjacent particles. A stronger binding interaction is observed for particles near the middle of the chain, and the dependence of the binding strength on incident laser power and number of particles in the chain is determined.
DOI
In this Letter, we demonstrate stable optical binding of spherical microparticles in counter-propagating evanescent optical fields formed by total reflection at a dielectric interface. The microspheres are observed to form one-dimensional chains oriented parallel to the direction of propagation of the beams. We characterize the strength of the optical binding interaction by measuring the extent of Brownian position fluctuations of the optically bound microspheres and relating this to a binding spring constant acting between adjacent particles. A stronger binding interaction is observed for particles near the middle of the chain, and the dependence of the binding strength on incident laser power and number of particles in the chain is determined.
DOI
Plasmonic random nanostructures on fiber tip for trapping live cells and colloidal particles
Jiajie Chen, Zhiwen Kang, Siu Kai Kong, and Ho-Pui Ho
We demonstrate optical trapping on a gold-coated single-mode fiber tip as excited by 980-nm laser radiation. The trapping force here is not due to common plasmonic localization, but dominated by the combined effect of thermophoresis and thermal convection. The reported scheme only requires simple thin-film deposition. More importantly, efficient broadband plasmonic absorption of the gold random nanostructures, aided by purely Gaussian excitation profile from the fiber core, has led to very low trapping-power threshold typically in hundreds of microwatts. This highly versatile fiber-based trapping scheme clearly offers many potential application possibilities in life sciences as well as engineering disciplines.
DOI
We demonstrate optical trapping on a gold-coated single-mode fiber tip as excited by 980-nm laser radiation. The trapping force here is not due to common plasmonic localization, but dominated by the combined effect of thermophoresis and thermal convection. The reported scheme only requires simple thin-film deposition. More importantly, efficient broadband plasmonic absorption of the gold random nanostructures, aided by purely Gaussian excitation profile from the fiber core, has led to very low trapping-power threshold typically in hundreds of microwatts. This highly versatile fiber-based trapping scheme clearly offers many potential application possibilities in life sciences as well as engineering disciplines.
DOI
Magnetically self-assembled colloidal 3D structures as cell growth scaffold
Gašper Kokot , Špela Zemljič-Jokhadar , Urška Batista , and Dusan Babic
Understanding the chemical and physical conditions for cell growth is important from biological and medical aspects. Many tissues and cell types (e.g. epithelial cells, neurons) naturally grow on surfaces that span in three-dimensions and offer structural or mechanical support. The scaffold surface has to promote adhesion and cell proliferation as well as support their weight and retain its structural integrity. Here we present a flexible method which uses self-assembly of micrometer superparamagnetic particles to produce appropriate scaffold surfaces with controllable general appearance in three dimensions, such as oriented membranes, branched structure or void network. As a proof of principle Chinese hamster ovary epithelial cell line was successfully grown for several days on inclined membranes. Robustness of the oriented membrane architecture was probed with optical tweezers. We measured the magnetic force holding one particle in a self-assembled upright hexagonal sheet and modeled it as a sum of pair interaction forces between spatially arrested static dipoles.
DOI
Understanding the chemical and physical conditions for cell growth is important from biological and medical aspects. Many tissues and cell types (e.g. epithelial cells, neurons) naturally grow on surfaces that span in three-dimensions and offer structural or mechanical support. The scaffold surface has to promote adhesion and cell proliferation as well as support their weight and retain its structural integrity. Here we present a flexible method which uses self-assembly of micrometer superparamagnetic particles to produce appropriate scaffold surfaces with controllable general appearance in three dimensions, such as oriented membranes, branched structure or void network. As a proof of principle Chinese hamster ovary epithelial cell line was successfully grown for several days on inclined membranes. Robustness of the oriented membrane architecture was probed with optical tweezers. We measured the magnetic force holding one particle in a self-assembled upright hexagonal sheet and modeled it as a sum of pair interaction forces between spatially arrested static dipoles.
DOI
Tuesday, August 18, 2015
Single-cell analysis based on lab on a chip fluidic system
Alireza Valizadeh and Ahmad Yari Khosroushahi
The combination of nano/microfabrication-based technologies with cell biology has laid the foundation for facilitating the spatiotemporal analysis of single cells under well-defined physiologically relevant conditions. This combined technology allows so far unachievable insights into simultaneous characterization and manipulation of cells. Nano/microfluidic technology provides cost-effective, integrated, and high-throughput systems that are promising substitutes for conventional biological laboratory methods going from fundamental research to point-of-care diagnosis. This inscription reviews the latest progresses regarding cell manipulation methods based on; dielectrophoretic, electrophoresis, optical, acoustical, encaging, hydrodynamic, magnetite, centrifugal cell trapping/sorting, and cell characterization based on mechanical, electrical, biochemical, and optical methods using fluidic systems.
DOI
The combination of nano/microfabrication-based technologies with cell biology has laid the foundation for facilitating the spatiotemporal analysis of single cells under well-defined physiologically relevant conditions. This combined technology allows so far unachievable insights into simultaneous characterization and manipulation of cells. Nano/microfluidic technology provides cost-effective, integrated, and high-throughput systems that are promising substitutes for conventional biological laboratory methods going from fundamental research to point-of-care diagnosis. This inscription reviews the latest progresses regarding cell manipulation methods based on; dielectrophoretic, electrophoresis, optical, acoustical, encaging, hydrodynamic, magnetite, centrifugal cell trapping/sorting, and cell characterization based on mechanical, electrical, biochemical, and optical methods using fluidic systems.
