Large-scale nanoarrays of single biomolecules enable high-throughput assays while unmasking the underlying heterogeneity within ensemble populations. Until recently, creating such grids which combine the advantages of microarrays and single-molecule experiments (SMEs) has been particularly challenging due to the mismatch between the size of these molecules and the resolution of top-down fabrication techniques. DNA origami placement (DOP) combines two powerful techniques to address this issue: (i) DNA origami, which provides a ∼100 nm self-assembled template for singlemolecule organization with 5 nm resolution and (ii) top-down lithography, which patterns these DNA nanostructures, transforming them into functional nanodevices via large-scale integration with arbitrary substrates. Presently, this technique relies on state-of-the-art infrastructure and highly trained personnel, making it prohibitively expensive for researchers. Here, we introduce a cleanroom-free, $1 benchtop technique to create meso-to-macro-scale DNA origami nanoarrays using self-assembled colloidal nanoparticles, thereby circumventing the need for top-down fabrication. We report a maximum yield of 74%, 2-fold higher than the statistical limit of 37% imposed on non-specific molecular loading alternatives. Furthermore, we provide a proof-of-principle for the ability of this nanoarray platform to transform traditionally low-throughput, stochastic, single-molecule assays into high-throughput, deterministic ones, without compromising data quality. Our approach has the potential to democratize single-molecule nanoarrays and demonstrates their utility as a tool for biophysical assays and diagnostics.

Self-assembly of artificial opals has garnered significant interest as a facile nanofabrication technique capable of producing highly ordered structures for optical, electrochemical, biomolecular, and thermal applications. In these applications, the optimum opal particle diameter can vary by several orders of magnitude because the properties of the resultant structures depend strongly on the feature size. However, current opal fabrication techniques only produce high-quality structures over a limited range of sphere sizes or require complex processes and equipment. In this work, the rational and simple fabrication of polycrystalline opals with diameters between 500 nm and 10 μm was demonstrated using slope self-assembly of colloids suspended in ethanol–water. The role of the various process parameters was elucidated through a scaling-based model that accurately captures the variations of opal substrate coverage for spheres of size 2 μm or smaller. For spheres of 10 μm and larger, capillary forces were shown to play a key role in the process dynamics. Based on these insights, millimeter-scale monolayered opals were successfully fabricated, while centimeter-scale opals were possible with sparse sphere stacking or small uncovered areas. These insights provide a guide for the simple and fast fabrication of opals that can be used as optical coatings, templates for high power density electrodes, molecule templates, and high-performance thermo-fluidic devices.

Large-scale nanoarrays of single biomolecules enable high-throughput assays while unmasking the underlying heterogeneity within ensemble populations. Until recently, creating such grids which combine the unique advantages of microarrays and single-molecule experiments (SMEs) has been particularly challenging due to the mismatch between the size of these molecules and the resolution of top-down fabrication techniques. DNA Origami Placement (DOP) combines two powerful techniques to address this issue: (i) DNA origami, which provides a 100-nm self-assembled template for single-molecule organization with 5 nm resolution, and (ii) top-down lithography, which patterns these DNA nanostructures, transforming them into functional nanodevices via large-scale integration with arbitrary substrates. Presently, this technique relies on state-of-the-art infrastructure and highly-trained personnel, making it prohibitively expensive for researchers. Here, we introduce a bench-top technique to create meso-to-macro-scale DNA origami nanoarrays using self-assembled colloidal nanoparticles, thereby circumventing the need for top-down fabrication. We report a maximum yield of 74%, two-fold higher than the statistical limit of 37% imposed on non-specific molecular loading alternatives. Furthermore, we provide a proof-of-principle for the ability of this nanoarray platform to transform traditionally low-throughput, stochastic, single-molecule assays into high-throughput, deterministic ones, without compromising data quality. Our approach has the potential to democratize single-molecule nanoarrays and demonstrates their utility as a tool for biophysical assays and diagnostics.

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We describe a method based on principles of computed tomography for 4D imaging of live cells with isotropic spatial resolution.

This paper presents three different microfabrication technologies for manufacturing out-of-plane, flat-bottomed, undercut trapezoidal structures for generating a fluidic microscale vortex (microvortex). The first method is based on anisotropic silicon etching and a ‘sandwich’ UV polymer casting assembly; the second method uses a backside diffuser photolithography technique; and the third method features a tilted backside photolithography technique. We discuss the advantages, limitations, and utility of each technique. We further demonstrate that the microvortex generated in the resultant undercut trapezoidal structures can be used to rotate biological microparticles, e.g. single, live cells for multiperspective, high resolution 3D imaging using computed tomography, and angularly resolved confocal imaging.

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We present a new approach for three-dimensional (3D) live single-cell imaging with isotropic sub-micron spatial resolution using fluorescence computed tomography (fCT). A thin, highly inclined and laminated optical (HILO) sheet of light is used for fluorescence excitation in live single cells that are rotated around an axis perpendicular to the optical axis. During a full rotation, 400-500 two-dimensional (2D) projection images of the cell are acquired from multiple viewing perspectives by rapidly scanning the HILO light sheet along the optical axis. We report technical characteristics of the HILO approach and the results of a quantitative comparison with conventional epi fCT, demonstrating that HILO fCT offers significantly (about 17 times) reduced photobleaching and a two-fold improvement in 3D imaging contrast. We discuss potential application areas of the method for cell structure studies in live single cells with isotropic 3D spatial resolution.