The Experts below are selected from a list of 4053 Experts worldwide ranked by ideXlab platform
Sabrina Leslie - One of the best experts on this subject based on the ideXlab platform.
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Single-cell analysis for drug development using Convex Lens-induced confinement imaging.
BioTechniques, 2019Co-Authors: Ndeye Khady Thiombane, Daniel Berard, Sabrina Leslie, Nicolas Coutin, Radin Tahvildari, Corey NislowAbstract:New technologies have powered rapid advances in cellular imaging, genomics and phenotypic analysis in life sciences. However, most of these methods operate at sample population levels and provide statistical averages of aggregated data that fail to capture single-cell heterogeneity, complicating drug discovery and development. Here we demonstrate a new single-cell approach based on Convex Lens-induced confinement (CLiC) microscopy. We validated CLiC on yeast cells, demonstrating subcellular localization with an enhanced signal-to-noise and fluorescent signal detection sensitivity compared with traditional imaging. In the live-cell CLiC assay, cellular proliferation times were consistent with flask culture. Using methotrexate, we provide drug response data showing a fivefold cell size increase following drug exposure. Taken together, CLiC enables high-quality imaging of single-cell drug response and proliferation for extended observation periods.
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Development of a platform for single cell genomics using Convex Lens-induced confinement.
Lab on a chip, 2015Co-Authors: Sara Mahshid, Daniel Berard, Mohammed Jalal Ahamed, Robert Sladek, Sabrina Leslie, Susan Amin, Walter ReisnerAbstract:We demonstrate a lab-on-a-chip that combines micro/nano-fabricated features with a Convex Lens-Induced Confinement (CLIC) device for the in situ analysis of single cells. A complete cycle of single cell analysis was achieved that includes: cell trapping, cell isolation, lysis, protein digestion, genomic DNA extraction and on-chip genomic DNA linearization. The ability to dynamically alter the flow-cell dimensions using the CLIC method was coupled with a flow-control mechanism for achieving efficient cell trapping, buffer exchange, and loading of long DNA molecules into nanofluidic arrays. Finite element simulation of fluid flow gives rise to optimized design parameters for overcoming the high hydraulic resistance present in the micro/nano-confinement region. By tuning design parameters such as the pressure gradient and CLIC confinement, an efficient on-chip single cell analysis protocol can be obtained. We demonstrate that we can extract Mbp long genomic DNA molecules from a single human lybphoblastoid cell and stretch these molecules in the nanochannels for optical interrogation.
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Convex Lens-induced nanoscale templating
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Daniel Berard, François Michaud, Sara Mahshid, Mohammed Jalal Ahamed, Christopher M. J. Mcfaul, Jason S. Leith, Pierre Bérubé, Robert Sladek, Walter Reisner, Sabrina LeslieAbstract:We demonstrate a new platform, Convex Lens-induced nanoscale templating (CLINT), for dynamic manipulation and trapping of single DNA molecules. In the CLINT technique, the curved surface of a Convex Lens is used to deform a flexible coverslip above a substrate containing embedded nanotopography, creating a nanoscale gap that can be adjusted during an experiment to confine molecules within the embedded nanostructures. Critically, CLINT has the capability of transforming a macroscale flow cell into a nanofluidic device without the need for permanent direct bonding, thus simplifying sample loading, providing greater accessibility of the surface for functionalization, and enabling dynamic manipulation of confinement during device operation. Moreover, as DNA molecules present in the gap are driven into the embedded topography from above, CLINT eliminates the need for the high pressures or electric fields required to load DNA into direct-bonded nanofluidic devices. To demonstrate the versatility of CLINT, we confine DNA to nanogroove and nanopit structures, demonstrating DNA nanochannel-based stretching, denaturation mapping, and partitioning/trapping of single molecules in multiple embedded cavities. In particular, using ionic strengths that are in line with typical biological buffers, we have successfully extended DNA in sub–30-nm nanochannels, achieving high stretching (90%) that is in good agreement with Odijk deflection theory, and we have mapped genomic features using denaturation analysis.