DOI
Generalized phase contrast-enhanced diffractive coupling to light-driven microtools
Mark Villangca ; Andrew Bañas ; Darwin Palima ; Jesper Glückstad
We have previously demonstrated on-demand dynamic coupling to optically manipulated microtools coined as wave-guided optical waveguides using diffractive techniques on a “point and shoot” approach. These microtools are extended microstructures fabricated using two-photon photopolymerization and function as free-floating optically trapped waveguides. Dynamic coupling of focused light via these structures being moved in three-dimensional space is done holographically. However, calculating the necessary holograms is not straightforward when using counter-propagating trapping geometry. The generation of the coupling spots is done in real time following the position of each microtool with the aid of an object tracking routine. This approach allows continuous coupling of light through the microtools which can be useful in a variety of biophotonics applications. To complement the targeted-light delivery capability of the microtools, the applied spatial light modulator has been illuminated with a properly matched input beam cross section based on the generalized phase contrast method. Our results show a significant gain in the output at the tip of each microtool as measured from the fluorescence signal of the trapping medium. The ability to switch from on-demand to continuous addressing with efficient illumination leverages our microtools for potential applications in stimulation and near-field-based biophotonics on cellular scales.
DOI
We have previously demonstrated on-demand dynamic coupling to optically manipulated microtools coined as wave-guided optical waveguides using diffractive techniques on a “point and shoot” approach. These microtools are extended microstructures fabricated using two-photon photopolymerization and function as free-floating optically trapped waveguides. Dynamic coupling of focused light via these structures being moved in three-dimensional space is done holographically. However, calculating the necessary holograms is not straightforward when using counter-propagating trapping geometry. The generation of the coupling spots is done in real time following the position of each microtool with the aid of an object tracking routine. This approach allows continuous coupling of light through the microtools which can be useful in a variety of biophotonics applications. To complement the targeted-light delivery capability of the microtools, the applied spatial light modulator has been illuminated with a properly matched input beam cross section based on the generalized phase contrast method. Our results show a significant gain in the output at the tip of each microtool as measured from the fluorescence signal of the trapping medium. The ability to switch from on-demand to continuous addressing with efficient illumination leverages our microtools for potential applications in stimulation and near-field-based biophotonics on cellular scales.
DOI
Curvilinear optical forces
Andrey Novitsky
Accelerating a light beam makes a particle move along a curvilinear trajectory. We show that the curvature of the trajectory causes a special kind of nonconservative force, which is called a curvilinear force. We obtain the expression for this force acting on a spherical particle in the Rayleigh approximation and determine its value for several types of accelerating fields including Bessel, Airy, and arbitrary-trajectory beams. We anticipate applications of the curvilinear forces in optical micromanipulation with accelerating beams.
DOI
Accelerating a light beam makes a particle move along a curvilinear trajectory. We show that the curvature of the trajectory causes a special kind of nonconservative force, which is called a curvilinear force. We obtain the expression for this force acting on a spherical particle in the Rayleigh approximation and determine its value for several types of accelerating fields including Bessel, Airy, and arbitrary-trajectory beams. We anticipate applications of the curvilinear forces in optical micromanipulation with accelerating beams.
DOI
Investigation of temperature effect on cell mechanics by optofluidic microchips
Tie Yang, Giovanni Nava, Paolo Minzioni, Manuela Veglione, Francesca Bragheri, Francesca Demetra Lelii, Rebeca Martinez Vazquez, Roberto Osellame, and Ilaria Cristiani
Here we present the results of a study concerning the effect of temperature on cell mechanical properties. Two different optofluidic microchips with external temperature control are used to investigate the temperature-induced changes of highly metastatic human melanoma cells (A375MC2) in the range of ~0 – 35 °C. By means of an integrated optical stretcher, we observe that cells’ optical deformability is strongly enhanced by increasing cell and buffer-fluid temperature. This finding is supported by the results obtained from a second device, which probes the cells’ ability to be squeezed through a constriction. Measured data demonstrate a marked dependence of cell mechanical properties on temperature, thus highlighting the importance of including a proper temperature-control system in the experimental apparatus.
DOI
Here we present the results of a study concerning the effect of temperature on cell mechanical properties. Two different optofluidic microchips with external temperature control are used to investigate the temperature-induced changes of highly metastatic human melanoma cells (A375MC2) in the range of ~0 – 35 °C. By means of an integrated optical stretcher, we observe that cells’ optical deformability is strongly enhanced by increasing cell and buffer-fluid temperature. This finding is supported by the results obtained from a second device, which probes the cells’ ability to be squeezed through a constriction. Measured data demonstrate a marked dependence of cell mechanical properties on temperature, thus highlighting the importance of including a proper temperature-control system in the experimental apparatus.
