Light-sheet microscopy with digital Fourier analysis measures transport properties over large field-of-view.

Using light-sheet microscopy combined with digital Fourier methods we probe the dynamics of colloidal samples and DNA molecules. This combination, referred to as selective-plane illumination differential dynamic microscopy (SPIDDM), has the benefit of optical sectioning to study, with minimal photobleaching, thick samples allowing us to measure the diffusivity of colloidal particles at high volume fractions. Further, SPIDDM exploits the inherent spatially-varying thickness of Gaussian light-sheets. Where the excitation sheet is most focused, we capture high spatial frequency dynamics as the signal-to-background is high. In thicker regions, we capture the slower dynamics as diffusion out of the sheet takes longer.

Effect of molecular architecture on ring polymer dynamics in semidilute linear polymer solutions.

Understanding the dynamics of ring polymers is a particularly challenging yet interesting problem in soft materials. Despite recent progress, a complete understanding of the nonequilibrium behavior of ring polymers has not yet been achieved. In this work, we directly observe the flow dynamics of DNA-based rings in semidilute linear polymer solutions using single molecule techniques. Our results reveal strikingly large conformational fluctuations of rings in extensional flow long after the initial transient stretching process has terminated, which is observed even at extremely low concentrations (0.025 c*) of linear polymers in the background solution. The magnitudes and characteristic timescales of ring conformational fluctuations are determined as functions of flow strength and polymer concentration. Our results suggest that ring conformational fluctuations arise due to transient threading of linear polymers through open ring chains stretching in flow.

Topology-dependent anomalous dynamics of ring and linear DNA are sensitive to cytoskeleton crosslinking.

Cytoskeletal crowding plays a key role in the diffusion of DNA molecules through the cell, acting as a barrier to effective intracellular transport and conformational stability required for processes such as transfection, viral infection, and gene therapy. Here, we elucidate the transport properties and conformational dynamics of linear and ring DNA molecules diffusing through entangled and crosslinked composite networks of actin and microtubules. We couple single-molecule conformational tracking with differential dynamic microscopy to reveal that ring and linear DNA exhibit unexpectedly distinct transport properties that are influenced differently by cytoskeleton crosslinking. Ring DNA coils are swollen and undergo heterogeneous and biphasic subdiffusion that is hindered by crosslinking. Conversely, crosslinking actually facilitates the single-mode subdiffusion that compacted linear chains exhibit. Our collective results demonstrate that transient threading by cytoskeleton filaments plays a key role in the dynamics of ring DNA, whereas the mobility of the cytoskeleton dictates transport of linear DNA.

Crowding Induces Entropically-Driven Changes to DNA Dynamics That Depend on Crowder Structure and Ionic Conditions.

Macromolecular crowding plays a principal role in a wide range of biological processes including gene expression, chromosomal compaction, and viral infection. However, the impact that crowding has on the dynamics of nucleic acids remains a topic of debate. To address this problem, we use single-molecule fluorescence microscopy and custom particle-tracking algorithms to investigate the impact of varying macromolecular crowding conditions on the transport and conformational dynamics of large DNA molecules. Specifically, we measure the mean-squared center-of-mass displacements, as well as the conformational size, shape, and fluctuations, of individual 115 kbp DNA molecules diffusing through various in vitro solutions of crowding polymers. We determine the role of crowder structure and concentration, as well as ionic conditions, on the diffusion and configurational dynamics of DNA. We find that branched, compact crowders (10 kDa PEG, 420 kDa Ficoll) drive DNA to compact, whereas linear, flexible crowders (10, 500 kDa dextran) cause DNA to elongate. Interestingly, the extent to which DNA mobility is reduced by increasing crowder concentrations appears largely insensitive to crowder structure (branched vs. linear), despite the highly different configurations DNA assumes in each case. We also characterize the role of ionic conditions on crowding-induced DNA dynamics. We show that both DNA diffusion and conformational size exhibit an emergent non-monotonic dependence on salt concentration that is not seen in the absence of crowders.