High throughput spectrally resolved super-resolution fluorescence microscopy with improved photon usage.

Single-molecule localization microscopy (SMLM), a type of super-resolution fluorescence microscopy, has become a strong technique in the toolbox of chemists, biologists, physicists, and engineers in recent years for its unique ability to resolve characteristic features at the nanoscopic level. It drastically improves the resolution of optical microscopes beyond the diffraction limit, with which previously unresolvable structures can now be studied. Spectrally resolved super-resolution fluorescence microscopy via multiplexing of different fluorophores is one of the greatest advancements among SMLM techniques. However, current spectrally resolved SMLM (SR-SMLM) methodologies present low spatial resolution due to loss of photons, low throughput due to spectral interferences, or require complex optical systems. Here, we overcome these drawbacks by developing a SR-SMLM methodology using a color glass filter. It enables high throughput and improved photon usage for hyperspectral imaging at the nanoscopic level. Our methodology can readily distinguish fluorophores of close spectral emission and achieves sub-10 nm localization and sub-5 nm spectral precisions.

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

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.

Tuning Chemical Interface Damping: Competition between Surface Damping Pathways in Amalgamated Gold Nanorods Coated with Mesoporous Silica Shells.

The mechanism of mercury (Hg) amalgamation in gold nanorods coated with a mesoporous silica shell (AuNRs@mSiO2) and the effect of chemical treatments on the localized surface plasmon resonance (LSPR) spectral changes in single amalgamated AuNRs@mSiO2 remains unclear. In this study, we investigated Hg amalgamation and inward Hg diffusion in single AuNRs@mSiO2 without structural deformation via dark-field scattering spectroscopy and X-ray photoelectron spectroscopy. Then, we investigated the chemisorption of thiol molecules on single amalgamated AuNRs@Hg-mSiO2. Unlike previous studies on single AuNRs, the thiolation on single AuNRs@Hg-mSiO2 resulted in a redshift and line width narrowing of the LSPR peak within 1 h. To determine the chemical effect, we investigated the competition between two surface damping pathways: metal interface damping (MID) and chemical interface damping (CID). When we exposed amalgamated AuNRs@Hg-mSiO2 to 1-alkanethiols with three different carbon chain lengths for 1 h, we observed an increase in the line width broadening with longer chain lengths owing to enhanced CID, demonstrating the tunability of CID and LSPR properties upon chemical treatments. We also investigated the competition between the two surface damping pathways as a function of the time-dependent Au-Hg surface properties in AuNRs@Hg-mSiO2. The 24-h Hg treatment resulted in increased line width broadening compared to the 1-h treatment for the same thiols, which was attributed to the predominance of CID. This was in contrast to the predominance of MID under the 1-h treatment, which formed a core-shell structure. Therefore, this study provides new insights into the Hg amalgamation process, the effect of chemical treatments, competition between surface decay pathways, and LSPR control in AuNRs@mSiO2.

Retraction: Single gold nanostars with multiple branches as multispectral orientation probes in single-particle rotational tracking.

Retraction of 'Single gold nanostars with multiple branches as multispectral orientation probes in single-particle rotational tracking' by Geun Wan Kim et al., Chem. Commun., 2021, 57, 3263-3266, https://doi.org/10.1039/D1CC00731A.

Single-Particle Study on Hg Amalgamation Mechanism and Slow Inward Diffusion in Mesoporous Silica-Coated Gold Nanorods without Structural Deformation.

This paper presents the structural and spectral variations of individual mesoporous silica-coated gold nanorods (AuNRs@mSiO2) compared to bare AuNRs upon Hg-Au amalgamation. First, the aspect ratio of AuNRs@mSiO2 exposed to Hg solutions was unchanged because the deformation related to the cores of AuNR was suppressed by the silica shell. Second, dark-field microscopy and spectroscopy revealed a blue shift of the localized surface plasmon resonance (LSPR) wavelength peak and strong plasmon damping in the individual AuNRs@mSiO2 scattering spectra, exposed to Hg solutions. Furthermore, we investigated time-dependent adsorption kinetics and spectral changes during the formation of Au-Hg amalgam in single AuNRs@mSiO2 over a long time frame without any disturbance from the structural deformation. The inward Hg diffusion into the AuNR core caused a gradual red shift and line width narrowing of the LSPR peak when AuNRs@mSiO2 were withdrawn from Hg solution. Thus, this paper provides new insights into the relationship among amalgamation process, morphological change, the role of silica shell, Hg inward diffusion, LSPR peak, and line width at the single-particle level.

