Silke R. Kirchner, Spas Nedev, Sol Carretero-Palacios, Andreas Mader, Madeleine Opitz, Theobald Lohmüller and Jochen Feldmann
We report on a highly sensitive approach to measure and quantify the time dependent changes of the flow generated by the flagella bundle rotation of single bacterial cells. This is achieved by observing the interactions between a silica particle and a bacterium, which are both trapped next to each other in a dual beam optical tweezer. In this configuration, the particle serves as a sensitive detector where the fast-Fourier analysis of the particle trajectory renders, it possible to access information about changes of bacterial activity.
DOI
Wednesday, March 12, 2014
Accurate position tracking of optically trapped live cells
Niall McAlinden, David G. Glass, Owain R. Millington, and Amanda J. Wright
Optical trapping is a powerful tool in Life Science research and is becoming common place in many microscopy laboratories and facilities. There is a growing need to directly trap the cells of interest rather than introduce beads to the sample that can affect the fundamental biological functions of the sample and impact on the very properties the user wishes to observe and measure. However, instabilities while tracking large inhomogeneous objects, such as cells, can make tracking position, calibrating trap strength and making reliable measurements challenging. These instabilities often manifest themselves as cell roll or re-orientation and can occur as a result of viscous drag forces and thermal convection, as well as spontaneously due to Brownian forces. In this paper we discuss and mathematically model the cause of this roll and present several experimental approaches for tackling these issues, including using a novel beam profile consisting of three closely spaced traps and tracking a trapped object by analysing fluorescence images. The approaches presented here trap T cells which form part of the adaptive immune response system, but in principle can be applied to a wide range of samples where the size and inhomogeneous nature of the trapped object can hinder particle tracking experiments.
DOI
Optical trapping is a powerful tool in Life Science research and is becoming common place in many microscopy laboratories and facilities. There is a growing need to directly trap the cells of interest rather than introduce beads to the sample that can affect the fundamental biological functions of the sample and impact on the very properties the user wishes to observe and measure. However, instabilities while tracking large inhomogeneous objects, such as cells, can make tracking position, calibrating trap strength and making reliable measurements challenging. These instabilities often manifest themselves as cell roll or re-orientation and can occur as a result of viscous drag forces and thermal convection, as well as spontaneously due to Brownian forces. In this paper we discuss and mathematically model the cause of this roll and present several experimental approaches for tackling these issues, including using a novel beam profile consisting of three closely spaced traps and tracking a trapped object by analysing fluorescence images. The approaches presented here trap T cells which form part of the adaptive immune response system, but in principle can be applied to a wide range of samples where the size and inhomogeneous nature of the trapped object can hinder particle tracking experiments.
DOI
Monday, March 10, 2014
Three-dimensional manipulation with scanning near-field optical nanotweezers
J. Berthelot, S. S. Aćimović, M. L. Juan, M. P. Kreuzer, J. Renger & R. Quidant
Recent advances in nanotechnologies have prompted the need for tools to accurately and non-invasively manipulate individual nano-objects1. Among the possible strategies, optical forces have been predicted to provide researchers with nano-optical tweezers capable of trapping a specimen and moving it in three dimensions2, 3, 4. In practice, however, the combination of weak optical forces and photothermal issues has thus far prevented their experimental realization. Here, we demonstrate the first three-dimensional optical manipulation of single 50 nm dielectric objects with near-field nanotweezers. The nano-optical trap is built by engineering a bowtie plasmonic aperture at the extremity of a tapered metal-coated optical fibre. Both the trapping operation and monitoring are performed through the optical fibre, making these nanotweezers totally autonomous and free of bulky optical elements. The achieved trapping performances allow for the trapped specimen to be moved over tens of micrometres over a period of several minutes with very low in-trap intensities. This non-invasive approach is foreseen to open new horizons in nanosciences by offering an unprecedented level of control of nanosized objects, including heat-sensitive biospecimens.