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single molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero mode waveguides zmws and Convex Lens induced confinement clic
Optics Express, 2013Co-Authors: Mary Williard Elting, Christopher M. J. Mcfaul, Jason S. Leith, Sabrina Leslie, Adam E. Cohen, Stirling L Churchman, Jonas Korlach, Michael J Levene, James A SpudichAbstract:Resolving single fluorescent molecules in the presence of high fluorophore concentrations remains a challenge in single-molecule biophysics that limits our understanding of weak molecular interactions. Total internal reflection fluorescence (TIRF) imaging, the workhorse of single-molecule fluorescence microscopy, enables experiments at concentrations up to about 100 nM, but many biological interactions have considerably weaker affinities, and thus require at least one species to be at micromolar or higher concentration. Current alternatives to TIRF often require three-dimensional confinement, and thus can be problematic for extended substrates, such as cytoskeletal filaments. To address this challenge, we have demonstrated and applied two new single-molecule fluorescence microscopy techniques, linear zero-mode waveguides (ZMWs) and Convex Lens induced confinement (CLIC), for imaging the processive motion of molecular motors myosin V and VI along actin filaments. Both technologies will allow imaging in the presence of higher fluorophore concentrations than TIRF microscopy. They will enable new biophysical measurements of a wide range of processive molecular motors that move along filamentous tracks, such as other myosins, dynein, and kinesin. A particularly salient application of these technologies will be to examine chemomechanical coupling by directly imaging fluorescent nucleotide molecules interacting with processive motors as they traverse their actin or microtubule tracks.
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Precision platform for Convex Lens-induced confinement microscopy.
The Review of scientific instruments, 2013Co-Authors: Daniel Berard, François Michaud, Christopher M. J. Mcfaul, Jason S. Leith, Adriel Arsenault, Sabrina LeslieAbstract:We present the conception, fabrication, and demonstration of a versatile, computer-controlled microscopy device which transforms a standard inverted fluorescence microscope into a precision single-molecule imaging station. The device uses the principle of Convex Lens-induced confinement [S. R. Leslie, A. P. Fields, and A. E. Cohen, Anal. Chem. 82, 6224 (2010)], which employs a tunable imaging chamber to enhance background rejection and extend diffusion-limited observation periods. Using nanopositioning stages, this device achieves repeatable and dynamic control over the geometry of the sample chamber on scales as small as the size of individual molecules, enabling regulation of their configurations and dynamics. Using microfluidics, this device enables serial insertion as well as sample recovery, facilitating temporally controlled, high-throughput measurements of multiple reagents. We report on the simulation and experimental characterization of this tunable chamber geometry, and its influence upon the diffusion and conformations of DNA molecules over extended observation periods. This new microscopy platform has the potential to capture, probe, and influence the configurations of single molecules, with dramatically improved imaging conditions in comparison to existing technologies. These capabilities are of immediate interest to a wide range of research and industry sectors in biotechnology, biophysics, materials, and chemistry.
Salvatore Torquato - One of the best experts on this subject based on the ideXlab platform.
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Hard Convex Lens-shaped particles: Characterization of dense disordered packings.
