Mapping complex profiles of light intensity with interferometric lithography.

Solving Maxwell's equations numerically to map electromagnetic fields in the vicinity of nanostructured metal surfaces can be a daunting task when studying non-periodic, extended patterns. However, for many nanophotonic applications such as sensing or photovoltaics it is often important to have an accurate description of the actual, experimental spatial field distributions near device surfaces. In this article, we show that the complex light intensity patterns formed by closely-spaced multiple apertures in a metal film can be faithfully mapped with sub-wavelength resolution, from near-field to far-field, in the form of a 3D solid replica of isointensity surfaces. The permittivity of the metal film plays a role in shaping of the isointensity surfaces, over the entire examined spatial range, which is captured by simulations and confirmed experimentally.

Real-Time Optical Measurements of Nanoparticle-Induced Melting and Resolidification Dynamics.

The photothermally induced nanoscale dynamics of rapid melting and resolidification of a thin layer of molecular material surrounding a nanoparticle is examined in real time by an all-optical approach. The method employs pulsed periodic modulation of the medium's dielectric constant through absorption of a low-duty-cycle laser pulse train by a single nanoparticle that acts as a localized heating source. Interpretation of experimental data, including inference of a phase change and of the liquid/solid interface dynamics, is obtained by comparing experimental data with results from coupled optical-thermal numerical simulations. The combined experimental/computational workflow presented in this proof-of-principle study will enable future explorations of material parameters at nanoscale, which are often different from their bulk values and in many cases difficult to infer from macroscopic measurements.

Orbital dynamics at atmospheric pressure in a lensed dual-beam optical trap.

Orbital dynamics of a dielectric microparticle in air using a lensed counter-propagating dual-beam trap was studied experimentally and by numerical simulations. Relationships between the dynamic parameters, trap geometry, and optical power were examined both experimentally and computationally. We found that this scheme can provide narrow bandwidth (δν/ν≈10-3) detection that is at least two orders of magnitude below typical values attainable with previously studied geometries. We predict that this characteristic makes the approach suitable for ultrasensitive in-situ detection of particle mass changes. In our experimental conditions, silica microspheres orbited on trajectories spanning tens of µm, at frequencies of up to ∼2kHz, at atmospheric pressure. With the help of simulations, we briefly discuss how the dual-beam lensed orbital trap approach can be further enhanced to gain unmatched capabilities to measure changes in the physical parameters associated with a particle interacting with its surrounding medium.

Ultrafast Collective Excited-State Dynamics of a Virus-Supported Fluorophore Antenna.

Radiation brightening was recently observed in a multifluorophore-conjugated brome mosaic virus (BMV) particle at room temperature under pulsed excitation. On the basis of its nonlinear dependence on the number of chromophores, the origins of the phenomenon were attributed to a collective relaxation. However, the mechanism remains unknown. We present ultrafast transient absorption and fluorescence spectroscopic studies which shed new light on the collective nature of the relaxation dynamics in such radiation-brightened, multifluorophore particles. Our findings indicate that the emission dynamics is consistent with a superradiance mechanism. The ratio between the rates of competing radiative and nonradiative relaxation pathways depends on the number of chromophores per virus. The findings suggest that small icosahedral virus shells provide a unique biological scaffold for developing nonclassical, deep subwavelength light sources and may open new avenues for the development of photonic probes for medical imaging applications.

Virus Assembly Pathways Inside a Host Cell.

Simple RNA viruses self-assemble spontaneously and encapsulate their genome into a shell called the capsid. This process is mainly driven by the attractive electrostatics interaction between the positive charges on capsid proteins and the negative charges on the genome. Despite its importance and many decades of intense research, how the virus selects and packages its native RNA inside the crowded environment of a host cell cytoplasm in the presence of an abundance of nonviral RNA and other anionic polymers has remained a mystery. In this paper, we perform a series of simulations to monitor the growth of viral shells and find the mechanism by which cargo-coat protein interactions can impact the structure and stability of the viral shells. We show that coat protein subunits can assemble around a globular nucleic acid core by forming nonicosahedral cages, which have been recently observed in assembly experiments involving small pieces of RNA. We find that the resulting cages are strained and can easily be split into fragments along stress lines. This suggests that such metastable nonicosahedral intermediates could be easily reassembled into the stable native icosahedral shells if the larger wild-type genome becomes available, despite the presence of a myriad of nonviral RNAs.

Hydrophobic Cargo Encapsulation into Virus Protein Cages by Self-Assembly in an Aprotic Organic Solvent.