DOI
Wednesday, August 12, 2015
Pharmacological targeting of membrane rigidity: implications on cancer cell migration and invasion
Simone Braig, B U Sebastian Schmidt, Katharina Stoiber, Chris Händel, Till Möhn, Oliver Werz, Rolf Müller, Stefan Zahler, Andreas Koeberle, Josef A Käs
The invasive potential of cancer cells strongly depends on cellular stiffness, a physical quantity that is not only regulated by the mechanical impact of the cytoskeleton but also influenced by the membrane rigidity. To analyze the specific role of membrane rigidity in cancer progression, we treated cancer cells with the Acetyl-CoA carboxylase inhibitor Soraphen A and revealed an alteration of the phospholipidome via mass spectrometry. Migration, invasion, and cell death assays were employed to relate this alteration to functional consequences, and a decrease of migration and invasion without significant impact on cell death has been recorded. Fourier fluctuation analysis of giant plasma membrane vesicles showed that Soraphen A increases membrane rigidity of carcinoma cell membranes. Mechanical measurements of the creep deformation response of whole intact cells were performed using the optical stretcher. The increase in membrane rigidity was observed in one cell line without changing the creep deformation response indicating no restructuring of the cytoskeleton. These data indicate that the increase of membrane rigidity alone is sufficient to inhibit invasiveness of cancer cells, thus disclosing the eminent role of membrane rigidity in migratory processes.
DOI
The invasive potential of cancer cells strongly depends on cellular stiffness, a physical quantity that is not only regulated by the mechanical impact of the cytoskeleton but also influenced by the membrane rigidity. To analyze the specific role of membrane rigidity in cancer progression, we treated cancer cells with the Acetyl-CoA carboxylase inhibitor Soraphen A and revealed an alteration of the phospholipidome via mass spectrometry. Migration, invasion, and cell death assays were employed to relate this alteration to functional consequences, and a decrease of migration and invasion without significant impact on cell death has been recorded. Fourier fluctuation analysis of giant plasma membrane vesicles showed that Soraphen A increases membrane rigidity of carcinoma cell membranes. Mechanical measurements of the creep deformation response of whole intact cells were performed using the optical stretcher. The increase in membrane rigidity was observed in one cell line without changing the creep deformation response indicating no restructuring of the cytoskeleton. These data indicate that the increase of membrane rigidity alone is sufficient to inhibit invasiveness of cancer cells, thus disclosing the eminent role of membrane rigidity in migratory processes.
DOI
Probing differentiation in cancer cell lines by single-cell micro-Raman spectroscopy
Surekha Barkur; Aseefhali Bankapur; Madhura Pradhan; Santhosh Chidangil; Deepak Mathur; Uma Ladiwala
Single-cell micro-Raman spectroscopy has been applied to explore cell differentiation in single, live, and malignant cells from two tumor cell lines. The spectra of differentiated cells exhibit substantial enhancement primarily in the intensities of protein peaks with concomitant decrease in intensities of O−P−O asymmetric stretching peaks in DNA/RNA. Principal component analyses show that the spectral score of differentiated cells tends to asymptotically approach that of spectra obtained from normal neural stem cells/progenitors. This lends credence to the notion that the observed spectral changes are specific to differentiation, since upon differentiation, malignant cells become less malignant and tend toward benignity.
DOI
Single-cell micro-Raman spectroscopy has been applied to explore cell differentiation in single, live, and malignant cells from two tumor cell lines. The spectra of differentiated cells exhibit substantial enhancement primarily in the intensities of protein peaks with concomitant decrease in intensities of O−P−O asymmetric stretching peaks in DNA/RNA. Principal component analyses show that the spectral score of differentiated cells tends to asymptotically approach that of spectra obtained from normal neural stem cells/progenitors. This lends credence to the notion that the observed spectral changes are specific to differentiation, since upon differentiation, malignant cells become less malignant and tend toward benignity.
DOI
Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK
Daniela Bauer, Dale R. Merz, Benjamin Pelz, Kelly E. Theisen, Gail Yacyshyn, Dejana Mokranjac, Ruxandra I. Dima, Matthias Rief, and Gabriel Žoldák
The regulation of protein function through ligand-induced conformational changes is crucial for many signal transduction processes. The binding of a ligand alters the delicate energy balance within the protein structure, eventually leading to such conformational changes. In this study, we elucidate the energetic and mechanical changes within the subdomains of the nucleotide binding domain (NBD) of the heat shock protein of 70 kDa (Hsp70) chaperone DnaK upon nucleotide binding. In an integrated approach using single molecule optical tweezer experiments, loop insertions, and steered coarse-grained molecular simulations, we find that the C-terminal helix of the NBD is the major determinant of mechanical stability, acting as a glue between the two lobes. After helix unraveling, the relative stability of the two separated lobes is regulated by ATP/ADP binding. We find that the nucleotide stays strongly bound to lobe II, thus reversing the mechanical hierarchy between the two lobes. Our results offer general insights into the nucleotide-induced signal transduction within members of the actin/sugar kinase superfamily.
DOI
The regulation of protein function through ligand-induced conformational changes is crucial for many signal transduction processes. The binding of a ligand alters the delicate energy balance within the protein structure, eventually leading to such conformational changes. In this study, we elucidate the energetic and mechanical changes within the subdomains of the nucleotide binding domain (NBD) of the heat shock protein of 70 kDa (Hsp70) chaperone DnaK upon nucleotide binding. In an integrated approach using single molecule optical tweezer experiments, loop insertions, and steered coarse-grained molecular simulations, we find that the C-terminal helix of the NBD is the major determinant of mechanical stability, acting as a glue between the two lobes. After helix unraveling, the relative stability of the two separated lobes is regulated by ATP/ADP binding. We find that the nucleotide stays strongly bound to lobe II, thus reversing the mechanical hierarchy between the two lobes. Our results offer general insights into the nucleotide-induced signal transduction within members of the actin/sugar kinase superfamily.
