ABSTRACT
Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)-for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.
Subject(s)
Bromovirus , RNA Viruses , Bromovirus/genetics , Bromovirus/metabolism , Capsid/metabolism , RNA Viruses/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , Virion/genetics , Virion/metabolismABSTRACT
Interferometric scattering microscopy can image the dynamics of nanometer-scale systems. The typical approach to analyzing interferometric images involves intensive processing, which discards data and limits the precision of measurements. We demonstrate an alternative approach: modeling the interferometric point spread function and fitting this model to data within a Bayesian framework. This approach yields best-fit parameters, including the particle's three-dimensional position and polarizability, as well as uncertainties and correlations between these parameters. Building on recent work, we develop a model that is parameterized for rapid fitting. The model is designed to work with Hamiltonian Monte Carlo techniques that leverage automatic differentiation. We validate this approach by fitting the model to interferometric images of colloidal nanoparticles. We apply the method to track a diffusing particle in three dimensions, to directly infer the diffusion coefficient of a nanoparticle without calculating a mean-square displacement, and to quantify the ejection of DNA from an individual lambda phage virus, demonstrating that the approach can be used to infer both static and dynamic properties of nanoscale systems.
ABSTRACT
Self-assembly is widely used by biological systems to build functional nanostructures, such as the protein capsids of RNA viruses. But because assembly is a collective phenomenon involving many weakly interacting subunits and a broad range of timescales, measurements of the assembly pathways have been elusive. We use interferometric scattering microscopy to measure the assembly kinetics of individual MS2 bacteriophage capsids around MS2 RNA. By recording how many coat proteins bind to each of many individual RNA strands, we find that assembly proceeds by nucleation followed by monotonic growth. Our measurements reveal the assembly pathways in quantitative detail and also show their failure modes. We use these results to critically examine models of the assembly process.
Subject(s)
Capsid/metabolism , Levivirus/physiology , RNA Viruses/physiology , RNA, Viral/genetics , Virion/physiology , Virus Assembly , Capsid/chemistry , Capsid Proteins/chemistry , Capsid Proteins/genetics , Capsid Proteins/metabolism , Genome, Viral , Kinetics , Levivirus/chemistry , Levivirus/genetics , Levivirus/growth & development , RNA Viruses/chemistry , RNA Viruses/genetics , RNA Viruses/growth & development , RNA, Viral/chemistry , RNA, Viral/metabolism , Virion/chemistry , Virion/geneticsABSTRACT
The technique of phase contrast imaging, combined with tomographic reconstructions, can rapidly measure ultrasonic fields propagating in water, including ultrasonic fields with complex wavefront shapes, which are difficult to characterize with standard hydrophone measurements. Furthermore, the technique can measure the absolute pressure amplitudes of ultrasonic fields without requiring a pressure calibration. Absolute pressure measurements have been previously demonstrated using optical imaging methods for ultrasonic frequencies below 2.5 MHz. The present work demonstrates that phase contrast imaging can accurately measure ultrasonic fields with frequencies up to 20 MHz and pressure amplitudes near 10 kPa. Accurate measurements at high ultrasonic frequencies are performed by tailoring the measurement conditions to limit optical diffraction as guided by a simple dimensionless parameter. In some situations, differences between high frequency measurements made with the phase contrast method and a calibrated hydrophone become apparent, and the reasons for these differences are discussed. Extending optical imaging measurements to high ultrasonic frequencies could facilitate quantitative applications of ultrasound measurements in nondestructive testing and medical therapeutics and diagnostics such as photoacoustic imaging.
ABSTRACT
Significance: Mueller matrix polarimetry (MMP) and spatial frequency domain imaging (SFDI) are wide-field optical imaging modalities that differentiate tissue primarily by structure alignment and photon transport coefficient, respectively. Because these effects can be related, combining MMP and SFDI may enhance tissue differentiation beyond the capability of each modality alone. Aim: An instrument was developed to combine MMP and SFDI with the goal of testing whether it enhances contrast of features in reflection mode. Approach: The instrument was constructed using liquid crystal elements for polarization control, a digital light processing projector for generating sinusoidal illumination patterns, and a digital camera for imaging. A theoretical analysis shows that the SFD Mueller matrix is complex-valued and does not follow the same behavior as a regular Mueller matrix. Images were acquired from an anisotropic tissue phantom, an optical fiber bundle, and cerebellum, thalamus, and cerebrum tissues. Results: The measurement results suggest that singly scattered, few scattered, and diffusely scattered photon paths can be distinguished in some of the samples investigated. The combined imaging modality yields additional spatial frequency phase information, which highlights paths having only a few scattering events. Conclusions: The combination of MMP and SFDI offers contrast mechanisms inaccessible by each modality used alone.