Single-particle study: effects of mercury amalgamation on morphological and spectral changes in anisotropic gold nanorods.

This study investigated the amalgamation of gold nanorods (AuNRs) exposed to Hg(II) solution and its effects on structural and spectral changes in single AuNRs using scanning electron microscopy and total internal reflection scattering microscopy. First, Hg adsorption on AuNR surfaces formed AuNRs@Hg core-shell structures. Afterwards, they transformed to AuNRs@AuHg alloy shell structures in air due to the slow inward diffusion of Hg over time. The aspect ratio (AR) of the AuNRs@AuHg formed by the amalgamation was significantly decreased compared to that of bare AuNRs. Furthermore, the Hg coating on AuNRs induced a dramatic blue shift of the localized surface plasmon resonance (LSPR) peak and linewidth broadening, followed by a red shift and linewidth narrowing of the LSPR peak due to inward diffusion of Hg into the AuNR core. Finally, we investigated the effects of oxygen plasma treatment on the structural changes of AuNRs@AuHg and found that their AR was a decreasing function of the plasma treatment time. More notably, a major structural change was observed 5 min after the plasma treatment. Therefore, fundamental information on the relationship among amalgamation process, plasma treatment time, structural change, and LSPR peak and linewidth is provided at the single-particle level.

Localized surface plasmon resonance inflection points for improved detection of chemisorption of 1-alkanethiols under total internal reflection scattering microscopy.

Plasmonic gold nanoparticles are widely used in localized surface plasmon resonance (LSPR) sensing. When target molecules adsorb to the nanoparticles, they induce a shift in the LSPR scattering spectrum. In conventional LSPR sensing, this shift is monitored at the maximum of the LSPR scattering peak. Herein, we describe the sensitivity of detecting chemisorption of 1-alkanethiols with different chain lengths (1-butanethiol and 1-haxanethiol) on single gold nanorods (AuNRs) of fixed diameter (25 nm) and three different aspect ratios under a total internal reflection scattering microscope. For single AuNRs of all sizes, the inflection point (IF) at the long-wavelength side (or low-energy side) of the LSPR scattering peak showed higher detection sensitivity than the traditionally used peak maximum. The improved sensitivity can be ascribed to the shape change of the LSPR peak when the local refractive index is increased by chemisorption. Our results demonstrate the usefulness of tracking the curvature shapes by monitoring the homogeneous LSPR IF at the red side of the scattering spectrum of single AuNRs.

Single gold nanostars with multiple branches as multispectral orientation probes in single-particle rotational tracking.

Herein, we performed a single-particle correlation study to characterize the optical properties of gold nanostars (AuNSs) with multiple sharp branches under dark-field (DF) and differential interference contrast (DIC) microscopy, and to examine their use as multispectral orientation probes. We presented the polarization-dependent, periodic DIC images and intensities of single AuNSs at their localized surface plasmon resonance (LSPR) wavelengths with high sensitivity. Furthermore, we demonstrated that single AuNSs protrude multiple branches that can be used as individual sensors with DIC polarization anisotropy. Thus, unlike conventional Au nanorod (AuNR) probes, single AuNSs were presented as multispectral optical sensors that can provide detailed information such as rotational motions and rotational speeds at different branches of their star-like structure in dynamic environments.

Mesoporous silica shell-coated single gold nanorods as multifunctional orientation probes in dynamic biological environments.

Mesoporous silica shell-coated gold nanorods (AuNRs@mSiO2) can be employed as promising multifunctional orientation probes in biological studies owing to their anisotropic optical properties, enhanced stability, excellent biocompatibility, etc. In this study, the optical properties of single AuNRs@mSiO2 are characterized under dark-field and differential interference contrast (DIC) microscopy. Furthermore, we presented polarization-dependent, periodic DIC images and intensities of single AuNRs@mSiO2 at their localized surface plasmon resonance wavelength and investigated their use as multifunctional orientation probes in dynamic biological environments. Moreover, the real-time rotational motions of the AuNRs@mSiO2 on the HeLa cell membranes were tracked with millisecond temporal resolution. Overall, AuNRs@mSiO2 demonstrated their capacity to act as multifunctional optical probes owing to the combined effect of the Au core, which can serve as an orientation probe and a local heat generator for phototherapy, and the mesoporous silica shell, which can be used as a reservoir of chemotherapeutics owing to its excellent loading capacity.

