The Optimal Aspect Ratio of Gold Nanorods for Plasmonic Bio-sensing

The plasmon resonance of metal nanoparticles shifts upon refractive index changes of the surrounding medium through the binding of analytes. The use of this principle allows one to build ultra-small plasmon sensors that can detect analytes (e.g., biomolecules) in volumes down to attoliters. We use simulations based on the boundary element method to determine the sensitivity of gold nanorods of various aspect ratios for plasmonic sensors and find values between 3 and 4 to be optimal. Experiments on single particles confirm these theoretical results. We are able to explain the optimum by showing a corresponding maximum for the quality factor of the plasmon resonance.     

Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular compartments of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. Here, we describe the design and operation of a superresolution two-photon microscope using pulsed excitation and STED lasers. We examine the depth dependence of STED imaging in acute tissue slices and find enhancement of 2P resolution ranging from approximately fivefold at 20 μm to approximately twofold at 90-μm deep. The depth dependence of resolution is found to be consistent with the depth dependence of depletion efficiency, suggesting resolution is limited by STED laser propagation through turbid tissue. Finally, we achieve live imaging of dendritic spines with 60-nm resolution and demonstrate that our technique allows accurate quantification of neuronal morphology up to 30-μm deep in living brain tissue.     

Far-Field Raman Imaging of Short-Wavelength Particle Plasmons on Gold Nanorods

The surface plasmon fields of gold nanorods with a diameter of 100 nm and lengths of 1–5 m are imaged by using far-field Raman scattering of methylene blue adsorbed on the rods. When optically exciting the nanorods under total internal reflection with wave vector and electric field vector orientations along the rod axis, the plasmon field intensity along this axis is observed to be periodically modulated. This modulation is attributable to a beating of the exciting light wave and the nanorod plasmon mode. The plasmon wavelength deduced from the beat frequency is 379 nm, which is considerably smaller than the exciting laser wavelength of 647 nm. In general, Raman imaging is shown to be a powerful technique to probe local plasmon fields using far-field spectroscopy.     

In Vitro Identification of Gold Nanorods through Hyperspectral Imaging

Due to their unique plasmonic and optical properties, gold nanorods (GNR) have shown tremendous potential for nano-based applications extending into a variety of fields including bioimaging, sensor development, electronics, and cancer therapy. These distinctive, shape-specific properties are strongly dependent upon the GNR aspect ratio, thus producing the ability to be targeted for an application by fine-tuning their physical parameters. It is owing to their characteristic spectral signature, which is vastly different from that of a cellular setting, that GNRs are emerging as an ideal candidate for nano-based imaging applications. However, one challenge that has emerged in the field of bioimaging is the need to account for the observed plasmon coupling effect that arises from GNR agglomeration in a physiological environment. In this study, GNRs with aspect ratios of 2.5 and 6.0 were actively identified in an in vitro setting through a hyperspectral imaging (HSI) analysis; which successfully recognized and separated the light scattering pattern of these particles from that of the surrounding cells. Through inclusion of agglomerated GNR spectral patterns in the HSI spectral library, this imaging technique was able to overcome the complication of plasmon coupling, though to varying degrees. These results demonstrate the tremendous potential of GNRs coupled with HSI analysis to advance the field of nano-based sensing and imaging mechanisms.     

RGD/FA双靶纳米金棒的制备及其在肿瘤细胞协同靶向成像与光热治疗中的应用

目的:通过制备RGD/FA双靶纳米金考察其与高表达整合素与叶酸受体B16细胞的协同靶向成像与热疗作用;方法:采用功能化PEG分子将靶向小分子RGD与叶酸通过强健Au-S键连接至纳米金棒表面,利用激光共聚焦与808 nm近红外激光器评价修饰纳米金的协同靶向作用;结果:RGD与叶酸分子被成功连接于纳米金表面,且双靶纳米金对小鼠黑色素瘤细胞具有较好的协同靶向作用;结论:同时靶向同一肿瘤细胞的不同表位,可克服单一靶向功能化纳米粒子难以在肿瘤位点有效积累的问题,本研究为多功能纳米金棒在临床肿瘤早期诊断与光热治疗中的应用提供研究基础。     

Reversible Tuning the Aspect Ratio and Plasmonic Shift of Gold Nanorods in Alkaline Environment: Growth,Etching and Rebuilding

Plasmonics - The well-developed seed-mediated growth of gold nanorods (GNRs) is under the weakly acidic condition, and the aggregation of the GNRs usually takes place when the pH value is increased...     

