Raghuveer Parthasarathy

Conducting nanowires built by controlled self-assembly of amyloid fibers and selective metal deposition

Recent research in the field of nanometer-scale electronics has focused on the operating principles of small-scale devices and schemes to realize useful circuits. In contrast to established “top-down” fabrication techniques, molecular self-assembly is emerging as a “bottom-up” approach for fabricating nanostructured materials. Biological macromolecules, especially proteins, provide many valuable properties, but poor physical stability and poor electrical characteristics have prevented their direct use in electrical circuits. Here we describe the use of self-assembling amyloid protein fibers to construct nanowire elements. Self-assembly of a prion determinant from Saccharomyces cerevisiae, the N-terminal and middle region (NM) of Sup35p, produced 10-nm-wide protein fibers that were stable under a wide variety of harsh physical conditions. Their lengths could be roughly controlled by assembly conditions in the range of 60 nm to several hundred micrometers. A genetically modified NM variant that presents reactive, surface-accessible cysteine residues was used to covalently link NM fibers to colloidal gold particles. These fibers were placed across gold electrodes, and additional metal was deposited by highly specific chemical enhancement of the colloidal gold by reductive deposition of metallic silver and gold from salts. The resulting silver and gold wires were ≈100 nm wide. These biotemplated metal wires demonstrated the conductive properties of a solid metal wire, such as low resistance and ohmic behavior. With such materials it should be possible to harness the extraordinary diversity and specificity of protein functions to nanoscale electrical circuitry.

Electronic Transport in Metal Nanocrystal Arrays: The Effect of Structural Disorder on Scaling Behavior

We investigate the impact of structural disorder on electronic transport in gold nanocrystal monolayers. Arrays ranging from void-filled networks to well-ordered superlattices show clear voltage thresholds VT due to Coulomb blockade, and temperature-independent conduction indicative of quantum tunneling. Current-voltage characteristics of arrays with and without long-range structural order were found to collapse onto distinct scaling curves. The former follow a single power law: I V 2 VT z , z 2.25 6 0.1. The latter show additional structure, reflecting the underlying disordered topology.

Rapid, accurate particle tracking by calculation of radial symmetry centers.

I introduce an algorithm for subpixel localization of imaged objects based on an analytic, non-iterative calculation of the best-fit radial symmetry center. This approach yields tracking accuracies that are near theoretical limits, similarly to Gaussian fitting, but with orders-of-magnitude faster execution time, lower sensitivity to nearby particles and applicability to any radially symmetric intensity distribution. I demonstrate the method with several types of data, including super-resolution microscopy images.

Fluorescence Imaging of Membrane Dynamics

Imaging membrane dynamics is an important goal, motivated by the abundance of biochemical and biophysical events that are orchestrated at, or by, cellular membranes. The short length scales, fast timescales, and environmental requirements of membrane phenomena present challenges to imaging experiments. Several technical advances offer means to overcome these challenges, and we describe here three powerful techniques applicable to membrane imaging: total internal reflection fluorescence (TIRF) microscopy, fluorescence interference contrast (FLIC) microscopy, and fluorescence correlation spectroscopy (FCS). For each, we discuss the physics underpinning the approach, its practical implementation, and recent examples highlighting its achievements in exploring the membrane environment.

Curvature and spatial organization in biological membranes

Cellular membranes bend and curve into a multitude of shapes as they perform various functions. These deformations make use of the remarkable material properties of biological membranes inherent in their nature as two-dimensional fluids. The curvature of membranes is controlled by the constituent proteins and lipids, but conversely, curvature itself provides mechanisms for organizing mobile membrane molecules. In this article we survey recent experiments that have uncovered intriguing connections between mechanics and biochemistry at membranes, focusing on the influence of molecular shape on curvature, links between phase separation and curvature, and membrane bending at inter-cellular contacts. We describe the concepts that emerge from these studies, especially the existence of long-range, curvature-mediated mechanisms for spatial organization in membranes, and highlight open areas for future research.

