Convex Lens-induced Confinement Research Articles

Quantitative Visualization of Lipid Nanoparticle Fusion as a Function of Formulation and Process Parameters.

Lipid nanoparticles (LNPs) have proven to be promising delivery vehicles for RNA-based vaccines and therapeutics, particularly in LNP formulations containing ionizable cationic lipids that undergo protonation/deprotonation in response to buffer pH changes. These nanoparticles are typically formulated using a rapid mixing technique at low pH, followed by a return to physiological pH that triggers LNP-LNP fusion. A detailed understanding of these dynamic processes is crucial to optimize the overall performance and efficiency of LNPs. However, knowledge gaps persist regarding how particle formation mechanisms impact drug loading and delivery functions. In this work, we employ single-molecule Convex Lens-induced Confinement (CLiC) microscopy in combination with Förster resonance energy transfer (FRET) measurements to study LNP fusion dynamics in relation to various formulation parameters, including lipid concentration, buffer conditions, drug loading ratio, PEG-lipid concentrations, and ionizable lipid selection. Our results reveal a strong correlation between the measured fusion dynamics and the formulation parameters used; these findings are consistent with DLS and Cryo-TEM-based assays. These measurements offer a cost-effective method for characterizing and screening potential drug candidates and can provide additional insights into their design, with opportunities for optimization.

Studies of supercoiling-induced denaturation within DNA plasmids using single-molecule convex lens-induced confinement microscopy

Studies of supercoiling-induced denaturation within DNA plasmids using single-molecule convex lens-induced confinement microscopy

Determination of the interior pH of lipid nanoparticles using a pH-sensitive fluorescent dye-based DNA probe

Lipid nanoparticles (LNPs) containing ionizable cationic lipids are proven delivery systems for therapeutic nucleic acids, such as small interfering RNA (siRNA). It is important to understand the relationship between the interior pH of LNPs and the pH of the external environment to understand LNP formulation and function. Here, we developed a simple and rapid approach for determining the pH of the LNP core using a pH-sensitive fluorescent dye-based DNA probe. LNP siRNA systems containing pH-responsive DNA probes (LNP-siRNA&DNA) were generated by rapid mixing of lipids in ethanol and pH 4 aqueous buffer containing siRNA and DNA probes. We demonstrated that DNA probes were readily encapsulated in LNP systems and were sequestered into an environment at a high concentration as evidenced by an inter-probe FRET signal. It was shown that the pH of LNP encapsulated probes closely follows the pH increase or decrease of the external environment. This indicates that the clinically approved LNP RNA systems with similar lipid compositions (e.g., Onpattro and Comirnaty) are highly permeable to protons and that the pH of the interior environment closely mirrors the external environment. The pH-dependent response of the probe in LNPs was also confirmed under buffer conditions at various pHs. Furthermore, we showed that the pH-sensitive DNA probe can be incorporated into LNP systems at levels that allow the pH response to be monitored at a single LNP level using convex lens-induced confinement (CLiC) confocal microscopy. Direct visualization of the internal pH of single particles with the fluorescent DNA probe was achieved by CLiC for LNP-siRNA&DNA systems formulated under both high and normal ionic strength conditions.

Effects of Molecular Weight and Surface Interactions on Polymer Diffusion in Confinement

Understanding the transport and thermodynamics of polymers in confined spaces is helpful for many separation processes like water purification, drug delivery, and oil recovery. Specifically, for water purification, dextran has been used as a "model" foulant. Uncovering how these polymers interact in confinement can reduce the fouling of organic membranes and will lead to better separation processes overall. We have determined the diffusion coefficient, D, of dextran and sodium polyacrylate in convex lens-induced confinement using differential dynamic microscopy. In this setup, the gap height ranges continuously from 0.077-21.8 μm. It was found that polymer diffusion becomes slower in higher confinement, which is consistent with a change in the increase of the hydrodynamic resistance to macromolecule motion and depends on the surface properties. These findings indicate that dextran diffusion changes in confinement and can lead to a better understanding of separation processes.

Enhanced Facilitated Diffusion of Membrane-Associating Proteins under Symmetric Confinement.

The facilitated surface diffusion of transiently adsorbing molecules in a planar confined microenvironment (i.e., slit-like confinement) is highly relevant to biological phenomena, such as extracellular signaling, as well as numerous biotechnology systems. Here, we studied the surface diffusion of individual proteins confined between two symmetric lipid bilayer membranes, under a continuum of confinement heights, using single-molecule tracking and convex lens-induced confinement as well as hybrid, kinetic Monte Carlo simulations of a generalized continuous time random walk process. Surface diffusion was observed to vary non-monotonically with confinement height, exhibiting a maximum at a height of ∼750 nm, where diffusion was nearly 40% greater than that for a semi-infinite system. This demonstrated that planar confinement can, in fact, increase surface diffusion, qualitatively validating previous theoretical predictions. Simulations reproduced the experimental results and suggested that confinement enhancement of surface diffusion for symmetric systems is limited to cases where the adsorbate exhibits weak surface sticking.