Characterizing the non-paraxial Talbot effect of two-dimensional periodic arrays of plasmonic gold nanodisks by differential interference contrast microscopy.

Exploiting the working principle of conventional differential interference contrast (DIC) microscopy, we experimentally investigate the non-paraxial Talbot effect of two-dimensional periodic arrays of gold nanodisks (AuNDs) with a periodicity ao comparable to the excitation wavelength λ. In the non-paraxial regime, strongly contrasting self-image patterns at the Talbot and fractional Talbot distances appeared perpendicularly to the AuND array. The experimental self-image distances were comparable to the calculated non-paraxial Talbot distances at two excitation wavelengths (540 nm and 600 nm). Beyond the paraxial limit, Talbot distances were observed at positions smaller than the paraxial Talbot distance.

Single-particle spectroscopy and defocused imaging of anisotropic gold nanorods by total internal reflection scattering microscopy.

Total internal reflection scattering (TIRS) microscopy is based on evanescent field illumination at the interface. Compared to conventional dark-field (DF) microscopy, TIRS microscopy has been rarely applied to the spectroscopic studies of plasmonic nanoparticles. Furthermore, there has been no detailed correlation study on the characteristic optical properties of single gold nanorods (AuNRs) obtained by DF and TIRS microscopy. Herein, through a single-particle correlation study, we compare the spectroscopic and defocusing properties of single AuNRs obtained by DF and TIRS microscopy, which have different illumination geometries. Compared to DF microscopy, TIRS microscopy yielded almost identical single-particle scattering spectra and localized surface plasmon resonance (LSPR) linewidth for the same in-focus AuNRs. However, TIRS microscopy, which is based on evanescent field illumination at the interface, provided a higher signal-to-noise ratio in the defocused image of the same AuNRs compared to DF microscopy. Furthermore, the heavily reduced background noise clarified the defocused scattering patterns of TIRS microscopy, which provided more detailed and accurate angular information than that obtained by conventional DF microscopy.

Effect of chemisorbed thiophenols with an electron donating group on surface-enhanced Raman scattering of gold nanorods.

Surface-enhanced Raman scattering (SERS) is a powerful technique to amplify the weak Raman scattering intensity by molecules chemisorbed on a metallic surface. Herein, we present the interfacial electronic effect of para-substituted aromatic thiophenols (TPs) with an electron donating group (EDG) on SERS of anisotropic gold nanorods (AuNRs) under resonant conditions. Probe molecules with an EDG showed great SERS enhancement in AuNRs at the resonant excitation wavelength. We found that the SERS enhancement with an EDG is caused by the formation of aggregates through intermolecular interactions among probe molecules, such as dimerization with hydrogen bonding via an amino group (-NH2) of p-aminothiophenol (p-ATP) and hydroxyl group (-OH) of p-mercaptophenol (p-MP), resulting in hot-spots between AuNRs. Furthermore, SERS having a stronger EDG (-NH2, p-ATP) with the Hammett constant of -0.66 exhibited greater enhancement than p-MP having hydroxyl (-OH) groups with the Hammett constant of -0.37. We found that the greater enhancement is ascribed to the temporary formation of a positively charged electron withdrawing group (-NH3+) in p-ATP, unlike p-MP, via the interaction of the lone pair of the amino group (-NH2) with ethanol. Therefore, this investigation provides new insightful experimental observations on SERS enhancement of probe molecules with an EDG.

Single-particle study: effects of oxygen plasma treatment on structural and spectral changes of anisotropic gold nanorods.

Oxygen plasma treatment is a common method for removing the surfactant capping material from gold nanoparticles, improving their functionalization and lowering their cytotoxicity for biological studies. This single-particle study investigates the effects of oxygen plasma treatment on the structural and localized surface plasmon resonance (LSPR) spectral variations of anisotropic gold nanorods (AuNRs). Single AuNRs subjected to different plasma treatment times were characterized by scanning electron microscopy and dark-field microscopy. The AuNR length was a gradually decreasing function of plasma treatment time. After 120 s of plasma treatment, the aspect ratio of the AuNRs was reduced by a major structural deformation. Furthermore, increasing the plasma treatment time gradually broadened the LSPR linewidth of the single AuNRs. This trend was attributed to the decreased aspect ratio and the increased plasmon damping. These results provide vital and fundamental information on the relationship among plasma treatment time, structural change, and LSPR damping at the single-particle level.