DOI
Controlling local temperature in water using femtosecond optical tweezer
Dipankar Mondal and Debabrata Goswami
A novel method of directly observing the effect of temperature rise in water at the vicinity of optical trap center is presented. Our approach relies on changed values of corner frequency of the optical trap that, in turn, is realized from its power spectra. Our two color experiment is a unique combination of a non-heating femtosecond trapping laser at 780 nm, coupled to a femtosecond infrared heating laser at 1560 nm, which precisely controls temperature at focal volume of the trap center using low powers (100-800 µW) at high repetition rate. The geometric ray optics model quantitatively supports our experimental data.
DOI
A novel method of directly observing the effect of temperature rise in water at the vicinity of optical trap center is presented. Our approach relies on changed values of corner frequency of the optical trap that, in turn, is realized from its power spectra. Our two color experiment is a unique combination of a non-heating femtosecond trapping laser at 780 nm, coupled to a femtosecond infrared heating laser at 1560 nm, which precisely controls temperature at focal volume of the trap center using low powers (100-800 µW) at high repetition rate. The geometric ray optics model quantitatively supports our experimental data.
DOI
Tuesday, August 11, 2015
Raman Spectroscopy of Optically Trapped Single Biological Micro-Particles
Brandon Redding, Mark Schwab and Yong-le Pan
The combination of optical trapping with Raman spectroscopy provides a powerful method for the study, characterization, and identification of biological micro-particles. In essence, optical trapping helps to overcome the limitation imposed by the relative inefficiency of the Raman scattering process. This allows Raman spectroscopy to be applied to individual biological particles in air and in liquid, providing the potential for particle identification with high specificity, longitudinal studies of changes in particle composition, and characterization of the heterogeneity of individual particles in a population. In this review, we introduce the techniques used to integrate Raman spectroscopy with optical trapping in order to study individual biological particles in liquid and air. We then provide an overview of some of the most promising applications of this technique, highlighting the unique types of measurements enabled by the combination of Raman spectroscopy with optical trapping. Finally, we present a brief discussion of future research directions in the field.
DOI
The combination of optical trapping with Raman spectroscopy provides a powerful method for the study, characterization, and identification of biological micro-particles. In essence, optical trapping helps to overcome the limitation imposed by the relative inefficiency of the Raman scattering process. This allows Raman spectroscopy to be applied to individual biological particles in air and in liquid, providing the potential for particle identification with high specificity, longitudinal studies of changes in particle composition, and characterization of the heterogeneity of individual particles in a population. In this review, we introduce the techniques used to integrate Raman spectroscopy with optical trapping in order to study individual biological particles in liquid and air. We then provide an overview of some of the most promising applications of this technique, highlighting the unique types of measurements enabled by the combination of Raman spectroscopy with optical trapping. Finally, we present a brief discussion of future research directions in the field.
DOI
Dynein arms are strain-dependent direction-switching force generators
Chikako Shingyoji, Izumi Nakano, Yuichi Inoue and Hideo Higuchi
Dynein is a minus-end-directed motor that can generate (forward) force to move along the microtubule toward its minus end. In addition, axonemal dyneins were reported to oscillate in the generation of forward force, and cytoplasmic dynein is observed to generate bidirectional forces in response to defined chemical states. Both dyneins can also respond to mechanically applied force. To test whether axonemal dynein can switch direction of force generation, we measured force using an optical trap and UV-photolysis of caged ATP. We observed that isolated dynein could repeatedly generate force in both directions along the microtubule. Bidirectional force was also observed for dynein arms that are still attached on the doublet microtubules. Axonemal dynein generated force to move backward (∼4 pN) as well as forward (5-6 pN) along microtubules. Furthermore, backward force could be stimulated by plus-end directed external force applied to axonemal dynein prior to ATP application. The results show that axonemal dynein is unique exhibiting multiple modes of force generation including backward and forward force, oscillatory force and slow, repetitive bidirectional force. The results also demonstrate that mechanical strain is important for switching the directionality of force generation in axonemal dyneins. This article is protected by copyright. All rights reserved.
DOI
Dynein is a minus-end-directed motor that can generate (forward) force to move along the microtubule toward its minus end. In addition, axonemal dyneins were reported to oscillate in the generation of forward force, and cytoplasmic dynein is observed to generate bidirectional forces in response to defined chemical states. Both dyneins can also respond to mechanically applied force. To test whether axonemal dynein can switch direction of force generation, we measured force using an optical trap and UV-photolysis of caged ATP. We observed that isolated dynein could repeatedly generate force in both directions along the microtubule. Bidirectional force was also observed for dynein arms that are still attached on the doublet microtubules. Axonemal dynein generated force to move backward (∼4 pN) as well as forward (5-6 pN) along microtubules. Furthermore, backward force could be stimulated by plus-end directed external force applied to axonemal dynein prior to ATP application. The results show that axonemal dynein is unique exhibiting multiple modes of force generation including backward and forward force, oscillatory force and slow, repetitive bidirectional force. The results also demonstrate that mechanical strain is important for switching the directionality of force generation in axonemal dyneins. This article is protected by copyright. All rights reserved.