Non-equilibrium structural dynamics of supercoiled DNA plasmids exhibits asymmetrical relaxation.

Many cellular processes occur out of equilibrium. This includes site-specific unwinding in supercoiled DNA, which may play an important role in gene regulation. Here, we use the Convex Lens-induced Confinement (CLiC) single-molecule microscopy platform to study these processes with high-throughput and without artificial constraints on molecular structures or interactions. We use two model DNA plasmid systems, pFLIP-FUSE and pUC19, to study the dynamics of supercoiling-induced secondary structural transitions after perturbations away from equilibrium. We find that structural transitions can be slow, leading to long-lived structural states whose kinetics depend on the duration and direction of perturbation. Our findings highlight the importance of out-of-equilibrium studies when characterizing the complex structural dynamics of DNA and understanding the mechanisms of gene regulation.

Diffusive dynamics of charged nanoparticles in convex lens-induced confinement.

Transport through heterogeneous confined geometries is encountered in many processes and applications such as filtration, drug delivery, and enhanced oil recovery. We have used differential dynamic microscopy (DDM) and particle tracking to investigate dynamics of 36 nm negatively-charged polystyrene particles in convex lens-induced confinement (CLiC). The confinement gap height was controlled from 0.085 μm to 3.6 mm by sandwiching the aqueous particle solution between a glass coverslip and a convex lens using a homemade sample holder. With an inverted fluorescence microscope, sequences of micrographs were taken at various radial positions and gap heights for five particle concentrations (i.e. φ = 0.5 × 10-5, 1 × 10-5, 5 × 10-5, 10 × 10-5, 50 × 10-5) and ionic strengths ranging from 10-3 to 150 mM. The resulting image structure functions were fitted with a simple exponential model to extract the ensemble-averaged diffusive dynamics. It was found that particle diffusion was more hindered as a function of increased confinement. In addition, the ensemble-averaged diffusion coefficient was found to depend on the bulk concentration, and the concentration dependence increased as a function of confinement. Increasing particle and salt concentration led to confinement-dependent adsorption onto the geometry surface. Overall, we show that CLiC devices are simple and effective and can be used to study dynamics in continuous confinement from sub 100 nm to 100's of μm. These findings could lead to better understanding of separations and interactions in confining devices.

Simultaneous, Single-Particle Measurements of Size and Loading Give Insights into the Structure of Drug-Delivery Nanoparticles.

Nanoparticles are a promising solution for delivery of a wide range of medicines and vaccines. Optimizing their design depends on being able to resolve, understand, and predict biophysical and therapeutic properties, as a function of design parameters. While existing tools have made great progress, gaps in understanding remain because of the inability to make detailed measurements of multiple correlated properties. Typically, an average measurement is made across a heterogeneous population, obscuring potentially important information. In this work, we develop and apply a method for characterizing nanoparticles with single-particle resolution. We use convex lens-induced confinement (CLiC) microscopy to isolate and quantify the diffusive trajectories and fluorescent intensities of individual nanoparticles trapped in microwells for long times. First, we benchmark detailed measurements of fluorescent polystyrene nanoparticles against prior data to validate our approach. Second, we apply our method to investigate the size and loading properties of lipid nanoparticle (LNP) vehicles containing silencing RNA (siRNA), as a function of lipid formulation, solution pH, and drug-loading. By taking a comprehensive look at the correlation between the intensity and size measurements, we gain insights into LNP structure and how the siRNA is distributed in the LNP. Beyond introducing an analytic for size and loading, this work allows for future studies of dynamics with single-particle resolution, such as LNP fusion and drug-release kinetics. The prime contribution of this work is to better understand the connections between microscopic and macroscopic properties of drug-delivery vehicles, enabling and accelerating their discovery and development.

Diffusion of Short Semiflexible DNA Polymer Chains in Strong and Moderate Confinement.

In many technological applications, DNA is confined within nanoenvironments that are smaller than the size of the unconfined polymer in solution. However, the dependence of the diffusion coefficient on molecular weight and characteristic confinement dimension remains poorly understood in this regime. Here, convex lens-induced confinement (CLiC) was leveraged to examine how the diffusion of short DNA fragments varied as a function of slit height by using single-molecule fluorescence tracking microscopy. The diffusion coefficient followed approximate power law behavior versus confinement height, with exponents of 0.27 ± 0.01, 0.32 ± 0.02, and 0.42 ± 0.06 for 692, 1343, and 2686 base pair chains, respectively. The weak dependence on slit height suggests that shorter semiflexible chains may adopt increasingly rodlike conformations and therefore experience weaker excluded-volume interactions as the confinement dimension is reduced. The diffusion coefficient versus molecular weight also exhibited apparent power law behavior, with exponents that varied slightly (from -0.89 to -0.85) with slit height, consistent with hydrodynamic interactions intermediate between Rouse and Zimm model predictions.

