Improved Analysis for Determining Diffusion Coefficients from Short, Single-Molecule Trajectories with Photoblinking

Two maximum likelihood estimation (MLE) methods were developed for optimizing the analysis of single-molecule trajectories that include phenomena such as experimental noise, photoblinking, photobleaching, and translation or rotation out of the collection plane. In particular, short, single-molecule trajectories with photoblinking were studied, and our method was compared to existing analytical techniques applied to simulated data. The optimal method for various experimental cases was established, and the optimized MLE method was applied to a real experimental system: single-molecule diffusion of fluorescent molecular machines known as nanocars

Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging

Porous materials such as cellular cytosol, hydrogels, and block copolymers have nanoscale features that determine macroscale properties. Characterizing the structure of nanopores is difficult with current techniques due to imaging, sample preparation, and computational challenges. We produce a super-resolution optical image that simultaneously characterizes the nanometer dimensions of and diffusion dynamics within porous structures by correlating stochastic fluctuations from diffusing fluorescent probes in the pores of the sample, dubbed here as “fluorescence correlation spectroscopy super-resolution optical fluctuation imaging” or “fcsSOFI”. Simulations demonstrate that structural features and diffusion properties can be accurately obtained at sub-diffraction-limited resolution. We apply our technique to image agarose hydrogels and aqueous lyotropic liquid crystal gels. The heterogeneous pore resolution is improved by up to a factor of 2, and diffusion coefficients are accurately obtained through our method compared to diffraction-limited fluorescence imaging and single-particle tracking. Moreover, fcsSOFI allows for rapid and high-throughput characterization of porous materials. fcsSOFI could be applied to soft porous environments such hydrogels, polymers, and membranes in addition to hard materials such as zeolites and mesoporous silica

Super Temporal-Resolved Microscopy (STReM)

Super-resolution microscopy typically achieves high spatial resolution, but the temporal resolution remains low. We report super temporal-resolved microscopy (STReM) to improve the temporal resolution of 2D super-resolution microscopy by a factor of 20 compared to that of the traditional camera-limited frame rate. This is achieved by rotating a phase mask in the Fourier plane during data acquisition and then recovering the temporal information by fitting the point spread function (PSF) orientations. The feasibility of this technique is verified with both simulated and experimental 2D adsorption/desorption and 2D emitter transport. When STReM is applied to measure protein adsorption at a glass surface, previously unseen dynamics are revealed

Charge-Dependent Transport Switching of Single Molecular Ions in a Weak Polyelectrolyte Multilayer

The tunable nature of weak polyelectrolyte multilayers makes them ideal candidates for drug loading and delivery, water filtration, and separations, yet the lateral transport of charged molecules in these systems remains largely unexplored at the single molecule level. We report the direct measurement of the charge-dependent, pH-tunable, multimodal interaction of single charged molecules with a weak polyelectrolyte multilayer thin film, a 10 bilayer film of poly­(acrylic acid) and poly­(allylamine hydrochloride) PAA/PAH. Using fluorescence microscopy and single-molecule tracking, two modes of interaction were detected: (1) adsorption, characterized by the molecule remaining immobilized in a subresolution region and (2) diffusion trajectories characteristic of hopping (<i>D</i> ∼ 10^–9 cm²/s). Radius of gyration evolution analysis and comparison with simulated trajectories confirmed the coexistence of the two transport modes in the same single molecule trajectories. A mechanistic explanation for the probe and condition mediated dynamics is proposed based on a combination of electrostatics and a reversible, pH-induced alteration of the nanoscopic structure of the film. Our results are in good agreement with ensemble studies conducted on similar films, confirm a previously-unobserved hopping mechanism for charged molecules in polyelectrolyte multilayers, and demonstrate that single molecule spectroscopy can offer mechanistic insight into the role of electrostatics and nanoscale tunability of transport in weak polyelectrolyte multilayers

Excitonic Energy Migration in Conjugated Polymers: The Critical Role of Interchain Morphology

Excitonic energy migration was studied using single molecule spectroscopy of individual conjugated polymer (CP) chains and aggregates. To probe the effect of interchain morphology on energy migration in CP, tailored interchain morphologies were achieved using solvent vapor annealing to construct polymer aggregates, which were then studied with single aggregate spectroscopy. We report that highly ordered interchain packing in <i>regioregular</i> poly­(3-hexylthiophene) (<i>rr</i>-P3HT) enables long-range interchain energy migration, while disordered packing in <i>regiorandom</i> poly­(3-hexylthiophene) (<i>rra</i>-P3HT), even in aggregates of just a few chains, can dramatically impede the interchain mechanism. In contrast to <i>rr</i>-P3HT, interchain energy migration in poly­(3-(2′-methoxy-5′-octylphenyl)­thiophene) (POMeOPT), a polythiophene derivative with bulky side chains, can be completely inhibited. We use simulated structures to show that the reduction in interchain coupling is not due simply to increased packing distance between backbones of different chains, but reflects inhibition of stacking due to side-chain-induced twisting of the contours of individual chains. A competition from intrachain coupling has also been demonstrated by comparing POMeOPT aggregates with different polymer chain sizes

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. For resonantly coupled Alexa 647 and NRs, emission to the far field through the NR plasmon is highly prominent, resulting in underestimation of NR sizes. However, we demonstrate that it is possible to improve the accuracy of the emission localization when both the spectral and spatial separations between Alexa 647 and the LSPR are optimized