DOI
Compact optical tweezer with the capability of dynamic control
Alexander Korobtsov, Svetlana Kotova, Nikolay Losevsky, Aleksandra Mayorova, Sergey Samagin
The extension of capabilities towards the formation of controlled complex-shaped optical traps is demonstrated for the compact laser tweezer based on the four-channel LC modulator. The experimental results on the yeast cell manipulation, including the particles larger than 10 μm, are presented. The capture and confinement of the object with the dimensions 37 μm × 13 μm was realized by means of the use of the ellipse-shaped trap. The maximal escape velocity of this object was about 20 μm/s.
DOI
The extension of capabilities towards the formation of controlled complex-shaped optical traps is demonstrated for the compact laser tweezer based on the four-channel LC modulator. The experimental results on the yeast cell manipulation, including the particles larger than 10 μm, are presented. The capture and confinement of the object with the dimensions 37 μm × 13 μm was realized by means of the use of the ellipse-shaped trap. The maximal escape velocity of this object was about 20 μm/s.
DOI
Harmonic force spectroscopy measures load-dependent kinetics of individual human β-cardiac myosin molecules
Jongmin Sung, Suman Nag, Kim I. Mortensen, Christian L. Vestergaard, Shirley Sutton, Kathleen Ruppel, Henrik Flyvbjerg & James A. Spudich
Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using ‘harmonic force spectroscopy’. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method’s performance by measuring the force-dependent kinetics of individual human β-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule’s ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.
DOI
Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using ‘harmonic force spectroscopy’. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method’s performance by measuring the force-dependent kinetics of individual human β-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule’s ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.
DOI
Monday, August 10, 2015
Three-dimensional mapping of fluorescent nanoparticles using incoherent digital holography
Takumi Yanagawa, Ryosuke Abe, and Yoshio Hayasaki
Three-dimensional mapping of fluorescent nanoparticles was performed by using incoherent digital holography. The positions of the nanoparticles were quantitatively determined by using Gaussian fitting of the axial- and lateral-diffraction distributions through position calibration from the observation space to the sample space. It was found that the axial magnification was constant whereas the lateral magnification linearly depended on the axial position of the fluorescent nanoparticles. The mapping of multiple fluorescent nanoparticles fixed in gelatin and a single fluorescent nanoparticle manipulated with optical tweezers in water were demonstrated.
DOI
Three-dimensional mapping of fluorescent nanoparticles was performed by using incoherent digital holography. The positions of the nanoparticles were quantitatively determined by using Gaussian fitting of the axial- and lateral-diffraction distributions through position calibration from the observation space to the sample space. It was found that the axial magnification was constant whereas the lateral magnification linearly depended on the axial position of the fluorescent nanoparticles. The mapping of multiple fluorescent nanoparticles fixed in gelatin and a single fluorescent nanoparticle manipulated with optical tweezers in water were demonstrated.
DOI
Friday, August 7, 2015
Time-series methods in analysis of the optical tweezers recordings
Sławomir Drobczynski and Jakub Ślęzak
In this paper we treat optical tweezers as discrete-time linear filters and analyze the recorded trajectories of the trapped beads using time-series methods. Using these techniques we obtain a simple analytical formula for the aliased power-spectrum density. Moreover, we separate influences of the noise and blur induced by the video camera from the physical content of the measurements, providing simple tools to detect and account for these distortions. Finally, checking how our tools work on the real data, we identify what parameters of video camera calibration the blur is dominating and what the additive noise is dominating. We also detect a range where these two distortions cancel each other so that the data can be mistakenly classified as undisturbed.
DOI
In this paper we treat optical tweezers as discrete-time linear filters and analyze the recorded trajectories of the trapped beads using time-series methods. Using these techniques we obtain a simple analytical formula for the aliased power-spectrum density. Moreover, we separate influences of the noise and blur induced by the video camera from the physical content of the measurements, providing simple tools to detect and account for these distortions. Finally, checking how our tools work on the real data, we identify what parameters of video camera calibration the blur is dominating and what the additive noise is dominating. We also detect a range where these two distortions cancel each other so that the data can be mistakenly classified as undisturbed.
DOI
Exploring the physics of efficient optical trapping of dielectric nanoparticles with ultrafast pulsed excitation
Debjit Roy, Debabrata Goswami, and Arijit K. De
Stable optical trapping of dielectric nanoparticles with low power high-repetition-rate ultrafast pulsed excitation has received considerable attention in recent years. However, the exact role of such excitation has been quite illusive so far since, for dielectric micron-sized particles, the trapping efficiency turns out to be similar to that of continuous-wave excitation and independent of pulse chirping. In order to provide a coherent explanation of this apparently puzzling phenomenon, we justify the superior role of high-repetition-rate pulsed excitation in dielectric nanoparticle trapping which is otherwise not possible with continuous-wave excitation at a similar average power level. We quantitatively estimate the optimal combination of pulse peak power and pulse repetition rate leading to a stable trap and discuss the role of inertial response on the dependence of trapping efficiency on pulse width. In addition, we report gradual trapping of individual quantum dots detected by a stepwise rise in a two-photon fluorescence signal from the trapped quantum dots which conclusively proves individual particle trapping.