Polyelectrolyte Surface Diffusion in a Nanoslit Geometry

The surface diffusion of poly-l-lysine (PLL) in a planar nanoslit was studied using convex lens-induced confinement (CLiC) single-molecule tracking microscopy. Three surface chemistries were employ...

Biopolyelectrolyte Surface Diffusion Within a Planar Slit Geometry

The effective surface diffusion of biopolyelectrolytes within nanoscale confinement is integral to biosensing, biocatalysis, bioseparations (such as chromatography columns utilizing nanoporous media), as well as biophysical processes. Confinement influences the surface-mediated diffusion of biopolyelectrolyte chains due to the presence of electric double layers and the increased frequency of transient polymer/surface interactions. Here, the diffusion of poly-L-lysine (PLL) in a nanoslit was studied using Convex Lens-induced Confinement (CLiC) single-molecule tracking microscopy. Three surface chemistries were employed to understand and compare the effects of electrostatic and short-range interactions, respectively. In all cases, the effective surface diffusion coefficient increased rapidly with slit height until it saturated at its value for a semi-infinite interface for slit heights <30 nm. The evolution of the diffusion coefficient with slit height was faster for amine-functionalized surfaces with which PLL exhibited stronger short-range interactions. These hydrogen-bonding interactions influenced the intermittent random walk behavior by increasing the waiting times between flights and the re-adsorption probability (a.k.a. sticking coefficient) during flights. Intermittent random walks were simulated within a planar slit geometry, using the appropriate adsorption probabilities and waiting time distributions (obtained from diffusion at a single interface) to account for the characteristic short-range interactions between PLL and each type of surface chemistry. The simulated step-size distributions were in good agreement with experimental measurements, indicating that the fine details of diffusion in nanoscale confinement can be quantitatively described by a model that combines confined Brownian motion with transient surface adsorption and desorption, using parameters obtained only from the behavior at a single interface in a semi-infinite geometry. These results highlight the importance of short-range biopolyelectrolyte/surface interactions on diffusion within confined environments and moreover, demonstrate the complex relationship between electrostatics, confinement, and local polymer and surface chemistry on biopolyelectrolyte surface diffusion within a nanoslit.

Single-cell analysis for drug development using convex lens-induced confinement imaging.

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.

Standalone interferometry-based calibration of convex lens-induced confinement microscopy with nanoscale accuracy.

Strongly confined environments (confined dimensions between 1-100 nm) represent unique challenges and opportunities for understanding and manipulating molecular behavior due to the significant effects of electric double layers, high surface-area to volume ratios, and other phenomena at the nanoscale. Convex Lens-induced Confinement (CLiC) can be used to analyze the dynamics of individual molecules or particles confined in a planar slit geometry with continuously varying gap thickness. We describe an interferometry-based method for precise measurement of the slit pore geometry. Specifically, this approach permitted accurate characterization of separation distances as small as 5 nm, with 1 nm precision, without a priori knowledge or assumptions about the contact geometry, as well as a greatly simplified experimental setup that required only a lens, coverslip, and inverted microscope. The interferometry-based measurement of gap height offered a distinct advantage over conventional fluorescent dye-based methods; e.g., accurate interferometric height measurements were made at low gap heights regardless of solution conditions, while the concentration of fluorescent dye was significantly impacted by solution conditions such as ionic strength or pH. The accuracy of the interferometric measurements was demonstrated by comparing the experimentally measured concentration of a charged fluorescent dye as a function of gap thickness with dye concentration profiles calculated using Debye-Hückel theory. Accurate characterization of nanoscale gap thickness will enable researchers to study a variety of practical and biologically relevant systems within the CLiC geometry.

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 to 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.

Free Energy of a Polymer in Slit-like Confinement from the Odijk Regime to the Bulk

We directly measure the free energy of confinement for semiflexible polymers from the nanoscale to bulk regimes in slit-like confinement. We use convex lens-induced confinement (CLiC) microscopy of DNA to directly count molecules at equilibrium in a single chamber of smoothly increasing height. Our data, acquired across a continuum of confinement regimes, provide a bridge with which to connect scaling theories established for qualitatively different regimes. We present new experimental data and simulations that connect the Odijk theory describing sub-persistence-length confinement, the interpolation model by Chen and Sullivan extending Odijk to moderate confinement, and the Casassa theory describing the transition from moderate confinement to bulk. Further, this work establishes a robust, quantitative platform for understanding and manipulating biopolymers at the nanoscale, with key applications and insights toward emerging genomic analysis tools.

