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

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

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

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

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

Dual-Wavelength Detection of Rotational Diffusion of Single Anisotropic Nanocarriers on Live Cell Membranes

Single-particle rotational tracking is of great importance to monitor orientation changes of biomolecules and to understand their functions and mechanisms in biological systems. Differential interference contrast (DIC) microscopy has been found to be an excellent tool to measure polarization anisotropy for tracking rotational dynamics of gold nanorod (AuNR) probes. DIC polarization anisotropy can be conveniently obtained from the bright and dark intensities of a single DIC image of an AuNR. Here, DIC microscopy-based dual-wavelength detection of rotational motions of AuNRs at both transverse and longitudinal surface plasmon resonance (SPR) wavelengths is demonstrated. The transverse SPR mode was successfully used to track fast rotational dynamics of individual AuNRs on live cell membranes. This is important since the transverse SPR mode is mostly insensitive to the medium refractive index, AuNR aspect ratio, and adsorption of biomolecules. DIC polarization anisotropy was simultaneously obtained from the two SPR wavelengths during the dynamic process. Both wavelengths showed good agreement and provided accurate and reliable measurement of AuNR orientation

Detecting Plasmon Resonance Energy Transfer with Differential Interference Contrast Microscopy

Gold nanoparticles are ideal probes for studying intracellular environments and energy transfer mechanisms due to their plasmonic properties. Plasmon resonance energy transfer (PRET) relies on a plasmonic nanoparticle to donate energy to a nearby resonant acceptor molecule, a process which can be observed due to the plasmonic quenching of the donor nanoparticle. In this study, a gold nanosphere was used as the plasmonic donor, while the metalloprotein cytochrome c was used as the acceptor molecule. Differential interference contrast (DIC) microscopy allows for simultaneous monitoring of complex environments and noble metal nanoparticles in real time. Using DIC and specially designed microfluidic channels, we were able to monitor PRET at the single gold particle level and observe the reversibility of PRET upon the introduction of phosphate-buffered saline to the channel. In an additional experiment, single gold particles were internalized by HeLa cells and were subsequently observed undergoing PRET as the cell hosts underwent morphological changes brought about by ethanol-induced apoptosis

Plasmonic Behavior of Single Gold Dumbbells and Simple Dumbbell Geometries

Dumbbell-shaped nanoparticles are similar in size to their nanorod counterparts, but their optical properties have not been studied as extensively as the nanorod. In this paper, the spectra of a single dumbbell, several dumbbell dimers, and a pentamer were collected experimentally and compared with simulated spectra. Surface charge density plots were also obtained in order to elucidate the nature of the plasmonic modes. The dumbbell is shown to be a particle that acts as a transition from the nanorod to the nanosphere. Because the dumbbell shape allows adjacent particles to interlock like puzzle pieces, dumbbells can be thought of as optical building blocks that can combine into designs that are capable of supporting localized hot spots, Fano resonance, and tunable plasmon peaks