High-throughput in-focus differential interference contrast imaging of three-dimensional orientations of single gold nanorods coated with a mesoporous silica shell.

Plasmonic gold nanorods (AuNRs) have been widely applied as optical orientation probes in many biophysical studies. However, characterizing the various three-dimensional (3D) orientations of AuNRs in the same focal plane of the objective lens is a challenging task. To overcome this challenge, we fabricated single AuNRs (10 nm × 30 nm) coated with either an elliptical or spherical mesoporous silica shell (AuNRs@mSiO2). Unlike bare AuNRs and elliptical AuNRs@mSiO2, spherical AuNRs@mSiO2 contained randomly oriented AuNR cores in 3D space, which could be observed on the same focal plane within a single frame by differential interference contrast (DIC) microscopy. The spherical AuNRs@mSiO2 thus achieved high-throughput detection. The proposed approach can overcome the limitations of the current gel-matrix method, which requires vertical scanning of the embedded AuNRs to capture different focal planes.

Polarization- and wavelength-dependent defocused scattering imaging of single gold nanostars with multiple long branches.

The scattering properties of gold nanostars (AuNSs) are not completely understood. Furthermore, there have been no studies on the dark-field (DF) scattering patterns of a AuNS in defocused DF microscopy. Herein, we demonstrate the polarization- and wavelength-dependent defocused scattering properties at the localized surface plasmon resonance (LSPR) wavelengths of single AuNSs with multiple long branches protruding from their surfaces. The defocused scattering intensities of single AuNSs at two LSPR excitation wavelengths changed periodically as a function of the rotational angle of a polarizer, whereas the doughnut-shaped scattering pattern remained unaltered. Furthermore, the characteristic doughnut-shaped defocused scattering pattern enabled the resolution of the spatial field distributions of single dipoles on the same AuNS surface at two LSPR wavelengths. Finally, we tracked the real-time rotational dynamics of a AuNS rotating on a glass slide using defocused microscopy. These results provide a deeper understanding of the defocused scattering properties of single AuNSs with multiple sharp, long branches randomly protruding from their surfaces.

Tuning Chemical Interface Damping: Interfacial Electronic Effects of Adsorbate Molecules and Sharp Tips of Single Gold Bipyramids.

The optimization of the localized surface plasmon resonance (LSPR)-decaying channels of hot-electrons is essential for efficient optical and photochemical processes. Understanding and having the ability to control chemical interface damping (CID) channel contributions will bring about new possibilities for tuning the efficiency of plasmonic hot-electron energy transfer in artificial devices. In this scanning electron microscopy-correlated dark-field scattering study, the CID was controlled by focusing on the electronic nature of disubstituted benzene rings acting as adsorbates, as well as the effects of sharp tips on gold bipyramids (AuBPs) with similar aspect ratios to those of gold nanorods. The results showed that the sharp tips on single AuBPs, as well as the electronic effects of the adsorbate molecules, increase the interfacial contact between the nanoparticles and adsorbate molecules. Electron withdrawing groups (EWGs) on the adsorbates induce larger homogeneous LSPR line widths compared to those of electron donating groups (EDGs). Depending on the location (ortho, meta, and para) of the EDG, the effect of benzene rings with an EDG, which was considered to be induced by sulfur atoms bound to the nanoparticle surface, is weakened by the back transfer of electrons facilitated by the difference in the availability of the electrons of the EDG. Therefore, this study reports that the CID in the LSPR total decay channels can be tuned by controlling the electron withdrawing and electron donating features of adsorbate molecules with the surface topology of metal.

Direct Visualization of Wavelength-Dependent Single Dipoles Generated on Single Gold Nanourchins with Sharp Branches.

We present the optical properties of singe gold nanourchins (AuNUs) with sharp branches on their surfaces under dark-field (DF) microscopy and spectroscopy. The DF intensities of the single AuNUs were changed periodically as a function of the rotation angle at three localized surface plasmon resonance (LSPR) wavelengths. Furthermore, we demonstrate the generation of single dipoles with different LSPR wavelengths in multiple directions on the same AuNU surface. The multiple LSPR dipoles generated on the AuNU surface were further visualized under defocused DF microscopy and verified by characteristic doughnut-shaped defocused scattering field distributions. ᅟ.

