Molecular Approaches to Chromatography Using Single Molecule Spectroscopy

Kisley, Lydia; Landes, Christy F.

doi:10.1021/ac5039225

Cited by 36 publications

(36 citation statements)

References 231 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…(27, 63, 239, 240) There is also an interest in applications of single-molecule methods to bottom-up studies in polymer and materials science. (241–243) Here, we present selected studies that demonstrate the utility of accurate and precise 3D single-molecule localization for quantitative structural and dynamical measurements.…”

Section: Selected Applicationsmentioning

confidence: 99%

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

2017

View full text Add to dashboard Cite

Single-molecule super-resolution fluorescence microscopy and single-particle tracking are two imaging modalities that illuminate the properties of cells and materials on spatial scales down to tens of nanometers, or with dynamical information about nanoscale particle motion in the millisecond range, respectively. These methods generally use wide-field microscopes and two-dimensional camera detectors to localize molecules to much higher precision than the diffraction limit. Given the limited total photons available from each single-molecule label, both modalities require careful mathematical analysis and image processing. Much more information can be obtained about the system under study by extending to three-dimensional (3D) single-molecule localization: without this capability, visualization of structures or motions extending in the axial direction can easily be missed or confused, compromising scientific understanding. A variety of methods for obtaining both 3D super-resolution images and 3D tracking information have been devised, each with their own strengths and weaknesses. These include imaging of multiple focal planes, point-spread-function engineering, and interferometric detection. These methods may be compared based on their ability to provide accurate and precise position information of single-molecule emitters with limited photons. To successfully apply and further develop these methods, it is essential to consider many practical concerns, including the effects of optical aberrations, field-dependence in the imaging system, fluorophore labeling density, and registration between different color channels. Selected examples of 3D super-resolution imaging and tracking are described for illustration from a variety of biological contexts and with a variety of methods, demonstrating the power of 3D localization for understanding complex systems.

show abstract

Section: Selected Applicationsmentioning

confidence: 99%

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

2017

View full text Add to dashboard Cite

show abstract

“…Spectral features can be correlated to shape and structural changes due to metal-ion reduction, but the diffraction limit of light prevents accurate localization of where metal ions are reduced. 80 Because engineering reactive hot spots through local field enhancement is an exciting possibility for plasmonic nanoparticle electrocatalysts, 7 understanding where electrochemical reactions occur on a nanoparticle or nanoparticle aggregate is imperative for achieving plasmon-assisted reaction mechanisms.…”

Section: Lspr Tracking Of Metal-ion Reduction At the Nanoparticle Surfacementioning

confidence: 99%

Plasmonic Sensing and Control of Single-Nanoparticle Electrochemistry

Hoener

Kirchner

Heiderscheit

et al. 2018

Chem

Self Cite

104

View full text Add to dashboard Cite

Plasmonic nanoparticles offer promise in photoelectrochemistry by enhancing the rate and selectivity of reactions and in sensing by responding optically to local reactions. Optical electrochemical measurements at the single-particle level are necessary for understanding and eventually controlling the role of plasmons in such complex environments. Recently, researchers have developed techniques to optically measure electrochemical reactions at the surface of single nanoparticles and individual aggregates, allowing for the high-throughput screening necessary to resolve subpopulations of active nanoparticle catalysts and identify active sites on aggregate structures. This review highlights single-nanoparticle and nanoparticle aggregate electrochemical techniques and how they can be used to isolate and elucidate the role of surface plasmons in enhancing catalyst activity and sensing electrochemical processes at the nanoscale.

show abstract

“…Photophysics of the fluorophore label do not allow for comparison of the protein adsorption kinetics under variable pH. Adsorption kinetics at chromatographic interfaces have been studied by single‐molecule fluorescent imaging in the literature, where relatively fast desorption times on the orders of 100's of ms are typically observed . At slower desorption rates, resolving kinetics is complicated by the photophysics of the fluorophore label.…”

Section: Resultsmentioning

confidence: 99%

“…Total internal reflection fluorescence (TIRF) microscopy, coupled with single‐molecule tracking, extracts and quantifies analyte events in the ∼100 nm region of the stationary–mobile phase interface. TIRF microscopy has been used to study protein–stationary phase interactions for a range of chromatographic techniques , such as establishing the importance of long‐range charge interactions in CE and of hydrogen bonding and hydrophobic forces in biomolecule adsorption at the silica–water interface . Single analyte event analysis can be coupled with single‐molecule tracking , thereby revealing the relationships between surface chemistry and the heterogeneity present in molecular motion at interfaces for pH‐tunable polyelectrolyte multilayers and polymeric membrane surfaces .…”

Section: Introductionmentioning

confidence: 99%

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

Kisley

Poongavanam

Kourentzi

et al. 2015

J of Separation Science

Self Cite

View full text Add to dashboard Cite

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. The adsorption and diffusion of a model globular protein, α-lactalbumin, was studied by single-molecule microscopy at a silica-aqueous interface analogous to aqueous normal phase and hydrophilic interaction chromatography and capillary electrophoresis interfaces at varied pH. Electrostatic repulsion resulting in free diffusion was observed at pH above the isoelectric point of the protein. In contrast, at low pH strong adsorption and surface diffusion with either no (D ∼ 0.01 μm(2) /s) or translational (D ∼ 0.3 μm(2) /s) motion was observed where the protein likely interacted with the surface through electrostatic, hydrophobic, and hydrogen bonding forces. The fraction of proteins immobilized could be increased by lowering the pH. These results show that retention of proteins at the silica interface cannot be viewed solely as an adsorption/desorption process and that the type of surface diffusion, which ultimately leads to ensemble chromatographic separations, can be controlled by tuning long-range electrostatic and short-range hydrophobic and hydrogen bonding forces with pH.

show abstract

Molecular Approaches to Chromatography Using Single Molecule Spectroscopy

Cited by 36 publications

References 231 publications

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

Three-Dimensional Localization of Single Molecules for Super-Resolution Imaging and Single-Particle Tracking

Plasmonic Sensing and Control of Single-Nanoparticle Electrochemistry

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

Contact Info

Product

Resources

About