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). We describe and characterize how to change the angle of incidence to achieve HILO so that other researchers can easily incorporate this method onto their existing epi- or total internal reflection fluorescence microscopes. We show improvements up to a 32% in signal-to-background ratio and 118% in the number of single molecules detected within stationary phase particles when using HILO compared to epifluorescence. By controlling the objective position relative to the sample, we produce three-dimensional maps of molecule locations throughout entire stationary phase particles at nanoscale lateral and axial resolutions. The number of localized molecules remains constant axially throughout isolated stationary phase particles and between different particles, indicating that heterogeneity in a separation would not be caused by such affinity differences at microscales but instead kinetic differences at nanoscales on identifiable and distinct adsorption sites

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). We describe and characterize how to change the angle of incidence to achieve HILO so that other researchers can easily incorporate this method onto their existing epi- or total internal reflection fluorescence microscopes. We show improvements up to a 32% in signal-to-background ratio and 118% in the number of single molecules detected within stationary phase particles when using HILO compared to epifluorescence. By controlling the objective position relative to the sample, we produce three-dimensional maps of molecule locations throughout entire stationary phase particles at nanoscale lateral and axial resolutions. The number of localized molecules remains constant axially throughout isolated stationary phase particles and between different particles, indicating that heterogeneity in a separation would not be caused by such affinity differences at microscales but instead kinetic differences at nanoscales on identifiable and distinct adsorption sites

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. Our experiments investigated a crowding sensor protein (CrH2) and phosphoglycerate kinase (PGK), which undergoes cooperative unfolding. The crowding sensor quantifies the confinement effect of the cross-linked hydrogel: the 4% polyacrylamide hydrogel is similar to aqueous solution (no confinement), while the 10% hydrogel is strongly confining. FRAP measurements and protein concentration gradients in the 4% and 10% hydrogels further support this observation. PGK reveals that noncovalent interactions of the protein with the polymer surface are more important than confinement for determining protein properties in the gel: the mere presence of hydrogel increases protein stability, speeds up folding relaxation, and promotes irreversible binding to the polymer even at the solution–gel interface, whereas the difference between the 4% and the 10% hydrogels is negligible despite their large difference in confinement. The imaging capabilities of FReI, demonstrated to be diffraction limited, further revealed spatially homogeneous protein unfolding across the hydrogels at 500 nm length scales and revealed differences in protein properties at the gel–solution boundary

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

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

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