Homogeneous localized surface plasmon resonance inflection points for enhanced sensitivity and tracking plasmon damping in single gold bipyramids.

The most polarizable localized surface plasmon resonance (LSPR) longitudinal mode of anisotropic metallic nanoparticles, such as gold bipyramids (AuBPs), is of high prominence. This optical response has tremendous applications from spectroscopy to photonics and energy devices to sensing. In conventional LSPR-based sensing, broadening and asymmetry in peaks due to chemical and instrument noise hinder obtaining a precise insight on shift positions, accordingly limiting the effectiveness and impact of LSPR sensors. Further, when investigating LSPR properties, utilizing more simplistic frequency dependent dielectric-type models can aberrantly impact the reliability of fundamental properties used for designing and fabricating efficient optical devices. For instance, more approximations can effectively limit screening intra-band and inter-band (IB) electronic transition contributions and other related optical properties. With an aim to find alternative methods to further improve their efficiency, as a first report, we devoted a particular focus on LSPR scattering inflection points (IFs) of single AuBPs. The findings reveal that tracking LSPR IFs exhibit high sensitivity over their counterpart LSPR peak shift locations. In addition, we newly detected IB transition contributions near the resonance energy in the range (1.50 eV-2.00 eV) dominated by intra-band transitions. A small increase in the local RI effectively enhances the LSPR quality factor due to IB transitions. Therefore, while neglecting IB transitions in the range below 2.4 eV can work for local air refractive index (RI), in high local RI media it can be aberrantly underestimated. Demonstrated by the use of the dielectric function based on Kramers-Kronig consistent Lorentz oscillators, our findings are in good agreement with the enhancing RI sensitivity effect. The results of this investigation support the idea that tracking curvature changes of an optical signal can be effectively used for LSPR longitudinal peak RI sensing as well as damping in the local RI environment of a single AuBP.

Platinum-coated Core-Shell Gold Nanorods as Multifunctional Orientation Sensors in Differential Interference Contrast Microscopy.

We characterized the optical properties of single platinum-coated core-shell gold nanorods (Pt-AuNRs) under dark-field (DF) and differential interference contrast (DIC) microscopy. Furthermore, we examined their potential use as multifunctional orientation probes. Longitudinal surface plasmon resonance damping was observed for single Pt-AuNRs due to Pt metals coated on the AuNR surface under single-particle scattering spectroscopy. Despite the strong plasmon damping with a much-decreased scattering intensity, DIC microscopy allowed us to detect single Pt-AuNRs with much higher sensitivity. We found polarization-dependent DIC images and intensities of single Pt-AuNRs, which allowed us to determine their orientation angle under DIC microscopy. Therefore, we report that single Pt-AuNRs can be used to develop multifunctional orientation probes under DIC microscopy.

Three-dimensional defocused orientation sensing of single bimetallic core-shell gold nanorods as multifunctional optical probes.

Bimetallic core-shell gold nanorods (AuNRs) are promising multifunctional orientation probes that can be employed in biological and physical studies. This paper presents the optical properties of single AuNRs coated with palladium (Pd) and platinum (Pt) under scattering-based dark-field (DF) microscopy. Strong longitudinal plasmon damping was observed for the bimetallic AuNRs due to Pd and Pt metals on the AuNR surface. Despite the strong plasmon damping, the bimetallic AuNRs yielded characteristic doughnut-shaped scattering patterns under defocused DF microscopy. Interestingly, a solid bright spot appeared at the center of the defocused scattering patterns due to strong damping in the longitudinal plasmon and the increased contribution from the transverse dipoles to the image patterns, which was verified further by a simulation study. Furthermore, the defocused scattering field distributions enabled a determination of the three-dimensional (3D) orientations of single bimetallic AuNRs through a pattern-match analysis technique without angular degeneracy. Therefore, deeper insight into the optical properties and defocused scattering patterns of single bimetallic AuNRs is provided, which can be used to develop multifunctional optical probes that are capable of sensing of the 3D orientation of a probe, biomolecules based on LSPR shift, gas and humidity, etc.