Recent advances in nanotechnologies have prompted the need for tools to accurately and non-invasively manipulate individual nano-objects1. Among the possible strategies, optical forces have been predicted to provide researchers with nano-optical tweezers capable of trapping a specimen and moving it in three dimensions2, 3, 4. In practice, however, the combination of weak optical forces and photothermal issues has thus far prevented their experimental realization. Here, we demonstrate the first three-dimensional optical manipulation of single 50 nm dielectric objects with near-field nanotweezers. The nano-optical trap is built by engineering a bowtie plasmonic aperture at the extremity of a tapered metal-coated optical fibre. Both the trapping operation and monitoring are performed through the optical fibre, making these nanotweezers totally autonomous and free of bulky optical elements. The achieved trapping performances allow for the trapped specimen to be moved over tens of micrometres over a period of several minutes with very low in-trap intensities. This non-invasive approach is foreseen to open new horizons in nanosciences by offering an unprecedented level of control of nanosized objects, including heat-sensitive biospecimens.
Rotational Dynamics of Optically Trapped Human Spermatozoa
Elavarasan Subramani, Himanish Basu, Shyam Thangaraju, Sucheta Dandekar, Deepak Mathur, and Koel Chaudhury
Introduction. Optical trapping is a laser-based method for probing the physiological and mechanical properties of cells in a noninvasive manner. As sperm motility is an important criterion for assessing the male fertility potential, this technique is used to study sperm cell motility behavior and rotational dynamics. Methods and Patients. An integrated optical system with near-infrared laser beam has been used to analyze rotational dynamics of live sperm cells from oligozoospermic and asthenozoospermic cases and compared with controls. Results. The linear, translational motion of the sperm is converted into rotational motion on being optically trapped, without causing any adverse effect on spermatozoa. The rotational speed of sperm cells from infertile men is observed to be significantly less as compared to controls. Conclusions. Distinguishing normal and abnormal sperm cells on the basis of beat frequency above 5.6 Hz may be an important step in modern reproductive biology to sort and select good quality spermatozoa. The application of laser-assisted technique in biology has the potential to be a valuable tool for assessment of sperm fertilization capacity for improving assisted reproductive technology.
DOI
Introduction. Optical trapping is a laser-based method for probing the physiological and mechanical properties of cells in a noninvasive manner. As sperm motility is an important criterion for assessing the male fertility potential, this technique is used to study sperm cell motility behavior and rotational dynamics. Methods and Patients. An integrated optical system with near-infrared laser beam has been used to analyze rotational dynamics of live sperm cells from oligozoospermic and asthenozoospermic cases and compared with controls. Results. The linear, translational motion of the sperm is converted into rotational motion on being optically trapped, without causing any adverse effect on spermatozoa. The rotational speed of sperm cells from infertile men is observed to be significantly less as compared to controls. Conclusions. Distinguishing normal and abnormal sperm cells on the basis of beat frequency above 5.6 Hz may be an important step in modern reproductive biology to sort and select good quality spermatozoa. The application of laser-assisted technique in biology has the potential to be a valuable tool for assessment of sperm fertilization capacity for improving assisted reproductive technology.
DOI
Nano-opto-mechanical effects in plasmonic waveguides
Alexander S. Shalin, Pavel Ginzburg, Pavel A. Belov, Yuri S. Kivshar, Anatoly V. Zayats
In order to achieve interaction between light beams, a mediating material object is required. Nonlinear materials are commonly used for this purpose. Here a new approach to control light with light, based on a nano-opto-mechanical system integrated in a plasmonic waveguide is proposed. Optomechanics of a free-floating resonant nanoparticle in a subwavelength plasmonic V-groove waveguide is studied. It is shown that nanoparticle auto-oscillations in the waveguide induced by a control light result in the periodic modulation of a transmitted plasmonic signal. The modulation depth of 10% per single nanoparticle of 25 nm diameter with the clock frequencies of tens of MHz and the record low energy-per-bit energies of 10−18 J is observed. The frequency of auto-oscillations depends on the intensity of the continuous control light. The efficient modulation and deep-subwavelength dimensions make this nano-optomechanical system of significant interest for opto-electronic and opto-fluidic technologies.
