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Single-Molecule Imaging

Total internal reflection fluorescence microscopy (TIRFM)

allows us to image millions of individual molecules dynamically at solid/liquid and liquid/liquid interfaces.  As with all single-molecule experimental methods, these experiments go beyond understanding the average behavior of adsorbates and enable us to probe variability in surface chemistry, molecular conformations, and adsorbate dynamics.  Making such measurements, we often find that the behavior is much richer and more interesting than previously suspected.                

Molecular Transport at Interfaces

The dynamic behavior of molecules and nanoparticles at

interfaces leads to complex phenomena, where heterogeneity may arise from spatial variation of the interface itself, from molecular structures, or through inhomogeneous dynamic behavior. To obtain relevant information about these complex dynamics, we have developed highly multiplexed single-molecule/single-particle tracking methods that acquire large numbers of trajectories permitting rigorous analysis using statistical modeling and machine learning. A specific discovery that was enabled by these methods involves the ubiquitous intermittent motion (i.e. “hopping diffusion”) of molecules at interfaces, which was explicitly confirmed using 3D double-helix point spread function imaging. Ongoing research studies the impacts of interfacial dynamics on various technological applications, membrane biophysics, and separations processes.

Mapping Using Accumulated Probe

Trajectories (MAPT)

In MAPT, a time series of images is obtained using single-molecule TIFRM, and imaging processing is used to determine the locations of all the individual molecules. By tracking these locations through a time series, physical properties of probe/surface interactions (such as adsorption rates, local diffusion, desorption probability and surface coverage) are determined.  By distributing these events to appropriate locations on the surface, one can determine characteristic values as a function of position. The use of single-molecule localization makes the MAPT analysis intrinsically super-resolution and we have demonstrated resolution of ~100 nm in our un-optimized initial results.  In contrast with all previous super-resolution methods, MAPT can be used on any surface and provides absolute quantitative values of physical interaction properties. Homogenous surfaces can then be characterized and used as “calibration” points to connect small scale heterogeneities to specific surface chemistries.  Using such maps, we found the first experimental evidence for desorption-mediated diffusion at the solid-liquid interface. 

MAPT images of a photopatterned surface using multiple modes of contrast. (a) adsorption rate (1013 µm-2s-1M-1) (b) average diffusion coefficient (µm2/s) (c) desorption probability per 400 ms and (d) surface coverage/occupancy (1013 µm-2s-1M-1).

Amyloidogenic Protein Refolding in

Lipidic and Polymeric Environments

Amyloid fibrils are highly stable protein species that are correlated to numerous diseases, including Alzheimer's and Parkinson's. In the Schwartz Lab, we utilize functional biomaterials to stabilize amyloidogenic proteins, inhibit their fibrillization, and in some cases, even completely reverse that fibrillization. These functional materials, namely lipid vesicles and polymer brush functionalized nanoparticles, can be tuned to be optimally stabilizing for specific proteins. Numerous techniques including fluorescence spectroscopy, circular dichroism spectroscopy, and single molecule methods give insight into how these materials work.

Dynamics in Confined Environments

Understanding molecular behavior under confinement is critical to various applications including biosensing, material synthesis, intracellular and extracellular transport, and a number of industrial separation processes.  In order to understand these environments, we have investigated systems ranging from surface crowding at the solid-liquid interface to structurally-induced confinement within synthesized materials.  Based on single molecule trajectories, we have characterized dynamics using a variety of empirical and model dependent analyses.  For example, it was discovered that poly(ethylene glycol) chains exhibited behavior consistent with the generalized continuous-time random walk model over a large range of surface coverage, from the dilute to well above the surface overlap concentration.   Additionally, the polymer molecules had three distinct regimes of dynamics as a function of surface crowding, as shown by measurements of short-time diffusion coefficients, mean squared displacement, and surface waiting times.  In other experiments, nanoparticle trajectories in porous polymer membranes were analyzed in order to measure the tortuosity of the membrane, which was found to be a function of the operating conditions and not merely a constant value.  Moreover, distributions of the tortuosity indicated a large degree of spatiotemporal heterogeneity within the membranes, where anomalous, highly confined regions appeared to exist.  Further experiments investigating dynamics in inverse opal structures are currently underway, where individual particle trajectories are being investigated in the context of the narrow escape problem in order to provide a better understanding of confined diffusion in periodic nanostructures.