Direct Observation of In-Focus Plasmonic Cargos via Breaking Angular Degeneracy in Differential Interference Contrast Microscopy

Abstract

Breaking the angular degeneracy arising from the 2-fold optical symmetry of plasmonic anisotropic nanoprobes is critical in biological studies. In this study, we propose differential interference contrast (DIC) microscopy-based focused orientation and position imaging (dFOPI) to break the angular degeneracy of single gold nanorods (AuNRs). Single in-focus AuNRs (39 nm × 123 nm) within a spherical mesoporous silica shell were characterized with high throughput and produced distinct doughnut-shaped DIC image patterns featuring two lobes in the peripheral region, attributed to the scattering contribution of the AuNRs with large scattering cross sections. Interestingly, rotation of the lobes was observed in the focal plane for a large AuNR (>100 nm) tilted by more than ∼20° from the horizontal plane as the rotational stage was moved by 10° in a rotational study. From the rotation-dependent characteristic patterns, we directly visualized counterclockwise/clockwise rotations without the angular degeneracy at the localized surface plasmon resonance wavelength. Therefore, our dFOPI method can be applied for in vivo studies of important biological systems. To validate this claim, we tracked the three-dimensional rotational behavior of transferrin-modified in-focus AuNRs during clathrin-mediated endocytosis in real time without sacrificing the temporal and spatial resolution. In the invagination and scission stage, one or two directed twist motions of the AuNR cargos detached the AuNR-containing vesicles from the cell membrane. Furthermore, the dFOPI method directly visualized and revealed the right-handed twisting action along the dynamin helix in dynamin-catalyzed fission in live cells.

Figure 1

(A) Schematic of the spherical AuNRs@mSiO₂ particles with their AuNR cores randomly oriented in the mesoporous silica shell. Their projected lengths depend on their spatial orientations, which are freely available in 3D space. (B) TEM image of the spherical AuNRs@mSiO₂. (C) Overlaid UV–vis extinction spectra of the bare AuNRs (blue curve) and spherical AuNRs@mSiO₂ (red curve). The anisotropic AuNRs yielded two distinct LSPR peaks. (D) TEM image analysis based on a custom MATLAB script of the length (major axis) and diameter (minor axis) of the AuNRs with and without a spherical mesoporous silica shell. The inset shows the coefficient of variation (CV) values to compare the variation of the major length of both nanoparticles (AuNRs@mSiO₂ and bare AuNRs).

Figure 2

Single-particle correlation study between SEM and DF microscopy. (A) SEM image of three single spherical AuNRs@mSiO₂. (B) DF scattering image of the three AuNRs@mSiO₂ in (A). (C) Defocused scattering image of the AuNRs@mSiO₂. (D) Single-particle scattering spectra of the AuNRs@mSiO₂ within the yellow squares indicated in (B). (E) Polarization-dependent scattering intensities of the three AuNRs@mSiO₂ as a function of polarization angle from 0 to 360° with 10° steps.

Figure 3

Determination of 3D spatial orientations of the three AuNRs inside the spherical shell through defocused orientation imaging. (A–C) TEM images of spherical (A) AuNR1@mSiO₂, (B) AuNR3@mSiO₂, and (C) AuNR3@mSiO₂. (D–F) Measured defocused image patterns of the three AuNRs@mSiO₂. The white dashed line indicates the single dipole orientation. (G–I) Corresponding best-fit simulated scattering patterns for the three AuNRs@mSiO₂. (J–L) 3D spatial orientations of the three AuNRs@mSiO₂ determined through pattern match analysis. The red line shows the determined 3D orientation and the blue-dotted line is the corresponding in-plane projection.

Figure 4

Characterization of spherical AuNRs@mSiO₂ under DIC microscopy. (A) Definition of the 2D azimuthal angle φ and the 3D polar angle θ. (B) DIC images of AuNR1@mSiO₂ at azimuthal angles φ of 0, 30, 60, 90, 120, and 150° at the LSPR wavelength of 700 nm. A complete set of DIC images at the wavelength is provided in the Supporting Information. (C) DIC intensities (top) and the computed DIC polarization anisotropy P (bottom) of the AuNR at the LSPR wavelength as functions of the rotation angle. I_b and I’_b indicate the bright and dark background intensities, respectively.

Figure 5

Breaking the angular degeneracy in DIC microscopy. (A) Doughnut-shaped image pattern of AuNR1@mSiO₂ measured at 700 nm. The azimuthal angle is 180°. (B) DIC image patterns of an Anura as the azimuthal angle φ varies from 140 to 220° in 10° increments. The white dotted lines indicate the orientation of the AuNR. The azimuthal angle is easily distinguished as + φ or −φ with respect to the dark axis. (C) Doughnut-shaped image pattern of AuNR2@mSiO₂ at 700 nm. (D) DIC image patterns of the AuNR as φ varies from 140 to 220° in 10° steps.

Figure 6

DIC imaging of a transferrin-coated AuNR without the shell during clathrin-mediated endocytosis (CME). (A) Schematic of the complete CME process, which internalizes the AuNR in the A549 cell. (B) DIC image of AuNR3 landed on the cell membrane. (C) Changing DIC intensities of AuNR3 in the two polarization directions during the CME process. (D) Tracking the direction of AuNR3 during the second twisting action through the changing DIC pattern. The characteristic doughnut-shaped patterns of AuNR3 with two lobes rotate to the right over time.