DOI
Stable optical trapping of dielectric nanoparticles with low power high-repetition-rate ultrafast pulsed excitation has received considerable attention in recent years. However, the exact role of such excitation has been quite illusive so far since, for dielectric micron-sized particles, the trapping efficiency turns out to be similar to that of continuous-wave excitation and independent of pulse chirping. In order to provide a coherent explanation of this apparently puzzling phenomenon, we justify the superior role of high-repetition-rate pulsed excitation in dielectric nanoparticle trapping which is otherwise not possible with continuous-wave excitation at a similar average power level. We quantitatively estimate the optimal combination of pulse peak power and pulse repetition rate leading to a stable trap and discuss the role of inertial response on the dependence of trapping efficiency on pulse width. In addition, we report gradual trapping of individual quantum dots detected by a stepwise rise in a two-photon fluorescence signal from the trapped quantum dots which conclusively proves individual particle trapping.
DOI
Bridging cells of three colors with two bio-orthogonal click reactions
Yue Yuan, Di Li, Jia Zhang, Xianmin Chen, Chi Zhang, Zhanling Ding, Lin Wang, Xueqian Zhang, Junhua Yuan, Yinmei Li, Yan-Biao Kang and Gaolin Liang
Cell-cell interactions play a crucial role in the development and function of multicellular organisms. To study cell-cell interactions in vitro, it is a big challenge for researchers to artificially build up cell junctions to bridge different types of cells for this purpose. Herein, by employing two orthogonal click reactions, we rationally designed four click reagents Mal-CBT, Mal-Cys, Mal-Alkyne, and Mal-N3 and successfully applied them for bridging cells of three colors. Orthogonality between these two click reactions was validated in solution and characterized with HPLC and ESI-MS analyses. After modifications of fluorescent protein-expressing prokaryotic Escherichia coli (E. Coli) cells (or eukaryotic HEK 293T cells) of three colors with respective Mal-Cys, Mal-CBT and Mal-Alkyne, or Mal-N3, the cells were sequentially bridged. The HEK 293T cells showed higher efficiency of cell bridging than that of E. Coli cells. At last, using optical tweezers, we quantitatively measured the bridging probability between Mal-Cys-modified and Mal-CBT-modified HEK 293 cells, as well as the rupture force between two bridged cells. We found that the CBT-Cys click reaction markedly improved the efficiency of cell bridging and the rupture force between two bridged cells was measured to be 153.8 pN at a force-loading rate of 49 pN/s. Our results demonstrate that it is possible to use two (or n) orthogonal click reactions to bridge three (or n + 1) types of cells. Taken the biological importance of cell junctions into consideration, we anticipate our method of bridging three types of cells with two bio-orthogonal click reactions to be a useful tool for biologists to study cell-cell interactions with more convenience and efficiency.
DOI
Cell-cell interactions play a crucial role in the development and function of multicellular organisms. To study cell-cell interactions in vitro, it is a big challenge for researchers to artificially build up cell junctions to bridge different types of cells for this purpose. Herein, by employing two orthogonal click reactions, we rationally designed four click reagents Mal-CBT, Mal-Cys, Mal-Alkyne, and Mal-N3 and successfully applied them for bridging cells of three colors. Orthogonality between these two click reactions was validated in solution and characterized with HPLC and ESI-MS analyses. After modifications of fluorescent protein-expressing prokaryotic Escherichia coli (E. Coli) cells (or eukaryotic HEK 293T cells) of three colors with respective Mal-Cys, Mal-CBT and Mal-Alkyne, or Mal-N3, the cells were sequentially bridged. The HEK 293T cells showed higher efficiency of cell bridging than that of E. Coli cells. At last, using optical tweezers, we quantitatively measured the bridging probability between Mal-Cys-modified and Mal-CBT-modified HEK 293 cells, as well as the rupture force between two bridged cells. We found that the CBT-Cys click reaction markedly improved the efficiency of cell bridging and the rupture force between two bridged cells was measured to be 153.8 pN at a force-loading rate of 49 pN/s. Our results demonstrate that it is possible to use two (or n) orthogonal click reactions to bridge three (or n + 1) types of cells. Taken the biological importance of cell junctions into consideration, we anticipate our method of bridging three types of cells with two bio-orthogonal click reactions to be a useful tool for biologists to study cell-cell interactions with more convenience and efficiency.
DOI
Optical Epitaxial Growth of Gold Nanoparticle Arrays
Ningfeng Huang, Luis Javier Martínez, Eric Jaquay, Aiichiro Nakano, and Michelle L. Povinelli
We use an optical analogue of epitaxial growth to assemble gold nanoparticles into 2D arrays. Particles are attracted to a growth template via optical forces and interact through optical binding. Competition between effects determines the final particle arrangements. We use a Monte Carlo model to design a template that favors growth of hexagonal particle arrays. We experimentally demonstrate growth of a highly stable array of 50 gold particles with 200 nm diameter, spaced by 1.1 μm.
DOI
We use an optical analogue of epitaxial growth to assemble gold nanoparticles into 2D arrays. Particles are attracted to a growth template via optical forces and interact through optical binding. Competition between effects determines the final particle arrangements. We use a Monte Carlo model to design a template that favors growth of hexagonal particle arrays. We experimentally demonstrate growth of a highly stable array of 50 gold particles with 200 nm diameter, spaced by 1.1 μm.