Subdiffraction-Limit Two-Photon Fluorescence Microscopy for GFP-Tagged Cell Imaging

We report applications of two-photon excitation fluorescence (2PEF) microscopy with subdiffraction-limit resolution for green-fluorescent-protein-tagged cell imaging. The microscope integrates 2PEF microscopy and stimulated emission depletion microscopy in one microscope that has the benefits of both techniques: intrinsic three-dimensional resolution, confined photobleaching, and subdiffraction-limit resolution. The subdiffraction-limit resolution was demonstrated by resolving green-fluorescent-protein-tagged caveolar vesicles located within a distance shorter than the diffraction limit of a regular 2PEF microscope, which is ∼250 nm even with the best optics. The full width at half-maximum of the effective point-spread function for the 2PEF microscope was estimated to be ∼54 nm.     

Application of Gold Nanorods for Plasmonic and Magnetic Imaging of Cancer Cells

We report the use of biocompatible gold nanorods (GNRs) as multimodal (plasmonic and magnetic) probes for cancer cell labeling in vitro. These multifunctional and multimodal bioconjugates were prepared by replacing cetyltrimethylammonium bromide with a mixture of functionalized PEGylation molecules so that a variety of functionalities (e.g., magnetic resonance imaging agent gadolinium (Gd) and biorecognition molecule transferrin (Tf)) can be easily integrated using simple chemistry. It was shown that Gd incorporation did not interfere with the plasmonic properties of the GNRs and a strong T1 relaxivity was estimated (10.0 mM⁻¹ s⁻¹), which is more than twice that of the clinical MRI agent Gd-DTPA. The large observed T1 relaxivity was possibly due to the huge surface to volume ratio of GNR, which allowed huge amount of amine-terminated molecule to anchor on the surface, coupled with Gd (III) ions for the enhanced relaxation of water protons. Pancreatic cancer cell overexpressing the transferring receptor was served as the in vitro model, and the Tf-mediated uptake was demonstrated and confirmed by dark-field imaging and transmission electron microscopy. More importantly, cell viability (MTS) assay did not reveal any sign of toxicity in these treated cells, suggesting that PEGylated GNRs can serve as a biocompatible, multifunctional, and multimodal platform for variable bio-applications.     

Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching

We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca²⁺ events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca²⁺ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.     

Tuning Ultrafast Photoresponse of Gold Nanorods

Nondegenerate pump probe differential transmission experiments on gold nanorods with varying longitudinal surface plasmon resonance have revealed a new phenomenon where the polarity of the transient transmission signal can be reversibly switched between photo bleaching and photo-induced absorption by controlling probe fluence. Under the usual case where probe fluences are nominal, photo bleaching effect is observed for the nanorods with longitudinal surface plasmon resonance energy smaller than the probe photon energy. The laser-induced melting of the nanorods or change in their shape is ruled out for the observed optical switching effect. A quantitative understanding of the results is attempted by invoking a cascaded two-photon absorption dominant beyond a threshold probe fluence of ～75 μJ/cm².     

Optimal Aspect Ratio and Excitation Spectral Region of LSPR Sensors Using Individual Au Dimmeric Nanoplates

Plasmonics - Both the excitation wavelength and aspect ratio AR are key parameters controlling the refractive index sensitivity performances of plasmonic sensors of individual Au nanoplate dimmers....     

Surface Plasmon Dynamics of High-Aspect-Ratio Gold Nanorods

Ultrafast transient absorption studies are reported for high-aspect-ratio gold nanorods that were fabricated by electrochemical deposition in polycarbonate templates. The nanorods are 60 nm in diameter with distribution of lengths of up to 6 μm. The average aspect ratio was ∼50, resulting in a longitudinal surface plasmon resonance (SPR_L) band in the mid-IR, as well as a transverse (SPR_T) band in the visible. The rods were excited at 400 nm and probed at a range of wavelengths from the visible to the mid-IR to interrogate both SPR bands. The dynamics observed, including the electron–phonon coupling time and coherent acoustic breathing mode oscillations, closely resemble those previously reported for gold spherical nanoparticles and smaller-aspect-ratio nanorods. The electron–phonon coupling time was similarly determined to be 3.3 ± 0.2 ps for both of the SPR bands. Also, oscillations with a 32-ps period were observed for probing near the SPR_T band in the visible region due to impulsive coherent excitation of the acoustic breathing mode, which are consistent with the 60-nm diameter of the nanorods determined by scanning electron microscopy. The results demonstrate that the dynamics for long gold nanorods are similar to those for smaller nanoparticles. Gerald M. Sando is a NRL-ASEE Research Associate     

Functionalization of Gold Nanorods Toward Their Applications

The topics focusing on functionalization of gold nanorods have been reviewed with a view toward their advanced uses. In most cases, as-prepared gold nanorods are hydrophilic and protected by surfactants, since anisotropic growth of gold nanorods by chemical, electrochemical, and photo-induced methods is carried out in aqueous media in the presence of surfactants and additives. Since solvophilicity of gold nanorods predominantly affects on their optical properties, the control of dispersity of gold nanorods in matrices has been performed, without loss of their optical characters, by surface modification and hybridization with small molecules or polymers. As a result of the functionalization procedure, the capability of self-assembly of gold nanorods has been improved. Furthermore, the examples of application using gold nanorods demonstrate that gold nanorod is a promising material.     

