Effect of polyacrylamide gel elasticity on collagen type II fibril assembly.

Collagen type II fibrils provide structural integrity to the articular cartilage extracellular matrix. However, the conditions that control the fibril radial size scale, distribution, and formation inside of dense networks are not well understood. We have investigated how surrounding elastic networks affect fibril formation by observing the structure and dynamics of collagen type II in model polyacrylamide gels of varying moduli.

Quantitative Advantages of Corrosion Sensing Using Fluorescence, Microscopy, and Single-Molecule Detection.

The corrosion of metals and alloys is a fundamental issue in modern society. Understanding the mechanisms that cause and prevent corrosion is integral to saving millions of dollars each year and to ensure the safe use of infrastructure subject to the hazardous degrading effects of corrosion. Despite this, corrosion detection techniques have lacked precise, quantitative information, with industries taking a top-down, macroscale approach to analyzing corrosion with tests that span months to years and yield qualitative information.

Cross-Correlation Increases Sampling in Diffusion-Based Super-Resolution Optical Fluctuation Imaging.

Correlation signal processing of optical three-dimensional (, , ) data can produce super-resolution images. The second-order cross-correlation function has been documented to produce super-resolution imaging with static and blinking emitters but not for diffusing emitters. Here, we both analytically and numerically demonstrate cross-correlation analysis for diffusing particles.

A practical guide to light-sheet microscopy for nanoscale imaging: Looking beyond the cell.

We present a comprehensive guide to light-sheet microscopy (LSM) to assist scientists in navigating the practical implementation of this microscopy technique. Emphasizing the applicability of LSM to image both static microscale and nanoscale features, as well as diffusion dynamics, we present the fundamental concepts of microscopy, progressing through beam profile considerations, to image reconstruction. We outline key practical decisions in constructing a home-built system and provide insight into the alignment and calibration processes.

Cross-correlation increases sampling in diffusion-based super-resolution optical fluctuation imaging.

Correlation signal processing of optical three-dimensional (x, y, t) data can produce super-resolution images. The second order cross-correlation function has been documented to produce super-resolution imaging with static and blinking emitters but not for diffusing emitters. Here, we both analytically and numerically demonstrate cross-correlation analysis for diffusing particles.

Spatially Resolving Size Effects on Diffusivity in Nanoporous Extracellular Matrix-like Materials with Fluorescence Correlation Spectroscopy Super-Resolution Optical Fluctuation Imaging.

It is well documented that the nanoscale structures within porous microenvironments greatly impact the diffusion dynamics of molecules. However, how the interaction between the environment and molecules influences the diffusion dynamics has not been thoroughly explored. Here, we show that fluorescence correlation spectroscopy super-resolution optical fluctuation imaging (fcsSOFI) can be used to accurately measure the diffusion dynamics of molecules within varying matrices such as nanopatterned surfaces and porous agarose hydrogels.

Peroxisome biogenesis initiated by protein phase separation.

Peroxisomes are organelles that carry out β-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction. Among disease-causing variant genes are those required for protein transport into peroxisomes.

Native diffusion of fluorogenic turn-on dyes accurately report interfacial chemical reaction locations.

Single-molecule fluorescence microscopy with "turn-on" dyes that change fluorescent state after a reaction report on the chemistry of interfaces relevant to analytical and bioanalytical chemistry. Paramount to accurately understanding the phenomena at the ultimate detection limit of a single molecule is ensuring fluorophore properties such as diffusion do not obscure the chemical reaction of interest. Here, we develop Monte Carlo simulations of a dye that undergoes reduction to turn-on at the cathode of a corroded iron surface taking into account the diffusion of the dye molecules in a total internal reflection fluorescence (TIRF) excitation volume, location of the cathode, and chemical reactions.

Single-Molecule Imaging in Commercial Stationary Phase Particles Using Highly Inclined and Laminated Optical Sheet Microscopy.

We resolve the three-dimensional, nanoscale locations of single-molecule analytes within commercial stationary phase materials using highly inclined and laminated optical sheet (HILO) microscopy. Single-molecule fluorescence microscopy of chromatography can reveal the molecular heterogeneities that lead to peak broadening, but past work has focused on surfaces designed to mimic stationary phases, which have different physical and chemical properties than the three-dimensional materials used in real columns and membranes. To extend single-molecule measurements to commercial stationary phases, we immobilize individual stationary phase particles and modify our microscope for imaging at further depths with HILO, a method which was originally developed to resolve single molecules in cells of comparable size to column packing materials (∼5-10 μm).

A blind benchmark of analysis tools to infer kinetic rate constants from single-molecule FRET trajectories.

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking.

Super-resolution fluorescence imaging of extracellular environments.

The extracellular matrix (ECM) is an important biophysical environment that plays a role in a number of physiological processes. The ECM is highly dynamic, with changes occurring as local, nanoscale, physicochemical variations in physical confinement and chemistry from the perspective of biological molecules. The length and time scale of ECM dynamics are challenging to measure with current spectroscopic techniques.

Computationally-efficient spatiotemporal correlation analysis super-resolves anomalous diffusion.

Anomalous diffusion dynamics in confined nanoenvironments govern the macroscale properties and interactions of many biophysical and material systems. Currently, it is difficult to quantitatively link the nanoscale structure of porous media to anomalous diffusion within them. Fluorescence correlation spectroscopy super-resolution optical fluctuation imaging (fcsSOFI) has been shown to extract nanoscale structure and Brownian diffusion dynamics within gels, liquid crystals, and polymers, but has limitations which hinder its wider application to more diverse, biophysically-relevant datasets.