DOI
Calibration of optical tweezers with non-spherical probes via high-resolution detection of Brownian motion
A. Butykai, F.M. Mor, R. Gaál, P. Domínguez-García, L. Forró, S. Jeney
Optical tweezers are commonly used and powerful tools to perform force measurements on the piconewton scale and to detect nanometer-scaled displacements. However, the precision of these instruments relies to a great extent on the accuracy of the calibration method. A well-known calibration procedure is to record the stochastic motion of the trapped particle and compare its statistical behavior with the theory of the Brownian motion in a harmonic potential. Here we present an interactive calibration software which allows for the simultaneous fitting of three different statistical observables (power spectral density, mean square displacement and velocity autocorrelation function) calculated from the trajectory of the probe to enhance fitting accuracy. The fitted theory involves the hydrodynamic interactions experimentally observable at high sampling rates. Furthermore, a qualitative extension is included in our model to handle the thermal fluctuations in the orientation of optically trapped asymmetric objects. The presented calibration methodology requires no prior knowledge of the bead size and can be applied to non-spherical probes as well. The software was validated on synthetic and experimental data.
DOI
Optical tweezers are commonly used and powerful tools to perform force measurements on the piconewton scale and to detect nanometer-scaled displacements. However, the precision of these instruments relies to a great extent on the accuracy of the calibration method. A well-known calibration procedure is to record the stochastic motion of the trapped particle and compare its statistical behavior with the theory of the Brownian motion in a harmonic potential. Here we present an interactive calibration software which allows for the simultaneous fitting of three different statistical observables (power spectral density, mean square displacement and velocity autocorrelation function) calculated from the trajectory of the probe to enhance fitting accuracy. The fitted theory involves the hydrodynamic interactions experimentally observable at high sampling rates. Furthermore, a qualitative extension is included in our model to handle the thermal fluctuations in the orientation of optically trapped asymmetric objects. The presented calibration methodology requires no prior knowledge of the bead size and can be applied to non-spherical probes as well. The software was validated on synthetic and experimental data.
DOI
Tuesday, August 4, 2015
Reprint of: Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria
S. Suresh, J. Spatz, J.P. Mills, A. Micoulet, M. Dao, C.T. Lim, M. Beil, T. Seufferlein
We investigate connections between single-cell mechanical properties and subcellular structural reorganization from biochemical factors in the context of two distinctly different human diseases: gastrointestinal tumor and malaria. Although the cell lineages and the biochemical links to pathogenesis are vastly different in these two cases, we compare and contrast chemomechanical pathways whereby intracellular structural rearrangements lead to global changes in mechanical deformability of the cell. This single-cell biomechanical response, in turn, seems to mediate cell mobility and thereby facilitates disease progression in situations where the elastic modulus increases or decreases due to membrane or cytoskeleton reorganization. We first present new experiments on elastic response and energy dissipation under repeated tensile loading of epithelial pancreatic cancer cells in force- or displacement-control. Energy dissipation from repeated stretching significantly increases and the cell’s elastic modulus decreases after treatment of Panc-1 pancreatic cancer cells with sphingosylphosphorylcholine (SPC), a bioactive lipid that influences cancer metastasis. When the cell is treated instead with lysophosphatidic acid, which facilitates actin stress fiber formation, neither energy dissipation nor modulus is noticeably affected. Integrating recent studies with our new observations, we ascribe these trends to possible SPC-induced reorganization primarily of keratin network to perinuclear region of cell; the intermediate filament fraction of the cytoskeleton thus appears to dominate deformability of the epithelial cell. Possible consequences of these results to cell mobility and cancer metastasis are postulated. We then turn attention to progressive changes in mechanical properties of the human red blood cell (RBC) infected with the malaria parasite Plasmodium falciparum. We present, for the first time, continuous force–displacement curves obtained from in-vitro deformation of RBC with optical tweezers for different intracellular developmental stages of parasite. The shear modulus of RBC is found to increase up to 10-fold during parasite development, which is a noticeably greater effect than that from prior estimates. By integrating our new experimental results with published literature on deformability of Plasmodium-harbouring RBC, we examine the biochemical conditions mediating increases or decreases in modulus, and their implications for disease progression. Some general perspectives on connections among structure, single-cell mechanical properties and biological responses associated with pathogenic processes are also provided in the context of the two diseases considered in this work.
DOI
We investigate connections between single-cell mechanical properties and subcellular structural reorganization from biochemical factors in the context of two distinctly different human diseases: gastrointestinal tumor and malaria. Although the cell lineages and the biochemical links to pathogenesis are vastly different in these two cases, we compare and contrast chemomechanical pathways whereby intracellular structural rearrangements lead to global changes in mechanical deformability of the cell. This single-cell biomechanical response, in turn, seems to mediate cell mobility and thereby facilitates disease progression in situations where the elastic modulus increases or decreases due to membrane or cytoskeleton reorganization. We first present new experiments on elastic response and energy dissipation under repeated tensile loading of epithelial pancreatic cancer cells in force- or displacement-control. Energy dissipation from repeated stretching significantly increases and the cell’s elastic modulus decreases after treatment of Panc-1 pancreatic cancer cells with sphingosylphosphorylcholine (SPC), a bioactive lipid that influences cancer metastasis. When the cell is treated instead with lysophosphatidic acid, which facilitates actin stress fiber formation, neither energy dissipation nor modulus is noticeably affected. Integrating recent studies with our new observations, we ascribe these trends to possible SPC-induced reorganization primarily of keratin network to perinuclear region of cell; the intermediate filament fraction of the cytoskeleton thus appears to dominate deformability of the epithelial cell. Possible consequences of these results to cell mobility and cancer metastasis are postulated. We then turn attention to progressive changes in mechanical properties of the human red blood cell (RBC) infected with the malaria parasite Plasmodium falciparum. We present, for the first time, continuous force–displacement curves obtained from in-vitro deformation of RBC with optical tweezers for different intracellular developmental stages of parasite. The shear modulus of RBC is found to increase up to 10-fold during parasite development, which is a noticeably greater effect than that from prior estimates. By integrating our new experimental results with published literature on deformability of Plasmodium-harbouring RBC, we examine the biochemical conditions mediating increases or decreases in modulus, and their implications for disease progression. Some general perspectives on connections among structure, single-cell mechanical properties and biological responses associated with pathogenic processes are also provided in the context of the two diseases considered in this work.