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.

BioSpotlight / Citations

BioTechniquesVol. 61, No. 2 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114439AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit A reality check for dsRNAsA number of popular methods and commercial kits for single-step isolation of high-quality RNA use a concentrated solution of guanidinium thiocyanate (GuSCN)–a powerful chaotropic agent—to denature cellular proteins and inactivate RNases. Besides acting as a robust protein denaturant, a less well-known effect of concentrated GuSCN solutions is to promote DNA—DNA, RNA—DNA, and RNA—RNA hybridization. In this issue of BioTechniques, Triin Mölder and Mart Speek at the Tallinn University of Technology provide evidence suggesting that use of concentrated GuSCN during isolation of cellular RNA causes the formation of double-stranded RNAs (dsRNAs) from complementary single-stranded RNAs (ssRNAs). The authors demonstrated that incubation in a concentrated GuSCN solution of low concentrations of two in vitro—transcribed RNAs with a region of complementarity catalyzed their hybridization into dsRNA, in the absence or presence of total cellular RNA. This suggests the need to carefully verify that any putative novel dsRNAs identified from RNA samples isolated by GuSCN extraction are not actually artifacts caused by this effect.See “Accelerated RNA—RNA hybridization by concentrated guanidinium thiocyanate solution in single-step RNA isolation”Indenting multiple cellsStudies of the response of cells to mechanical force transduction have relied on instruments such as atomic force microscopes (AFMs) or cytoindentors, but these tools can only indent single cells one-by-one, which makes the collection of data from a sufficient number of cells a tedious and time-consuming affair. To overcome this problem, researchers at McGill University led by Claire Brown and Sabrina Leslie describe in this month's issue their development of a high-throughput, parallelized cytoindentor for the simultaneous local compression of multiple live cells. They modified their previously described convex lens—induced confinement (CLiC) set-up by adhering a film patterned with microposts to a microscope lens. This lens is then gradually lowered onto multiple cells maintained in a stage-top incubator to indent micrometer-sized areas on each cell while allowing imaging of those cells. By functionalizing the micropost arrays with an extracellular matrix protein such as fibronectin, it was also possible to pull on the cells. This novel parallelized cytoindentor will greatly facilitate studies of mechanobiology in living cells.See “Parallelized cytoindentation using convex micropatterned surfaces”Protein interactions: limiting diffusion on arraysProtein arrays are important tools for those exploring protein—protein interactions on a large scale. These arrays can be spotted with purified proteins, or cell-free protein synthesis can be performed on the arrays. Protein synthesis approaches such as the nucleic acid programmable protein array (NAPPA) streamline the building of large arrays, but they also produce large, diffuse protein spots that limit array density. Yazaki et al. solve this problem with their newly devised HaloTag- NAPPA technology, which produces higher-density protein spotting. To accomplish this, they exchanged the C-terminal GST fusion used in the original NAPPA for a HaloTag fusion that can be irreversibly bound to ligands co-spotted on the array. HaloTag-NAPPA captured synthesized proteins with significantly less diffusion compared to GST fusion proteins. To test their method, the authors spotted 12,000 Arabidopsis clones onto arrays, synthesized proteins, and then examined protein— protein interactions with 38 plant transcription factors. The new approach identified novel interactions with high sensitivity and low background.Yazaki, J. et al. 2016. Mapping transcription factor interactome networks using HaloTag protein arrays. Proc. Natl. Acad. Sci. USA. DOI: 10.1073/pnas.1603229113Gene expression profiling: it's all about locationProfiling gene expression is a powerful approach for understanding basic biology and human disease. Now, Stahl et al. introduce a new method that could significantly expand the scope of gene expression profiling by adding spatial resolution within tissue samples. In “spatial transcriptomics,” histological samples are placed onto slides with reverse transcription primers possessing positional barcodes immobilized on the slide surface. Following permeabilization, reverse transcription reagents are placed onto the tissues, where cDNA is synthesized and left coupled to the oligonucleotides on the slide. Once the tissue is enzymatically removed, the cDNAs are collected and sequenced, akin to traditional gene expression profiling. After sequencing, the locations of cDNA transcripts can be mapped back to the tissue using the incorporated positional barcodes. By adding only a few additional steps, scientists will now be able to obtain spatial information by transcriptional profiling, thus greatly enhancing gene expression data sets.Stahl, P.L., et al. 2016. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science. 353(6294):78-82.FiguresReferencesRelatedDetails Vol. 61, No. 2 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print August 2016 Information© 2016 Author(s)PDF download

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 drift and can alter molecular conformations and interactions. Taken together, this work opens doors to myriad biophysical studies and biotechnologies which operate on the nanoscale.