Three-Dimensional Orientation Determination of Stationary Anisotropic Nanoparticles with Sub-Degree Precision under Total Internal Reflection Scattering Microscopy

Single-particle and single-molecule orientation determination plays a vital role in deciphering nanoscale motion in complex environments. Previous attempts to determine the absolute three-dimensional orientation of anisotropic particles rely on subjective pattern matching and are inherently plagued by high degrees of uncertainty. Herein, we describe a method utilizing total internal reflection scattering microscopy to determine the 3D orientation of gold nanorods with subdegree uncertainty. The method is then applied to the biologically relevant system of microtubule cargo loading. Finally, we demonstrate the method holds potential for identifying single particles versus proximate neighbors within the diffraction limited area

Simultaneous Single-Particle Superlocalization and Rotational Tracking

Superlocalization of single molecules and nanoparticles has become an essential procedure to bring new insights into nanoscale structures and dynamics of biological systems. In the present study, superlocalization is combined with the newly introduced differential interference contrast (DIC) microscopy-based single-particle orientation and rotational tracking. The new technique overcomes the difficulty in localization of the antisymmetric DIC point spread function by using a dual-modality microscope configuration for simultaneous rotational tracking and localization of single gold nanorods with nanometer-scale precision. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment. This technique has great potential in the study of biological processes where both localization and rotational information are required

Behavior of Interacting Species in Capillary Electrophoresis Described by Mass Transfer Equation

Affinity capillary electrophoresis (ACE) has been used to estimate thermodynamic constants of binding interactions with linear or nonlinear regression methods. The accuracy of this approach relies heavily on the binding interaction mechanism, which is controlled by both the nature of the interaction and the experimental conditions. The development of a highly efficient computer-simulated ACE system makes it possible to demonstrate the detailed behavior of any interacting species of a given interaction under any conditions. The order of the mobilities of the complex and the two binding species in their free forms is a key factor to determine what molecules in what locations of the column are involved in the interaction, and the peak shape resulting from such interactions, of a given ACE experiment. In this paper and the supporting materials, 18 scenarios in 6 different combinations of migration orders of the free analyte, free additive, and complex formed are studied by a computer simulation program based on the mass transfer equation. From the study of these situations, we conclude high additive concentration (ensuring high capacity factor) and low analyte concentration (ensuring fast fill-in of the free additive in the analyte plug) are crucial for obtaining accurate results when using the regression methods. On the other hand, the approach to estimate binding constants with computer simulation can be much more accurate as long as accurate and efficient simulation models can be developed, especially when the ratio of the additive and analyte concentrations is not large enough

Simultaneous Single-Particle Superlocalization and Rotational Tracking

Superlocalization of single molecules and nanoparticles has become an essential procedure to bring new insights into nanoscale structures and dynamics of biological systems. In the present study, superlocalization is combined with the newly introduced differential interference contrast (DIC) microscopy-based single-particle orientation and rotational tracking. The new technique overcomes the difficulty in localization of the antisymmetric DIC point spread function by using a dual-modality microscope configuration for simultaneous rotational tracking and localization of single gold nanorods with nanometer-scale precision. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment. This technique has great potential in the study of biological processes where both localization and rotational information are required

Simultaneous Single-Particle Superlocalization and Rotational Tracking

Superlocalization of single molecules and nanoparticles has become an essential procedure to bring new insights into nanoscale structures and dynamics of biological systems. In the present study, superlocalization is combined with the newly introduced differential interference contrast (DIC) microscopy-based single-particle orientation and rotational tracking. The new technique overcomes the difficulty in localization of the antisymmetric DIC point spread function by using a dual-modality microscope configuration for simultaneous rotational tracking and localization of single gold nanorods with nanometer-scale precision. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment. This technique has great potential in the study of biological processes where both localization and rotational information are required

Quantitative Analysis of Systematic Errors Originated from Wall Adsorption and Sample Plug Lengths in Affinity Capillary Electrophoresis Using Two-Dimensional Simulation