Physical review. E, 2019Co-Authors: Giorgio Cinacchi, Salvatore TorquatoAbstract:Among the family of hard Convex Lens-shaped particles (Lenses), the one with aspect ratio equal to 2/3 is "optimal" in the sense that the maximally random jammed (MRJ) packings of such Lenses achieve the highest packing fraction ϕ_{MRJ}≃0.73 [G. Cinacchi and S. Torquato, Soft Matter 14, 8205 (2018)1744-683X10.1039/C8SM01519H]. This value is only a few percent lower than ϕ_{DKP}=0.76210⋯, the packing fraction of the corresponding densest-known crystalline (degenerate) packings [G. Cinacchi and S. Torquato, J. Chem. Phys. 143, 224506 (2015)JCPSA60021-960610.1063/1.4936938]. By exploiting the appreciably reduced propensity that a system of such optimal Lenses has to positionally and orientationally order, disordered packings of them are progressively generated by a Monte Carlo method-based procedure from the dilute equilibrium isotropic fluid phase to the dense nonequilibrium MRJ state. This allows us to closely monitor how the (micro)structure of these packings changes in the process of formation of the MRJ state. The gradual changes undergone by the many structural descriptors calculated here can coherently and consistently be traced back to the gradual increase in contacts between the hard particles until the isostatic mean value of ten contact neighbors per Lens is reached at the effectively hyperuniform MRJ state. Compared to the MRJ state of hard spheres, the MRJ state of such optimal Lenses is denser (less porous), more disordered, and rattler-free. This set of characteristics makes them good glass formers. It is possible that this conclusion may also hold for other hard Convex uniaxial particles with a correspondingly similar aspect ratio, be they oblate or prolate, and that, by using suitable biaxial variants of them, that set of characteristics might further improve.
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Hard Convex Lens-shaped particles: metastable, glassy and jammed states.
Soft matter, 2018Co-Authors: Giorgio Cinacchi, Salvatore TorquatoAbstract:We generate and study dense positionally and/or orientationally disordered, including jammed, monodisperse packings of hard Convex Lens-shaped particles (Lenses). Relatively dense isotropic fluid configurations of Lenses of various aspect ratios are slowly compressed via a Monte Carlo method based procedure. Under this compression protocol, while ‘flat’ Lenses form a nematic fluid phase (where particles are positionally disordered but orientationally ordered) and ‘globular’ Lenses form a plastic solid phase (where particles are positionally ordered but orientationally disordered), ‘intermediate’, neither ‘flat’ nor ‘globular’, Lenses do not form either mesophase. In general, a crystal solid phase (where particles are both positionally and orientationally ordered) does not spontaneously form during lengthy numerical simulation runs. In correspondence to those volume fractions at which a transition to the crystal solid phase would occur in equilibrium, a ‘downturn’ is observed in the inverse compressibility factor versus volume fraction curve beyond which this curve behaves essentially linearly. This allows us to estimate the volume fraction at jamming of the dense non-crystalline packings so generated. These packings are nematic for ‘flat’ Lenses and plastic for ‘globular’ Lenses, while they are robustly isotropic for ‘intermediate’ Lenses, as confirmed by the calculation of the τ order metric, among other quantities. The structure factors S(k) of the corresponding jammed states tend to zero as the wavenumber k goes to zero, indicating they are effectively hyperuniform (i.e., their infinite-wavelength density fluctuations are anomalously suppressed). Among all possible Lens shapes, ‘intermediate’ Lenses with aspect ratio around 2/3 are special because they are those that reach the highest volume fractions at jamming while being positionally and orientationally disordered and these volume fractions are as high as those reached by nematic jammed states of ‘flat’ Lenses and plastic jammed states of ‘globular’ Lenses. All of their attributes, taken together, make such ‘intermediate’ Lens packings particularly good glass-forming materials.