While extensive studies of virus capsid assembly in environments mimicking in vivo conditions have led to an understanding of the thermodynamic driving forces at work, applying this knowledge to virus assembly in other solvents than aqueous buffers has not been attempted yet. In this study, Brome mosaic virus (BMV) capsid proteins were shown to preserve their self-assembly abilities in an aprotic polar solvent, dimethyl sulfoxide (DMSO). This facilitated protein cage encapsulation of nanoparticles and dye molecules that favor organic solvents, such as ß-NaYF4-based upconversion nanoparticles and BODIPY dye. Assembly was found to be robust relative to a surprisingly broad range of DMSO concentrations. Cargos with poor initial stability in aqueous solutions were readily encapsulated at high DMSO concentrations and then transferred to aqueous solvents, where they remained stable and preserved their function for months.

Subset of Fluorophores Is Responsible for Radiation Brightening in Viromimetic Particles.

In certain conditions, dye-conjugated icosahedral virus shells exhibit suppression of concentration quenching. The recently observed radiation brightening at high fluorophore densities has been attributed to coherent emission, i.e., to a cooperative process occurring within a subset of the virus-supported fluorophores. Until now, the distribution of fluorophores among potential conjugation sites and the nature of the active subset remained unknown. With the help of mass spectrometry and molecular dynamics simulations, we found which conjugation sites in the brome mosaic virus capsid are accessible to fluorophores. Reactive external surface lysines but also those at the lumenal interface where the coat protein N-termini are located showed virtually unrestricted access to dyes. The third type of labeled lysines was situated at the intercapsomeric interfaces. Through limited proteolysis of flexible N-termini, it was determined that dyes bound to them are unlikely to be involved in the radiation brightening effect. At the same time, specific labeling of genetically inserted cysteines on the exterior capsid surface alone did not lead to radiation brightening. The results suggest that lysines situated within the more rigid structural part of the coat protein provide the chemical environments conducive to radiation brightening, and we discuss some of the characteristics of these environments.

Virus Mechanics under Molecular Crowding.

Viruses avoid exposure of the viral genome to harmful agents with the help of a protective protein shell known as the capsid. A secondary effect of this protective barrier is that macromolecules that may be in high concentration on the outside cannot freely diffuse across it. Therefore, inside the cell and possibly even outside, the intact virus is generally under a state of osmotic stress. Viruses deal with this type of stress in various ways. In some cases, they might harness it for infection. However, the magnitude and influence of osmotic stress on virus physical properties remains virtually unexplored for single-stranded RNA viruses-the most abundant class of viruses. Here, we report on how a model system for the positive-sense RNA icosahedral viruses, brome mosaic virus (BMV), responds to osmotic pressure. Specifically, we study the mechanical properties and structural stability of BMV under controlled molecular crowding conditions. We show that BMV is mechanically reinforced under a small external osmotic pressure but starts to yield after a threshold pressure is reached. We explain this mechanochemical behavior as an effect of the molecular crowding on the entropy of the "breathing" fluctuation modes of the virus shell. The experimental results are consistent with the viral RNA imposing a small negative internal osmotic pressure that prestresses the capsid. Our findings add a new line of inquiry to be considered when addressing the mechanisms of viral disassembly inside the crowded environment of the cell.

Virus Assembly Pathways: Straying Away but Not Too Far.

Non-enveloped RNA viruses pervade all domains of life. In a cell, they co-assemble from viral RNA and capsid proteins. Virus-like particles can form in vitro where virtually any non-cognate polyanionic cargo can be packaged. How only viral RNA gets selected for packaging in vivo, in presence of myriad other polyanionic species, has been a puzzle. Through a combination of charge detection mass spectrometry and cryo-electron microscopy, it is determined that co-assembling brome mosaic virus (BMV) coat proteins and nucleic acid oligomers results in capsid structures and stoichiometries that differ from the icosahedral virion. These previously unknown shell structures are strained and less stable than the native one. However, they contain large native structure fragments that can be recycled to form BMV virions, should a viral genome become available. The existence of such structures suggest the possibility of a previously unknown regulatory pathway for the packaging process inside cells.

Disassembly Intermediates of the Brome Mosaic Virus Identified by Charge Detection Mass Spectrometry.

