Daniel Berard

Convex lens-induced nanoscale templating

Significance Convex lens-induced nanoscale templating (CLINT) represents a conceptual breakthrough in nanofluidic technology for single-molecule manipulation. CLINT solves a key challenge faced by the nanofluidics field by bridging the multiple-length scales required to efficiently bring single-molecule analytes from the pipette tip to the nanofluidic channel. To do this, CLINT loads single-molecule analytes into embedded nanofeatures via dynamic control of applied vertical confinement, which we have demonstrated by loading and extending DNA within nanochannels. CLINT offers unique advantages in single-molecule DNA mapping by facilitating surface passivation, increasing loading efficiency, obviating the need for applied pressure or electric fields, and enhancing compatibility with physiological buffers and long DNA molecules extracted from complex genomes. 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.

Precision platform for convex lens-induced confinement microscopy

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.

Formatting and ligating biopolymers using adjustable nanoconfinement

Sensitive visualization and conformational control of long, delicate biopolymers present critical challenges to emerging biotechnologies and biophysical studies. Next-generation nanofluidic manipulation platforms strive to maintain the structural integrity of genomic DNA prior to analysis but can face challenges in device clogging, molecular breakage, and single-label detection. We address these challenges by integrating the Convex Lens-induced Confinement (CLiC) technique with a suite of nanotopographies embedded within thin-glass nanofluidic chambers. We gently load DNA polymers into open-face nanogrooves in linear, concentric circular, and ring array formats and perform imaging with single-fluorophore sensitivity. We use ring-shaped nanogrooves to access and visualize confinement-enhanced self-ligation of long DNA polymers. We use concentric circular nanogrooves to enable hour-long observations of polymers at constant confinement in a geometry which eliminates the confinement gradient which causes drif...

Visualizing structure-mediated interactions in supercoiled DNA molecules

Abstract We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid’s structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

Rotation-Induced Macromolecular Spooling of DNA

Genetic information is stored in a linear sequence of base-pairs; however, thermal fluctuations and complex DNA conformations such as folds and loops make it challenging to order genomic material for in vitro analysis. In this work, we discover that rotation-induced macromolecular spooling of DNA around a rotating microwire can monotonically order genomic bases, overcoming this challenge. We use single-molecule fluorescence microscopy to directly visualize long DNA strands deforming and elongating in shear flow near a rotating microwire, in agreement with numerical simulations. While untethered DNA is observed to elongate substantially, in agreement with our theory and numerical simulations, strong extension of DNA becomes possible by introducing tethering. For the case of tethered polymers, we show that increasing the rotation rate can deterministically spool a substantial portion of the chain into a fully stretched, single-file conformation. When applied to DNA, the fraction of genetic information sequentially ordered on the microwire surface will increase with the contour length, despite the increased entropy. This ability to handle long strands of DNA is in contrast to modern DNA sample preparation technologies for sequencing and mapping, which are typically restricted to comparatively short strands resulting in challenges in reconstructing the genome. Thus, in addition to discovering new rotation-induced macromolecular dynamics, this work inspires new approaches to handling genomic-length DNA strands.

Open-frame system for single-molecule microscopy

We present the design and construction of a versatile, open frame inverted microscope system for wide-field fluorescence and single molecule imaging. The microscope chassis and modular design allow for customization, expansion, and experimental flexibility. We present two components which are included with the microscope which extend its basic capabilities and together create a powerful microscopy system: A Convex Lens-induced Confinement device provides the system with single-molecule imaging capabilities, and a two-color imaging system provides the option of imaging multiple molecular species simultaneously. The flexibility of the open-framed chassis combined with accessible single-molecule, multi-species imaging technology supports a wide range of new measurements in the health, nanotechnology, and materials science research sectors.

Single-molecule microscopy using tunable nanoscale confinement

We present the design, construction and implementation of a modular microscopy device that transforms a basic inverted fluorescence microscope into a versatile single-molecule imaging system. The device uses Convex Lens- Induced Confinement (CLIC) to improve background rejection and extend diffusion-limited observation time. To facilitate its integration into a wide range of laboratories, this implementation of the CLIC device can use a standard flow-cell, into which the sample is loaded. By mechanically deforming the flow-cell, the device creates a tunable, wedge-shaped imaging chamber which we have modeled using finite element analysis simulations and characterized experimentally using interferometry. A powerful feature of CLIC imaging technology is the ability to examine single molecules under a continuum of applied confinement, from the nanometer to the micrometer scale. We demonstrate, using freely diffusing λ-phage DNA, that when the imposed confinement is on the scale of individual molecules their molecular conformations and diffusivity are altered significantly. To improve the flow-cell stiffness, seal, and re-usability, we have innovated the fabrication of thin PDMS-bonded flow-cells. The presented flow-cell CLIC technology can be combined with surface-lithography to provide an accessible and powerful approach to tune, trap, and image individual molecules under an extended range of imaging conditions. It is well-suited to tackling open problems in biophysics, biotechnology, nanotechnology, materials science, and chemistry.