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Hard Convex Lens-shaped particles: Densest-known packings and phase behavior
The Journal of chemical physics, 2015Co-Authors: Giorgio Cinacchi, Salvatore TorquatoAbstract:By using theoretical methods and Monte Carlo simulations, this work investigates dense ordered packings and equilibrium phase behavior (from the low-density isotropic fluid regime to the high-density crystalline solid regime) of monodisperse systems of hard Convex Lens-shaped particles as defined by the volume common to two intersecting congruent spheres. We show that, while the overall similarity of their shape to that of hard oblate ellipsoids is reflected in a qualitatively similar phase diagram, differences are more pronounced in the high-density crystal phase up to the densest-known packings determined here. In contrast to those non-(Bravais)-lattice two-particle basis crystals that are the densest-known packings of hard (oblate) ellipsoids, hard Convex Lens-shaped particles pack more densely in two types of degenerate crystalline structures: (i) non-(Bravais)-lattice two-particle basis body-centered-orthorhombic-like crystals and (ii) (Bravais) lattice monoclinic crystals. By stacking at will, regularly or irregularly, laminae of these two crystals, infinitely degenerate, generally non-periodic in the stacking direction, dense packings can be constructed that are consistent with recent organizing principles. While deferring the assessment of which of these dense ordered structures is thermodynamically stable in the high-density crystalline solid regime, the degeneracy of their densest-known packings strongly suggests that colloidal Convex Lens-shaped particles could be better glass formers than colloidal spheres because of the additional rotational degrees of freedom.
Daniel Berard - One of the best experts on this subject based on the ideXlab platform.
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Single-cell analysis for drug development using Convex Lens-induced confinement imaging.
BioTechniques, 2019Co-Authors: Ndeye Khady Thiombane, Daniel Berard, Sabrina Leslie, Nicolas Coutin, Radin Tahvildari, Corey NislowAbstract:New technologies have powered rapid advances in cellular imaging, genomics and phenotypic analysis in life sciences. However, most of these methods operate at sample population levels and provide statistical averages of aggregated data that fail to capture single-cell heterogeneity, complicating drug discovery and development. Here we demonstrate a new single-cell approach based on Convex Lens-induced confinement (CLiC) microscopy. We validated CLiC on yeast cells, demonstrating subcellular localization with an enhanced signal-to-noise and fluorescent signal detection sensitivity compared with traditional imaging. In the live-cell CLiC assay, cellular proliferation times were consistent with flask culture. Using methotrexate, we provide drug response data showing a fivefold cell size increase following drug exposure. Taken together, CLiC enables high-quality imaging of single-cell drug response and proliferation for extended observation periods.
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Development of a platform for single cell genomics using Convex Lens-induced confinement.
Lab on a chip, 2015Co-Authors: Sara Mahshid, Daniel Berard, Mohammed Jalal Ahamed, Robert Sladek, Sabrina Leslie, Susan Amin, Walter ReisnerAbstract:We demonstrate a lab-on-a-chip that combines micro/nano-fabricated features with a Convex Lens-Induced Confinement (CLIC) device for the in situ analysis of single cells. A complete cycle of single cell analysis was achieved that includes: cell trapping, cell isolation, lysis, protein digestion, genomic DNA extraction and on-chip genomic DNA linearization. The ability to dynamically alter the flow-cell dimensions using the CLIC method was coupled with a flow-control mechanism for achieving efficient cell trapping, buffer exchange, and loading of long DNA molecules into nanofluidic arrays. Finite element simulation of fluid flow gives rise to optimized design parameters for overcoming the high hydraulic resistance present in the micro/nano-confinement region. By tuning design parameters such as the pressure gradient and CLIC confinement, an efficient on-chip single cell analysis protocol can be obtained. We demonstrate that we can extract Mbp long genomic DNA molecules from a single human lybphoblastoid cell and stretch these molecules in the nanochannels for optical interrogation.