Fluorophores "Turned-On" by Corrosion Reactions Can Be Detected at the Single-Molecule Level.

We demonstrate that fluorogenic molecules that "turn-on" upon redox reactions can sense the corrosion of iron at the single-molecule scale. We first observe the cathodic reduction of nonfluorescent resazurin to fluorescent resorufin in the presence of iron in bulk solution. The progression of corrosion is seen as a color change that is quantified as an increase in fluorescence emission intensity.

Transforming Separation Science with Single-Molecule Methods.

Empirical optimization of the multiscale parameters underlying chromatographic and membrane separations leads to enormous resource waste and production costs. A bottom-up approach to understand the physical phenomena underlying challenges in separations is possible with single-molecule observations of solute-stationary phase interactions. We outline single-molecule fluorescence techniques that can identify key interactions under ambient conditions.

Real-Time Measurement of Polymer Brush Dynamics Using Silicon Photonic Microring Resonators: Analyte Partitioning and Interior Brush Kinetics.

Polymer brushes are found in biomedical and industrial technologies, where they exhibit functionalities considerably dependent on polymer brush-solvent-analyte interactions. It remains a difficult challenge to quickly analyze solvent-swollen polymer brushes, both at the solvent-polymer brush interface and in the brush interior, as well as to monitor the kinetics of interaction of solvent-swollen brushes with key analytes. Here, we demonstrate the novel use of silicon photonic microring resonators to characterize swollen polymer brush-analyte interactions.

Probing Inhomogeneous Diffusion in the Microenvironments of Phase-Separated Polymers under Confinement.

Biomolecular condensates formed by liquid-liquid phase separation of proteins and nucleic acids have been recently discovered to be prevalent in biology. These dynamic condensates behave like biochemical reaction vessels, but little is known about their structural organization and biophysical properties, which are likely related to condensate size. Thus, it is critical that we study them on scales found in vivo.

Soluble Zwitterionic Poly(sulfobetaine) Destabilizes Proteins.

The widespread interest in neutral, water-soluble polymers such as poly(ethylene glycol) (PEG) and poly(zwitterions) such as poly(sulfobetaine) (pSB) for biomedical applications is due to their widely assumed low protein binding. Here we demonstrate that pSB chains in solution can interact with proteins directly. Moreover, pSB can reduce the thermal stability and increase the protein folding cooperativity relative to proteins in buffer or in PEG solutions.

Competitive multicomponent anion exchange adsorption of proteins at the single molecule level.

We use single molecule spectroscopy to study a multicomponent, competitive protein adsorption system. Fluorescently-labeled α-lactalbumin proteins are super-resolved adsorbing to cationic anion-exchange ligands in the presence of a competitor, insulin. We find that the competitor reduces the number of binding events by blocking ligands throughout the observed measurement time while the single-site adsorption kinetics are unchanged.

Direct Imaging of Protein Stability and Folding Kinetics in Hydrogels.

We apply fast relaxation imaging (FReI) as a novel technique for investigating the folding stability and dynamics of proteins within polyacrylamide hydrogels, which have diverse and widespread uses in biotechnology. FReI detects protein unfolding in situ by imaging changes in fluorescence resonance energy transfer (FRET) after temperature jump perturbations. Unlike bulk measurements, diffraction-limited epifluorescence imaging combined with fast temperature perturbations reveals the impact of local environment effects on protein-biomaterial compatibility.

Optimization of Spectral and Spatial Conditions to Improve Super-Resolution Imaging of Plasmonic Nanoparticles.

Interactions between fluorophores and plasmonic nanoparticles modify the fluorescence intensity, shape, and position of the observed emission pattern, thus inhibiting efforts to optically super-resolve plasmonic nanoparticles. Herein, we investigate the accuracy of localizing dye fluorescence as a function of the spectral and spatial separations between fluorophores (Alexa 647) and gold nanorods (NRs). The distance at which Alexa 647 interacts with NRs is varied by layer-by-layer polyelectrolyte deposition while the spectral separation is tuned by using NRs with varying localized surface plasmon resonance (LSPR) maxima.

Single-Molecule Kinetics of Protein Adsorption on Thin Nylon-6,6 Films.

Understanding and controlling protein adsorption on surfaces is critical to a range of biological and materials applications. Kinetic details that provide the equilibrium and nonequilibrium mechanisms are difficult to acquire. In this work, single-molecule fluorescence microscopy was used to study the adsorption of Alexa 555 labeled α-lactalbumin (α-LA) on two chemically identical but morphologically different polymer surfaces: flat and porous nylon-6,6 thin films.

Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation.

The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration.

pH-dependence of single-protein adsorption and diffusion at a liquid chromatographic interface.

pH is a common mobile phase variable used to control protein separations due to the tunable nature of amino acid and adsorbent charge. Like other column variables such as column density and ligand loading density, pH is usually optimized empirically. Single-molecule spectroscopy extracts molecular-scale data to provide a framework for mechanistic optimization of pH.

Ensemble and single-molecule biophysical characterization of D17.4 DNA aptamer-IgE interactions.

Background: The IgE-binding DNA aptamer 17.4 is known to inhibit the interaction of IgE with the high-affinity IgE Fc receptor FcεRI. While this and other aptamers have been widely used and studied, there has been relatively little investigation of the kinetics and energetics of their interactions with their targets, by either single-molecule or ensemble methods.