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.
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.
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-ofconcept 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.
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.
The formation of a viral capsidthe highly-ordered protein shell that surrounds the genome of a virusis the canonical example of self-assembly 1 . The capsids of many positive-sense RNA viruses spontaneously assemble from in vitro mixtures of the coat protein and RNA 2 . The high yield of proper capsids that assemble is 10 remarkable, given their structural complexity: 180 identical proteins must arrange into three distinct local congurations to form an icosahedral capsid with a triangulation number of 3 (T = 3) 1 . Despite a wealth of data from structural studies 35 and simulations 610 , even the most fundamental questions about how these 15 structures assemble remain unresolved. Experiments have not determined whether the assembly pathway involves aggregation or nucleation, or how the RNA controls the process. Here we use interferometric scattering microscopy 11,12 to directly observe the in vitro assembly kinetics of individual, unlabeled capsids of bac-20 teriophage MS2. By measuring how many coat proteins bind to each of many individual MS2 RNA strands on time scales from 1 ms to 900 s, we nd that the start of assembly is broadly distributed in time and is followed by a rapid increase in the number of bound proteins. These measurements provide strong evidence 25 for a nucleation-and-growth pathway. We also nd that malformed structures assemble when multiple nuclei appear on the same RNA before the rst nucleus has nished growing. Our measurements reveal the complex assembly pathways for viral capsids around RNA in quantitative detail, including the nucleation threshold, nu-30 cleation time, growth time, and constraints on the critical nucleus size. These results may inform strategies for engineering synthetic capsids 13 or for derailing the assembly of pathogenic viruses 14 .
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