DOI
In order to achieve interaction between light beams, a mediating material object is required. Nonlinear materials are commonly used for this purpose. Here a new approach to control light with light, based on a nano-opto-mechanical system integrated in a plasmonic waveguide is proposed. Optomechanics of a free-floating resonant nanoparticle in a subwavelength plasmonic V-groove waveguide is studied. It is shown that nanoparticle auto-oscillations in the waveguide induced by a control light result in the periodic modulation of a transmitted plasmonic signal. The modulation depth of 10% per single nanoparticle of 25 nm diameter with the clock frequencies of tens of MHz and the record low energy-per-bit energies of 10−18 J is observed. The frequency of auto-oscillations depends on the intensity of the continuous control light. The efficient modulation and deep-subwavelength dimensions make this nano-optomechanical system of significant interest for opto-electronic and opto-fluidic technologies.
DOI
Improved Laser Manipulation for On-chip Fabricated Microstructures Based on Solution Replacement and Its Application in Single Cell Analysis
Tao Yue, Masahiro Nakajima, Masaru Takeuchi and Toshio Fukuda
In this paper, we present the fabrication and assembly of microstructures inside a microfluidic device based on a photocrosslinkable resin and optical tweezers. We also report a method of solution replacement inside the microfluidic channel in order to improve the manipulation performance and apply the assembled microstructures for single cell cultivation. By the illumination of patterned ultraviolet (UV) through a microscope, microstructures of arbitrary shape were fabricated by the photocrosslinkable resin inside a microfluidic channel. Based on the microfluidic channel with both glass and polydimethylsiloxane (PDMS) surfaces, immovable and movable microstructures were fabricated and manipulated. The microstructures were fabricated at the desired places and manipulated by the optical tweezers. A rotational microstructure including a microgear and a rotation axis was assembled and rotated in demonstrating this technique. The improved laser manipulation of microstructures was achieved based on the on-chip solution replacement method. The manipulation speed of the microstructures increased when the viscosity of the solvent decreased. The movement efficiency of the fabricated microstructures inside the lower viscosity solvent was evaluated and compared with those microstructures inside the former high viscosity solvent. A novel cell cage was fabricated and the cultivation of a single yeast cell (w303) was demonstrated in the cell cage, inside the microfluidic device.
DOI
In this paper, we present the fabrication and assembly of microstructures inside a microfluidic device based on a photocrosslinkable resin and optical tweezers. We also report a method of solution replacement inside the microfluidic channel in order to improve the manipulation performance and apply the assembled microstructures for single cell cultivation. By the illumination of patterned ultraviolet (UV) through a microscope, microstructures of arbitrary shape were fabricated by the photocrosslinkable resin inside a microfluidic channel. Based on the microfluidic channel with both glass and polydimethylsiloxane (PDMS) surfaces, immovable and movable microstructures were fabricated and manipulated. The microstructures were fabricated at the desired places and manipulated by the optical tweezers. A rotational microstructure including a microgear and a rotation axis was assembled and rotated in demonstrating this technique. The improved laser manipulation of microstructures was achieved based on the on-chip solution replacement method. The manipulation speed of the microstructures increased when the viscosity of the solvent decreased. The movement efficiency of the fabricated microstructures inside the lower viscosity solvent was evaluated and compared with those microstructures inside the former high viscosity solvent. A novel cell cage was fabricated and the cultivation of a single yeast cell (w303) was demonstrated in the cell cage, inside the microfluidic device.
DOI
Wednesday, March 5, 2014
Unraveling secrets of telomeres: One molecule at a time
Jiangguo Lin, Parminder Kaur, Preston Countryman, Patricia L. Opresko, Hong Wang
Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure–function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins.
DOI
Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure–function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins.