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Convex Lens-induced nanoscale templating
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Daniel Berard, François Michaud, Sara Mahshid, Mohammed Jalal Ahamed, Christopher M. J. Mcfaul, Jason S. Leith, Pierre Bérubé, Robert Sladek, Walter Reisner, Sabrina LeslieAbstract:We demonstrate a new platform, Convex Lens-induced nanoscale templating (CLINT), for dynamic manipulation and trapping of single DNA molecules. In the CLINT technique, the curved surface of a Convex Lens is used to deform a flexible coverslip above a substrate containing embedded nanotopography, creating a nanoscale gap that can be adjusted during an experiment to confine molecules within the embedded nanostructures. Critically, CLINT has the capability of transforming a macroscale flow cell into a nanofluidic device without the need for permanent direct bonding, thus simplifying sample loading, providing greater accessibility of the surface for functionalization, and enabling dynamic manipulation of confinement during device operation. Moreover, as DNA molecules present in the gap are driven into the embedded topography from above, CLINT eliminates the need for the high pressures or electric fields required to load DNA into direct-bonded nanofluidic devices. To demonstrate the versatility of CLINT, we confine DNA to nanogroove and nanopit structures, demonstrating DNA nanochannel-based stretching, denaturation mapping, and partitioning/trapping of single molecules in multiple embedded cavities. In particular, using ionic strengths that are in line with typical biological buffers, we have successfully extended DNA in sub–30-nm nanochannels, achieving high stretching (90%) that is in good agreement with Odijk deflection theory, and we have mapped genomic features using denaturation analysis.
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Precision platform for Convex Lens-induced confinement microscopy.
The Review of scientific instruments, 2013Co-Authors: Daniel Berard, François Michaud, Christopher M. J. Mcfaul, Jason S. Leith, Adriel Arsenault, Sabrina LeslieAbstract:We present the conception, fabrication, and demonstration of a versatile, computer-controlled microscopy device which transforms a standard inverted fluorescence microscope into a precision single-molecule imaging station. The device uses the principle of Convex Lens-induced confinement [S. R. Leslie, A. P. Fields, and A. E. Cohen, Anal. Chem. 82, 6224 (2010)], which employs a tunable imaging chamber to enhance background rejection and extend diffusion-limited observation periods. Using nanopositioning stages, this device achieves repeatable and dynamic control over the geometry of the sample chamber on scales as small as the size of individual molecules, enabling regulation of their configurations and dynamics. Using microfluidics, this device enables serial insertion as well as sample recovery, facilitating temporally controlled, high-throughput measurements of multiple reagents. We report on the simulation and experimental characterization of this tunable chamber geometry, and its influence upon the diffusion and conformations of DNA molecules over extended observation periods. This new microscopy platform has the potential to capture, probe, and influence the configurations of single molecules, with dramatically improved imaging conditions in comparison to existing technologies. These capabilities are of immediate interest to a wide range of research and industry sectors in biotechnology, biophysics, materials, and chemistry.
Adam E. Cohen - One of the best experts on this subject based on the ideXlab platform.
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single molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero mode waveguides zmws and Convex Lens induced confinement clic
Optics Express, 2013Co-Authors: Mary Williard Elting, Christopher M. J. Mcfaul, Jason S. Leith, Sabrina Leslie, Adam E. Cohen, Stirling L Churchman, Jonas Korlach, Michael J Levene, James A SpudichAbstract:Resolving single fluorescent molecules in the presence of high fluorophore concentrations remains a challenge in single-molecule biophysics that limits our understanding of weak molecular interactions. Total internal reflection fluorescence (TIRF) imaging, the workhorse of single-molecule fluorescence microscopy, enables experiments at concentrations up to about 100 nM, but many biological interactions have considerably weaker affinities, and thus require at least one species to be at micromolar or higher concentration. Current alternatives to TIRF often require three-dimensional confinement, and thus can be problematic for extended substrates, such as cytoskeletal filaments. To address this challenge, we have demonstrated and applied two new single-molecule fluorescence microscopy techniques, linear zero-mode waveguides (ZMWs) and Convex Lens induced confinement (CLIC), for imaging the processive motion of molecular motors myosin V and VI along actin filaments. Both technologies will allow imaging in the presence of higher fluorophore concentrations than TIRF microscopy. They will enable new biophysical measurements of a wide range of processive molecular motors that move along filamentous tracks, such as other myosins, dynein, and kinesin. A particularly salient application of these technologies will be to examine chemomechanical coupling by directly imaging fluorescent nucleotide molecules interacting with processive motors as they traverse their actin or microtubule tracks.
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Convex Lens-induced confinement for imaging single molecules.