Subject(s)
Optical Imaging , Phantoms, Imaging , Optical Imaging/methods , Anisotropy , Spectrum AnalysisABSTRACT
SIGNIFICANCE: The polymer, polydimethylsiloxane (PDMS), has been increasingly used to make tissue simulating phantoms due to its excellent processability, durability, flexibility, and limited tunability of optical, mechanical, and thermal properties. We report on a robust technique to fabricate PDMS-based tissue-mimicking phantoms where the broad range of scattering and absorption properties are independently adjustable in the visible- to near-infrared wavelength range from 500 to 850 nm. We also report on an analysis method to concisely quantify the phantoms' broadband characteristics with four parameters. AIM: We report on techniques to manufacture and characterize solid tissue-mimicking phantoms of PDMS polymers. Tunability of the absorption (µa ( λ ) ) and reduced scattering coefficient spectra (µs'(λ)) in the wavelength range of 500 to 850 nm is demonstrated by adjusting the concentrations of light absorbing carbon black powder (CBP) and light scattering titanium dioxide powder (TDP) added into the PDMS base material. APPROACH: The µa ( λ ) and µs'(λ) of the phantoms were obtained through measurements with a broadband integrating sphere system and by applying an inverse adding doubling algorithm. Analyses of µa ( λ ) and µs'(λ) of the phantoms, by fitting them to linear and power law functions, respectively, demonstrate that independent control of µa ( λ ) and µs'(λ) is possible by systematically varying the concentrations of CBP and TDP. RESULTS: Our technique quantifies the phantoms with four simple fitting parameters enabling a concise tabulation of their broadband optical properties as well as comparisons to the optical properties of biological tissues. We demonstrate that, to a limited extent, the scattering properties of our phantoms mimic those of human tissues of various types. A possible way to overcome this limitation is demonstrated with phantoms that incorporate polystyrene microbead scatterers. CONCLUSIONS: Our manufacturing and analysis techniques may further promote the application of PDMS-based tissue-mimicking phantoms and may enable robust quality control and quality checks of the phantoms.
Subject(s)
Dimethylpolysiloxanes , Humans , Phantoms, ImagingABSTRACT
Tracking and analyzing the individual diffusion of nanoscale objects such as proteins and viruses is an important methodology in life science. Here, we show a sensor that combines the efficiency of light line illumination with the advantages of fluidic confinement. Tracking of freely diffusing nano-objects inside water-filled hollow core fibers with core diameters of tens of micrometers using elastically scattered light from the core mode allows retrieving information about the Brownian motion and the size of each particle of the investigated ensemble individually using standard tracking algorithms and the mean squared displacement analysis. Specifically, we successfully measure the diameter of every gold nanosphere in an ensemble that consists of several hundreds of 40 nm particles, with an individual precision below 17% (±8 nm). In addition, we confirm the relevance of our approach with respect to bioanalytics by analyzing 70 nm λ-phages. Overall these features, together with the strongly reduced demand for memory space, principally allows us to record thousands of frames and to achieve high frame rates for high precision tracking of nanoscale objects.
Subject(s)
Gold , Metal Nanoparticles , Motion , Nanospheres , Bacteriophage lambda , DiffusionABSTRACT
Spectrally resolved photoacoustic imaging is promising for label-free imaging in optically scattering materials. However, this technique often requires acquisition of a separate image at each wavelength of interest. This reduces imaging speeds and causes errors if the sample changes in time between images acquired at different wavelengths. We demonstrate a solution to this problem by using dual-comb spectroscopy for photoacoustic measurements. This approach enables a photoacoustic measurement at thousands of wavelengths simultaneously. In this technique, two optical-frequency combs are interfered on a sample and the resulting pressure wave is measured with an ultrasound transducer. This acoustic signal is processed in the frequency-domain to obtain an optical absorption spectrum. For a proof-of-concept demonstration, we measure photoacoustic signals from polymer films. The absorption spectra obtained from these measurements agree with those measured using a spectrophotometer. Improving the signal-to-noise ratio of the dual-comb photoacoustic spectrometer could enable high-speed spectrally resolved photoacoustic imaging.
ABSTRACT
Particles bound to fluid-fluid interfaces are widely used to study self-assembly and to make materials such as Pickering emulsions. In both contexts, the lateral interactions between such particles have been studied extensively. However, much less is known about the normal interactions between a particle and the interface prior to contact. We use digital holographic microscopy to measure the dynamics of individual micrometer-size colloidal particles as they approach an interface between an aqueous phase and oil. Our measurements show that the interaction between the particle and interface changes nonmonotonically as a function of salt concentration, from repulsive at 1 mM to attractive at tens of mM to negligible at 100 mM and attractive again above 200 mM. In the attractive regimes, the particles can bind to the interface at nanometer-scale separation without breaching it. Classical Derjaguin-Landau-Verwey-Overbeek theory does not explain these observations. However, a theory that accounts for nonlinear screening and correlations between the ions does predict the nonmonotonic dependence on salt concentration and produces trajectories that agree with experimental data. We further show that the normal interactions determine the lateral interactions between particles that are bound to the interface. Because the interactions we observe occur at salt concentrations used to make Pickering emulsions and other particle-laden interfaces, our results suggest that particle arrangements at the interface are likely out of equilibrium on experimental timescales.
ABSTRACT
A complete understanding of the cellular pathways involved in viral infections will ultimately require a diverse arsenal of experimental techniques, including methods for tracking individual viruses and their interactions with the host. Here we demonstrate the use of holographic microscopy to track the position, orientation, and DNA content of unlabeled bacteriophages (phages) in solution near a planar, functionalized glass surface. We simultaneously track over 100 individual λ phages at a rate of 100 Hz across a 33 µm × 33 µm portion of the surface. The technique determines the in-plane motion of the phage to nanometer precision, and the height of the phage above the surface to 100 nm precision. Additionally, we track the DNA content of individual phages as they eject their genome following the addition of detergent-solubilized LamB receptor. The technique determines the fraction of DNA remaining in the phage to within 10% of the total 48.5 kilobase pairs. Analysis of the data reveals that under certain conditions, λ phages move along the surface with their heads down and intermittently stick to the surface by their tails, causing them to stand up. Furthermore, we find that in buffer containing high concentrations of both monovalent and divalent salts, λ phages eject their entire DNA in about 7 s. Taken together, these measurements highlight the potential of holographic microscopy to resolve the fast kinetics of the early stages of phage infection.