Capsid disassembly and genome release are critical steps in the lifecycle of a virus. However, their mechanisms are poorly understood, both in vivo and in vitro. Here, we have identified two in vitro disassembly pathways of the brome mosaic virus (BMV) by charge detection mass spectrometry and transmission electron microscopy. When subjected to a pH jump to a basic environment at low ionic strength, protein-RNA interactions are disrupted. Under these conditions, BMV appears to disassemble mainly through a global cleavage event into two main fragments: a near complete capsid that has released the RNA and the released RNA complexed to a small number of the capsid proteins. Upon slow buffer exchange to remove divalent cations at neutral pH, capsid protein interactions are disrupted. The BMV virions swell but there is no measurable loss of the RNA. Some of the virions break into small fragments, leading to an increase in the abundance of species with masses less than 1 MDa. The peak attributed to the BMV virion shifts to a higher mass with time. The mass increase is attributed to additional capsid proteins associating with the disrupted capsid protein-RNA complex, where the RNA is presumably partially exposed. It is likely that this pathway is more closely related to how the capsid disassembles in vivo, as it offers the advantage of protecting the RNA with the capsid protein until translation begins.

Photothermal microspectroscopy with Bessel-Gauss beams and reflective objectives.

Here, we investigate scanning photothermal microspectroscopic imaging of metal nanoparticles with reflective objectives. We show that correction-less collection of spectra from single spherical nanoparticles embedded in a polymer is possible over a wide spectral band, with large depth of focus, long working distance, and high lateral spatial resolution. We posit that these beneficial characteristics are inherent of the Bessel-Gauss character of the focused beam. When compared with other types of optical microscopy, the combination of these characteristics give photothermal imaging with reflective objectives unique appeal for material characterization applications.

Radiation Brightening from Virus-like Particles.

Concentration quenching is a well-known challenge in many fluorescence imaging applications. Here, we show that the optical emission from hundreds of chromophores confined onto the surface of a 28 nm diameter virus particle can be recovered under pulsed irradiation. We have found that as one increases the number of chromophores tightly bound to the virus surface, fluorescence quenching ensues at first, but when the number of chromophores per particle is nearing the maximum number of surface sites allowable, a sudden brightening of the emitted light and a shortening of the excited-state lifetime are observed. This radiation brightening occurs only under short pulse excitation; steady-state excitation is characterized by conventional concentration quenching for any number of chromophores per particle. The observed suppression of fluorescence quenching is consistent with efficient, collective relaxation at room temperature. Interestingly, radiation brightening disappears when the emitters' spatial and/or dynamic heterogeneity is increased, suggesting that the template structural properties may play a role that could be instrumental in developing virus-enabled imaging vectors that have optical properties qualitatively different than those of state-of-the-art biophotonic agents.

Intermittency of Deformation and the Elastic Limit of an Icosahedral Virus under Compression.

Viruses undergo mesoscopic morphological changes as they interact with host interfaces and in response to chemical cues. The dynamics of these changes, over the entire temporal range relevant to virus processes, are unclear. Here, we report on creep compliance experiments on a small icosahedral virus under uniaxial constant stress. We find that even at small stresses, well below the yielding point and generally thought to induce a Hookean response, strain continues to develop in time via sparse, randomly distributed, relatively rapid plastic events. The intermittent character of mechanical compliance only appears above a loading threshold, similar to situations encountered in granular flows and the plastic deformation of crystalline solids. The threshold load is much smaller for the empty capsids of the brome mosaic virus than for the wild-type virions. The difference highlights the involvement of RNA in stabilizing the assembly interface. Numerical simulations of spherical crystal deformation suggest intermittency is mediated by lattice defect dynamics and identify the type of compression-induced defect that nucleates the transition to plasticity.

In Vitro Assembly of Virus-Derived Designer Shells Around Inorganic Nanoparticles.

Nanoparticle-templated assembly of virus shells provides a promising approach to the production of hybrid nanomaterials and a potential avenue toward new mechanistic insights in virus phenomena originating in many-body effects, which cannot be understood from examining the properties of molecular subunits alone. This approach complements the successful molecular biology perspective traditionally used in virology, and promises a deeper understanding of viruses and virus-like particles through an expanded methodological toolbox. Here we present protocols for forming a virus coat protein shell around functionalized inorganic nanoparticles.

Self-assembly of convex particles on spherocylindrical surfaces.