DOI
Impact of heating on passive and active biomechanics of suspended cells
C. J. Chan, G. Whyte, L. Boyde, G. Salbreux and J. Guck
A cell is a complex material whose mechanical properties are essential for its normal functions. Heating can have a dramatic effect on these mechanical properties, similar to its impact on the dynamics of artificial polymer networks. We investigated such mechanical changes by the use of a microfluidic optical stretcher, which allowed us to probe cell mechanics when the cells were subjected to different heating conditions at different time scales. We find that HL60/S4 myeloid precursor cells become mechanically more compliant and fluid-like when subjected to either a sudden laser-induced temperature increase or prolonged exposure to higher ambient temperature. Above a critical temperature of 52 ± 1°C, we observed active cell contraction, which was strongly correlated with calcium influx through temperature-sensitive transient receptor potential vanilloid 2 (TRPV2) ion channels, followed by a subsequent expansion in cell volume. The change from passive to active cellular response can be effectively described by a mechanical model incorporating both active stress and viscoelastic components. Our work highlights the role of TRPV2 in regulating the thermomechanical response of cells. It also offers insights into how cortical tension and osmotic pressure govern cell mechanics and regulate cell-shape changes in response to heat and mechanical stress.
DOI
A cell is a complex material whose mechanical properties are essential for its normal functions. Heating can have a dramatic effect on these mechanical properties, similar to its impact on the dynamics of artificial polymer networks. We investigated such mechanical changes by the use of a microfluidic optical stretcher, which allowed us to probe cell mechanics when the cells were subjected to different heating conditions at different time scales. We find that HL60/S4 myeloid precursor cells become mechanically more compliant and fluid-like when subjected to either a sudden laser-induced temperature increase or prolonged exposure to higher ambient temperature. Above a critical temperature of 52 ± 1°C, we observed active cell contraction, which was strongly correlated with calcium influx through temperature-sensitive transient receptor potential vanilloid 2 (TRPV2) ion channels, followed by a subsequent expansion in cell volume. The change from passive to active cellular response can be effectively described by a mechanical model incorporating both active stress and viscoelastic components. Our work highlights the role of TRPV2 in regulating the thermomechanical response of cells. It also offers insights into how cortical tension and osmotic pressure govern cell mechanics and regulate cell-shape changes in response to heat and mechanical stress.
DOI
Torque Measurement at the Single-Molecule Level
Scott Forth, Maxim Y. Sheinin, James Inman, and Michelle D. Wang
Methods for exerting and measuring forces on single molecules have revolutionized the study of the physics of biology. However, it is often the case that biological processes involve rotation or torque generation, and these parameters have been more difficult to access experimentally. Recent advances in the single-molecule field have led to the development of techniques that add the capability of torque measurement. By combining force, displacement, torque, and rotational data, a more comprehensive description of the mechanics of a biomolecule can be achieved. In this review, we highlight a number of biological processes for which torque plays a key mechanical role. We describe the various techniques that have been developed to directly probe the torque experienced by a single molecule, and detail a variety of measurements made to date using these new technologies. We conclude by discussing a number of open questions and propose systems of study that would be well suited for analysis with torsional measurement techniques.
DOI
Methods for exerting and measuring forces on single molecules have revolutionized the study of the physics of biology. However, it is often the case that biological processes involve rotation or torque generation, and these parameters have been more difficult to access experimentally. Recent advances in the single-molecule field have led to the development of techniques that add the capability of torque measurement. By combining force, displacement, torque, and rotational data, a more comprehensive description of the mechanics of a biomolecule can be achieved. In this review, we highlight a number of biological processes for which torque plays a key mechanical role. We describe the various techniques that have been developed to directly probe the torque experienced by a single molecule, and detail a variety of measurements made to date using these new technologies. We conclude by discussing a number of open questions and propose systems of study that would be well suited for analysis with torsional measurement techniques.
DOI
Two steps forward, one step back: Determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers
Maria Spies
XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5′–3′ polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interactions within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein–DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).
DOI
XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5′–3′ polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interactions within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein–DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).
DOI
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