Percolating through networks of random thresholds: Finite temperature electron tunneling in metal nanocrystal arrays

We investigate how temperature affects transport through large networks of nonlinear conductances with distributed thresholds. In monolayers of weakly coupled gold nanocrystals, quenched charge disorder produces a range of local thresholds for the onset of electron tunneling. Our measurements delineate two regimes separated by a crossover temperature T*. Up to T* the nonlinear zero-temperature shape of the current-voltage curves survives, but with a threshold voltage for conduction that decreases linearly with temperature. Above T* the threshold vanishes and the low-bias conductance increases rapidly with temperature. We develop a model that accounts for these findings and predicts T*.

A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy

Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems.

Direct patterning of self-assembled nanocrystal monolayers by electron beams

We demonstrate a method for laterally patterning metal nanocrystal monolayers. Extended monolayers are first self-assembled onto a solid substrate. Direct electron-beam exposure is then used to strip the dodecanethiol ligand coating from the nanocrystal cores, enabling the cores to stick to the underlying substrate. During a subsequent washing step in a solvent mixture, nanocrystals from the unexposed regions are removed and floated off, leaving behind the desired pattern.

Quantum Phase Transition of a Magnet in a Spin Bath

The excitation spectrum of a model magnetic system, LiHoF4, was studied with the use of neutron spectroscopy as the system was tuned to its quantum critical point by an applied magnetic field. The electronic mode softening expected for a quantum phase transition was forestalled by hyperfine coupling to the nuclear spins. We found that interactions with the nuclear spin bath controlled the length scale over which the excitations could be entangled. This generic result places a limit on our ability to observe intrinsic electronic quantum criticality.

Spatial and Temporal Features of the Growth of a Bacterial Species Colonizing the Zebrafish Gut

ABSTRACT The vertebrate intestine is home to microbial ecosystems that play key roles in host development and health. Little is known about the spatial and temporal dynamics of these microbial communities, limiting our understanding of fundamental properties, such as their mechanisms of growth, propagation, and persistence. To address this, we inoculated initially germ-free zebrafish larvae with fluorescently labeled strains of an Aeromonas species, representing an abundant genus in the zebrafish gut. Using light sheet fluorescence microscopy to obtain three-dimensional images spanning the gut, we quantified the entire bacterial load, as founding populations grew from tens to tens of thousands of cells over several hours. The data yield the first ever measurements of the growth kinetics of a microbial species inside a live vertebrate intestine and show dynamics that robustly fit a logistic growth model. Intriguingly, bacteria were nonuniformly distributed throughout the gut, and bacterial aggregates showed considerably higher growth rates than did discrete individuals. The form of aggregate growth indicates intrinsically higher division rates for clustered bacteria, rather than surface-mediated agglomeration onto clusters. Thus, the spatial organization of gut bacteria both relative to the host and to each other impacts overall growth kinetics, suggesting that spatial characterizations will be an important input to predictive models of host-associated microbial community assembly. IMPORTANCE Our intestines are home to vast numbers of microbes that influence many aspects of health and disease. Though we now know a great deal about the constituents of the gut microbiota, we understand very little about their spatial structure and temporal dynamics in humans or in any animal: how microbial populations establish themselves, grow, fluctuate, and persist. To address this, we made use of a model organism, the zebrafish, and a new optical imaging technique, light sheet fluorescence microscopy, to visualize for the first time the colonization of a live, vertebrate gut by specific bacteria with sufficient resolution to quantify the population over a range from a few individuals to tens of thousands of bacterial cells. Our results provide unprecedented measures of bacterial growth kinetics and also show the influence of spatial structure on bacterial populations, which can be revealed only by direct imaging. Our intestines are home to vast numbers of microbes that influence many aspects of health and disease. Though we now know a great deal about the constituents of the gut microbiota, we understand very little about their spatial structure and temporal dynamics in humans or in any animal: how microbial populations establish themselves, grow, fluctuate, and persist. To address this, we made use of a model organism, the zebrafish, and a new optical imaging technique, light sheet fluorescence microscopy, to visualize for the first time the colonization of a live, vertebrate gut by specific bacteria with sufficient resolution to quantify the population over a range from a few individuals to tens of thousands of bacterial cells. Our results provide unprecedented measures of bacterial growth kinetics and also show the influence of spatial structure on bacterial populations, which can be revealed only by direct imaging.