The precise control of assembly and packing of proteins and colloids on curved surfaces has fundamental implications in nanotechnology. In this paper, we describe dynamical simulations of the self-assembly of conical subunits around a spherocylindrical template, and a continuum theory for the bending energy of a triangular lattice with spontaneous curvature on a surface with arbitrary curvature. We find that assembly depends sensitively on mismatches between subunit spontaneous curvature and the mean curvature of the template, as well as anisotropic curvature of the template (mismatch between the two principal curvatures). Our simulations predict assembly morphologies that closely resemble those observed in experiments in which virus capsid proteins self-assemble around metal nanorods. Below a threshold curvature mismatch, our simulations identify a regime of optimal assembly leading to complete, symmetrical particles. Outside of this regime we observe defective particles, whose morphologies depend on the degree of curvature mismatch. To learn how assembly is affected by the nonuniform curvature of a spherocylinder, we also study the simpler cases of assembly around spherical and cylindrical cores. Our results show that both the intrinsic (Gaussian) and extrinsic (mean) curvatures of a template play significant roles in guiding the assembly of anisotropic subunits, providing a rich design space for the formation of nanoscale materials.

Defects and Chirality in the Nanoparticle-Directed Assembly of Spherocylindrical Shells of Virus Coat Proteins.

Virus coat proteins of small isometric plant viruses readily assemble into symmetric, icosahedral cages encapsulating noncognate cargo, provided the cargo meets a minimal set of chemical and physical requirements. While this capability has been intensely explored for certain virus-enabled nanotechnologies, additional applications require lower symmetry than that of an icosahedron. Here, we show that the coat proteins of an icosahedral virus can efficiently assemble around metal nanorods into spherocylindrical closed shells with hexagonally close-packed bodies and icosahedral caps. Comparison of chiral angles and packing defects observed by in situ atomic force microscopy with those obtained from molecular dynamics models offers insight into the mechanism of growth, and the influence of stresses associated with intrinsic curvature and assembly pathways.

Layered Structure and Complex Mechanochemistry Underlie Strength and Versatility in a Bacterial Adhesive.

While designing synthetic adhesives that perform in aqueous environments has proven challenging, microorganisms commonly produce bioadhesives that efficiently attach to a variety of substrates, including wet surfaces. The aquatic bacterium Caulobacter crescentus uses a discrete polysaccharide complex, the holdfast, to strongly attach to surfaces and resist flow. The holdfast is extremely versatile and has impressive adhesive strength. Here, we used atomic force microscopy in conjunction with superresolution microscopy and enzymatic assays to unravel the complex structure of the holdfast and to characterize its chemical constituents and their role in adhesion. Our data support a model whereby the holdfast is a heterogeneous material organized as two layers: a stiffer nanoscopic core layer wrapped into a sparse, far-reaching, flexible brush layer. Moreover, we found that the elastic response of the holdfast evolves after surface contact from initially heterogeneous to more homogeneous. From a composition point of view, besides N-acetyl-d-glucosamine (NAG), the only component that had been identified to date, our data show that the holdfast contains peptides and DNA. We hypothesize that, while polypeptides are the most important components for adhesive force, the presence of DNA mainly impacts the brush layer and the strength of initial adhesion, with NAG playing a primarily structural role within the core. The unanticipated complexity of both the structure and composition of the holdfast likely underlies its versatility as a wet adhesive and its distinctive strength. Continued improvements in understanding of the mechanochemistry of this bioadhesive could provide new insights into how bacteria attach to surfaces and could inform the development of new adhesives.IMPORTANCE There is an urgent need for strong, biocompatible bioadhesives that perform underwater. To strongly adhere to surfaces and resist flow underwater, the bacterium Caulobacter crescentus produces an adhesive called the holdfast, the mechanochemistry of which remains undefined. We show that the holdfast is a layered structure with a stiff core layer and a polymeric brush layer and consists of polysaccharides, polypeptides, and DNA. The DNA appears to play a role in the structure of the brush layer and initial adhesion, the peptides in adhesive strength, and the polysaccharides in the structure of the core. The complex, multilayer organization and diverse chemistry described here underlie the distinctive adhesive properties of the holdfast and will provide important insights into the mechanisms of bacterial adhesion and bioadhesive applications.

Contact Mechanics of a Small Icosahedral Virus.

A virus binding to a surface causes stress of the virus cage near the contact area. Here, we investigate the potential role of substrate-induced structural perturbation in the mechanical response of virus particles to adsorption. This is particularly relevant to the broad category of viruses stabilized by weak noncovalent interactions. We utilize atomic force microscopy to measure height distributions of the brome mosaic virus upon adsorption from solution on atomically flat substrates and present a continuum model that captures our observations and provides estimates of elastic properties and of the interfacial energy of the virus, without recourse to indentation.