Biophysical Reviews 'Meet the Editors Series' - a profile of Sabrina Leslie.

It is my pleasure to introduce myself to the readers of Biophysical Reviews as part of the 'Meet the Editors Series'.

Probing Inhomogeneous Diffusion in the Microenvironments of Phase-Separated Polymers under Confinement.

Biomolecular condensates formed by liquid-liquid phase separation of proteins and nucleic acids have been recently discovered to be prevalent in biology. These dynamic condensates behave like biochemical reaction vessels, but little is known about their structural organization and biophysical properties, which are likely related to condensate size. Thus, it is critical that we study them on scales found in vivo. However, previous in vitro studies of condensate assembly and physical properties have involved condensates up to 1000 times larger than those found in vivo. Here, we apply confinement microscopy to visualize condensates and control their sizes by creating appropriate confinement length scales relevant to the cell environment. We observe anomalous diffusion of probe particles embedded within confined condensates, as well as heterogeneous dynamics in condensates formed from PEG/dextran and in ribonucleoprotein complexes of RNA and the RNA-binding protein Dhh1. We propose that the observed non-Gaussian dynamics indicate a hopping diffusion mechanism inside condensates. We also observe that, for dextran-rich condensates, but not for ribonucleo condensates, probe particle diffusion depends on condensate size.

Miniaturized flow cell with pneumatically-actuated vertical nanoconfinement for single-molecule imaging and manipulation.

Convex Lens-induced Confinement (CLiC) is a single-molecule imaging technique that uses a deformable glass flow cell to gently trap, manipulate, and visualize single molecules within micro- and nano-structures, to enable a wide range of applications. Here, we miniaturize the CLiC flow cell, from 25 × 25 to 3 × 3 mm 2 and introduce pneumatic control of the confinement. Miniaturization of the flow cell improves fabrication throughput by almost two orders of magnitude and, advantageous for pharmaceutical and diagnostic applications where samples are precious, significantly lowers the internal volume from microliters to nanoliters. Pneumatic control of the device reduces the confinement gradient and improves mechanical stability while maintaining low autofluorescence and refractive index-matching with oil-immersion objectives. To demonstrate our "mini CLiC" system, we confine and image DNA in sub-50 nm nanogrooves, with high DNA extension consistent with the Odijk confinement regime.

Visualizing structure-mediated interactions in supercoiled DNA molecules.

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.

Manipulating and Visualizing Molecular Interactions in Customized Nanoscale Spaces.

We present a dynamically adjustable nanofluidic platform for formatting the conformations of and visualizing the interaction kinetics between biomolecules in solution, offering new time resolution and control of the reaction processes. This platform extends convex lens-induced confinement (CLiC), a technique for imaging molecules under confinement, by introducing a system for in situ modification of the chemical environment; this system uses a deep microchannel to diffusively exchange reagents within the nanoscale imaging region, whose height is fixed by a nanopost array. To illustrate, we visualize and manipulate salt-induced, surfactant-induced, and enzyme-induced reactions between small-molecule reagents and DNA molecules, where the conformations of the DNA molecules are formatted by the imposed nanoscale confinement. In response to dynamically modifying the local salt concentration, we report two salt-induced transitions in DNA molecules which occur on separate time scales: a rapid change in polymer extension due to modified local ionic screening and a gradual change in polymer brightness, reflecting release of intercalated YOYO-1 dye. Our time-resolved measurements provide new insights into the influence of YOYO-1 dye on polymer stiffness. In response to introducing cationic surfactants in solution, we temporally resolve single-molecule compaction trajectories of DNA polymers, guided by the confining nanogroove environment; this is in contrast to the uncontrolled collapse which would occur in free solution under similar conditions. In the presence of restriction enzymes, we directly visualize the cleavage of multiple DNA sites under adjustable nanoscale confinement. By using nanofabricated, nonabsorbing, low-background glass walls to confine biomolecules, our nanofluidic platform facilitates quantitative exploration of physiologically and biotechnologically relevant processes at the nanoscale. This device provides new kinetic information about dynamic chemical processes at the single-molecule level, using advancements in the CLiC design including a microchannel-based diffuser and postarray-based dialysis slit.

Parallelized cytoindentation using convex micropatterned surfaces.

Here we present a high-throughput, parallelized cytoindentor for local compression of live cells. The cytoindentor uses convex lens-induced confinement (CLiC) to indent micrometer-sized areas in single cells and/or populations of cells with submicron precision. This is accomplished using micropatterned poly(dimethylsiloxane) (PDMS) films that are adhered to a convex lens to create arrays of extrusions referred to here as "posts." These posts caused local deformation of subcellular regions without any evidence of cell lysis upon CLiC indentation. Our micropost arrays were also functionalized with glycoproteins, such as fibronectin, to both pull and compress cells under customized confinement geometries. Measurements of Chinese hamster ovary (CHO-K1) cell migration trajectories and oxidative stress showed that the CLiC device did not damage or significantly stress the cells. Our novel tool opens a new area of investigation for visualizing mechanobiology and mechanochemistry within living cells, and the high-throughput nature of the technique will streamline investigations as current tools for mechanically probing material properties and molecular dynamics within cells, such as traditional cytoindentors and atomic force microscopy (AFM), are typically restricted to single-cell manipulation.

Development of a platform for single cell genomics using convex lens-induced confinement.

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.

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.

Convex lens-induced nanoscale templating.

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.

Single-molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC).

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.

Convex lens-induced confinement for imaging single molecules.

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 diffusion-limited observation time per molecule than is achieved with confocal fluorescence correlation spectroscopy. The CLIC system also provides a new means to determine molecular size. The CLIC system does not require any nanofabrication, nor any custom optics, electronics, or computer control.