DOI
Dissection of Axial-Pore Loop Function during Unfolding and Translocation by a AAA+ Proteolytic Machine
Ohad Iosefson, Adrian O. Olivares, Tania A. Baker, Robert T. Sauer
In the axial channels of ClpX and related hexameric AAA+ protein-remodeling rings, the pore-1 loops are thought to play important roles in engaging, mechanically unfolding, and translocating protein substrates. How these loops perform these functions and whether they also prevent substrate dissociation to ensure processive degradation by AAA+ proteases are open questions. Using ClpX pore-1-loop variants, single-molecule force spectroscopy, and ensemble assays, we find that the six pore-1 loops function synchronously to grip and unfold protein substrates during a power stroke but are not important in preventing substrate slipping between power strokes. The importance of grip strength is task dependent. ClpX variants with multiple mutant pore-1 loops translocate substrates as well as the wild-type enzyme against a resisting force but show unfolding defects and a higher frequency of substrate release. These problems are magnified for more mechanically stable target proteins, supporting a threshold model of substrate gripping.
DOI
In the axial channels of ClpX and related hexameric AAA+ protein-remodeling rings, the pore-1 loops are thought to play important roles in engaging, mechanically unfolding, and translocating protein substrates. How these loops perform these functions and whether they also prevent substrate dissociation to ensure processive degradation by AAA+ proteases are open questions. Using ClpX pore-1-loop variants, single-molecule force spectroscopy, and ensemble assays, we find that the six pore-1 loops function synchronously to grip and unfold protein substrates during a power stroke but are not important in preventing substrate slipping between power strokes. The importance of grip strength is task dependent. ClpX variants with multiple mutant pore-1 loops translocate substrates as well as the wild-type enzyme against a resisting force but show unfolding defects and a higher frequency of substrate release. These problems are magnified for more mechanically stable target proteins, supporting a threshold model of substrate gripping.
DOI
A Simplified Configuration for Obtaining a Variable Focal Spot for Optical Tweezers
Yanghui Li, Zhaoyi Shi, Le Wang
We propose a simplified setup to generate a tunable focal spot. To make a flexible conversion between a doughnut focal spot and a peak-centered one, we introduce a dual ramp phase mask and realize the focal spot manipulation by changing the modulation coefficient. In this way, this configuration enables the trapping and controlling of the particles with diverse refractive index. Further we present an analysis of the system performance and investigate explicitly some key aspects that may affect the quality of the focal spot generated, including the polarization purity and the accuracy of the phase modulation.
DOI
We propose a simplified setup to generate a tunable focal spot. To make a flexible conversion between a doughnut focal spot and a peak-centered one, we introduce a dual ramp phase mask and realize the focal spot manipulation by changing the modulation coefficient. In this way, this configuration enables the trapping and controlling of the particles with diverse refractive index. Further we present an analysis of the system performance and investigate explicitly some key aspects that may affect the quality of the focal spot generated, including the polarization purity and the accuracy of the phase modulation.
DOI
Helical tractor beam: analytical solution of Rayleigh particle dynamics
Luis Carretero, Pablo Acebal, Celia Garcia, and Salvador Blaya
We analyze particle dynamics in an optical force field generated by helical tractor beams obtained by the interference of a cylindrical beam with a topological charge and a co-propagating temporally de-phased plane wave. We show that, for standard experimental conditions, it is possible to obtain analytical solutions for the trajectories of particles in such force field by using of some approximations. These solutions show that, in contrast to other tractor beams described before, the intensity becomes a key parameter for the control of particle trajectories. Therefore, by tuning the intensity value the particle can describe helical trajectories upstream and downstream, a circular trajectory in a fixed plane, or a linear displacement in the propagation direction. The approximated analytical solutions show good agreement to the corresponding numerical solutions of the exact dynamical differential equations.
DOI
We analyze particle dynamics in an optical force field generated by helical tractor beams obtained by the interference of a cylindrical beam with a topological charge and a co-propagating temporally de-phased plane wave. We show that, for standard experimental conditions, it is possible to obtain analytical solutions for the trajectories of particles in such force field by using of some approximations. These solutions show that, in contrast to other tractor beams described before, the intensity becomes a key parameter for the control of particle trajectories. Therefore, by tuning the intensity value the particle can describe helical trajectories upstream and downstream, a circular trajectory in a fixed plane, or a linear displacement in the propagation direction. The approximated analytical solutions show good agreement to the corresponding numerical solutions of the exact dynamical differential equations.
DOI
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