Two-dimensional (2D) simulation of capillary electrophoresis is developed to model affinity interaction and wall adsorption simultaneously. Finite difference schemes are used to evaluate the mass-transfer equation in cylindrical coordinates. A Langmuir second-order kinetic law is applied to regulate the wall adsorption and desorption processes. Contributions from the simulation parameters are investigated extensively, and parameters for accurate and efficient simulation are identified. With the 2D model, capillary zone electrophoresis and affinity capillary electrophoresis (ACE) in the presence of strong or weak wall adsorption are simulated to elucidate peak distortions. Finite sample injection length/amount and wall adsorption that lead to systematic errors in the estimated binding constants in ACE are quantified for the first time with both actual experiments and computer simulation. Methods for correcting the estimated binding constants are proposed to extend the usefulness of ACE

Three-Dimensional High-Resolution Rotational Tracking with Superlocalization Reveals Conformations of Surface-Bound Anisotropic Nanoparticles

The ability to directly follow three-dimensional rotational movement of anisotropic nanoparticles will greatly enhance our understanding of the way nanoparticles interact with surfaces. Herein, we demonstrate dual-color total internal reflection scattering microscopy as a tool to probe the interactions of plasmonic gold nanorods with functional surfaces. By taking advantage of both the short and long axis surface plasmon resonance scattering enhancement, we are able to decipher both in-plane and out-of-plane gold nanorod motion relative to the sample surface with equally high resolution. In combination with superlocalization through point spread function fitting, we overcome the four-quadrant angular degeneracy of gold nanorods in the focal plane of the objective and resolve conformations of surface-bound anisotropic nanoparticles in unprecedented detail

Simultaneous Single-Particle Superlocalization and Rotational Tracking

Superlocalization of single molecules and nanoparticles has become an essential procedure to bring new insights into nanoscale structures and dynamics of biological systems. In the present study, superlocalization is combined with the newly introduced differential interference contrast (DIC) microscopy-based single-particle orientation and rotational tracking. The new technique overcomes the difficulty in localization of the antisymmetric DIC point spread function by using a dual-modality microscope configuration for simultaneous rotational tracking and localization of single gold nanorods with nanometer-scale precision. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment. This technique has great potential in the study of biological processes where both localization and rotational information are required

Focused Orientation and Position Imaging (FOPI) of Single Anisotropic Plasmonic Nanoparticles by Total Internal Reflection Scattering Microscopy

The defocused orientation and position imaging (DOPI) and polarization-based in-focus imaging techniques have been widely used for detecting rotational motions with anisotropic gold nanorods (AuNRs) as orientation probes. However, these techniques have a number of significant limitations, such as the greatly reduced signal intensity and relatively low spatial and temporal resolutions for out-of-focus AuNRs and the angular degeneracy for in-focus AuNRs. Herein, we present a total internal reflection (TIR) scattering-based focused orientation and position imaging (FOPI) of AuNRs supported on a 50 nm thick gold film, which enables us to overcome the aforementioned limitations. Imaging AuNRs under the TIR scattering microscope provides excellent signal-to-noise ratio and results in no deteriorating images. The scattering patterns of AuNRs on the gold substrate are affected by the strong interaction of the excited dipole in the AuNR with the image dipole in the gold substrate. The doughnut-shaped scattering field distribution allows for high-throughput determination of the three-dimensional spatial orientation of in-focus AuNRs within a single frame without angular degeneracy. Therefore, the TIR scattering-based FOPI method is demonstrated to be an outstanding candidate for studying dynamics of functionalized nanoparticles on a large variety of functional surfaces

Single Particle Orientation and Rotation Tracking Discloses Distinctive Rotational Dynamics of Drug Delivery Vectors on Live Cell Membranes

Engineered nanoparticles have emerged as potentially revolutionary drug and gene delivery vectors. Using rod-shaped gold nanoparticles as a model, we studied for the first time the rotational dynamics of nanoparticle vectors on live cell membranes and its impact on the fate of these nanoparticle vectors. The rotational motions of gold nanorods with various surface modifiers were tracked continuously at 200 frames/s under a differential interference contrast microscope. We found that the rotational behaviors of gold nanorod vectors are strongly related to their surface charges. Specific surface functional groups and the availability of receptors on cell membranes also contribute to the rotational dynamics. The study of rotational Brownian motion of nanoparticles on cell membranes will lead to a better understanding of the mechanisms of drug delivery and provide guidance in designing surface modification strategies for drug delivery vectors under various circumstances