Analytical chemistry, 2010Co-Authors: Sabrina Leslie, Alexander P. Fields, Adam E. CohenAbstract:Fluorescence imaging is used to study the dynamics of a wide variety of single molecules in solution or attached to a surface. Two key challenges in this pursuit are (1) to image immobilized single molecules in the presence of a high level of fluorescent background and (2) to image freely diffusing single molecules for long times. Strategies that perform well by one measure often perform poorly by the other. Here, we present a simple modification to a wide-field fluorescence microscope that addresses both challenges and dramatically improves single-molecule imaging. The technique of Convex Lens-induced confinement (CLIC) restricts molecules to a wedge-shaped gap of nanoscale depth, formed between a plano-Convex Lens and a planar coverslip. The shallow depth of the imaging volume leads to 20-fold greater rejection of background fluorescence than is achieved with total internal reflection fluorescence (TIRF) imaging. Elimination of out-of-plane diffusion leads to an approximately 10 000-fold longer diffusio...
Walter Reisner - One of the best experts on this subject based on the ideXlab platform.
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Development of a platform for single cell genomics using Convex Lens-induced confinement.
Lab on a chip, 2015Co-Authors: Sara Mahshid, Daniel Berard, Mohammed Jalal Ahamed, Robert Sladek, Sabrina Leslie, Susan Amin, Walter ReisnerAbstract:We demonstrate a lab-on-a-chip that combines micro/nano-fabricated features with a Convex Lens-Induced Confinement (CLIC) device for the in situ analysis of single cells. A complete cycle of single cell analysis was achieved that includes: cell trapping, cell isolation, lysis, protein digestion, genomic DNA extraction and on-chip genomic DNA linearization. The ability to dynamically alter the flow-cell dimensions using the CLIC method was coupled with a flow-control mechanism for achieving efficient cell trapping, buffer exchange, and loading of long DNA molecules into nanofluidic arrays. Finite element simulation of fluid flow gives rise to optimized design parameters for overcoming the high hydraulic resistance present in the micro/nano-confinement region. By tuning design parameters such as the pressure gradient and CLIC confinement, an efficient on-chip single cell analysis protocol can be obtained. We demonstrate that we can extract Mbp long genomic DNA molecules from a single human lybphoblastoid cell and stretch these molecules in the nanochannels for optical interrogation.
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Convex Lens-induced nanoscale templating
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Daniel Berard, François Michaud, Sara Mahshid, Mohammed Jalal Ahamed, Christopher M. J. Mcfaul, Jason S. Leith, Pierre Bérubé, Robert Sladek, Walter Reisner, Sabrina LeslieAbstract:We demonstrate a new platform, Convex Lens-induced nanoscale templating (CLINT), for dynamic manipulation and trapping of single DNA molecules. In the CLINT technique, the curved surface of a Convex Lens is used to deform a flexible coverslip above a substrate containing embedded nanotopography, creating a nanoscale gap that can be adjusted during an experiment to confine molecules within the embedded nanostructures. Critically, CLINT has the capability of transforming a macroscale flow cell into a nanofluidic device without the need for permanent direct bonding, thus simplifying sample loading, providing greater accessibility of the surface for functionalization, and enabling dynamic manipulation of confinement during device operation. Moreover, as DNA molecules present in the gap are driven into the embedded topography from above, CLINT eliminates the need for the high pressures or electric fields required to load DNA into direct-bonded nanofluidic devices. To demonstrate the versatility of CLINT, we confine DNA to nanogroove and nanopit structures, demonstrating DNA nanochannel-based stretching, denaturation mapping, and partitioning/trapping of single molecules in multiple embedded cavities. In particular, using ionic strengths that are in line with typical biological buffers, we have successfully extended DNA in sub–30-nm nanochannels, achieving high stretching (90%) that is in good agreement with Odijk deflection theory, and we have mapped genomic features using denaturation analysis.