Simultaneous Live Cell Imaging Using Dual FRET Sensors with a Single Excitation Light

Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca²⁺ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.     

Modulation of Optical Properties of Gold Nanorods on Addition of KOH

Gold nanorods are known to exhibit two distinct surface plasmon oscillations namely, transverse and longitudinal bands corresponding to oscillations of conduction electrons along width and length of gold nanorods. Considerable changes in these surface plasmon resonance peak positions occurred when KOH was added to the nanorod solution. Nanorods with initial longitudinal plasmon band at 739, 796, and 895 nm are studied with variation in KOH concentration. While the longitudinal plasmon resonance peak showed blue shift, transverse plasmon resonance peak exhibited only intensity variations. Changes could be attributed to the shape transition of gold nanorods on variation of pH in the solution. Shape transition of gold nanorods is confirmed by transmission electron microscopy images.     

Pluronic Triblock Copolymer Encapsulated Gold Nanorods as Biocompatible Localized Plasmon Resonance-Enhanced Scattering Probes for Dark-Field Imaging of Cancer Cells

Gold nanorods (GNR) are synthesized using cetylmethylammonium bromide (CTAB) surfactants which function as structure-directing agents. However, CTAB forms a tightly bound cationic bilayer on GNR surface with the cationic trimethylammonium head group exposed to the aqueous media, which is known to be highly toxic in vitro and in vivo. Pluronic is a non-ionic triblock polymer, which can associate with CTAB and form stable CTAB?Cpolymer complexes due to hydrophobic interactions. In this work, two types of Pluronic triblock copolymers were used to encapsulate GNR to reduce their cytotoxicity and improve colloidal and optical stability for biological applications. These formulations were characterized by UV?Cvis absorption spectra analysis, transmission electron microscopy, cell viability studies, differential interference contrast microscopy and dark-field imaging.     

Facile and Rapid Detection of Microalbuminuria by Antibody-Functionalized Gold Nanorods

Plasmonics - Any excess or lack of human serum albumin (HSA) in urine should be considered a warning for renal or cardiovascular diseases. This report proposes a rapid and straightforward method...     

Distance-Dependent Fluorescence Quenching Efficiency of Gold Nanodisk: Effect of Aspect Ratio-Dependent Plasmonic Absorption

The fluorescence quenching efficiency of an emitter close to a gold nanodisk is investigated by theoretical calculation based on the modified quasi-static approximation and fluorescence energy transfer under dipole?Cdipole coupling. The calculation results show that the surface plasmon resonance (SPR) absorption is the key factor to affect the quenching efficiency. Because of the asymmetric shape of the gold disk, the light absorption depends on both particle volume and aspect ratio (AR). Thus, the AR cannot control the quenching efficiency of gold nanodisk alone. Whether the disk volume is fixed or not may bring different changing way of AR-controlled quenching efficiency. Increasing the AR leads to the quenching efficiency of perpendicular mode start to decrease at a farther distance when the disk volume is changed, but start to decrease at a nearer distance when the volume is fixed. At a given wavelength, one can find a distinct peak in the AR-dependent quenching efficiency curve of parallel mode when the volume is fixed, which is absent from the quenching efficiency curve when the volume is changed. All these tunable quenching efficiency characteristics have been explained by the changing of intensity, shift, and bandwidth of SPR absorption.     

Polarization-Dependent Confocal Imaging of Individual Ag Nanorods and Nanoparticles

Polarization-dependent inelastic optical scattering (IOS) of individual Ag nanorods and nanoparticles are studied by confocal imaging. Stronger IOS is observed at two ends of the nanorod with laser polarizing parallel to the rod long axis while the IOS images of Ag nanoparticles elongate along laser polarization direction. The correlation between the far-field IOS image and near-field spatial distributions of the nanostructures′ electric field can be obtained. The IOS imaging is demonstrated to be an effective technique to study the optical properties of metal nanostructures, which also provides information for their applications in surface-enhanced Raman scattering.     

Dichroic Optical Properties of Uniaxially Oriented Gold Nanorods in Polymer Films

Applications based on the optical excitation of the longitudinal surface plasmon resonance of gold nanorods (AuNRs) work at highest efficiency if all component AuNRs can be maximally excited simultaneously. This can be achieved in aligned AuNR structures, such as those embedded in uniaxially stretched polymer films. Since too high heating temperatures during film stretching cause reshaping and alteration of optical properties of the rods, a maximum allowable heating temperature is determined. The alignment of the rods is quantified by an orientational order parameter of 0.92 based on a statistically significant sample of assumed t distributed means and obtained by scanning electron microscopy. We show that a stretched AuNRs-PVA composite film has optical properties that approach the dichroic properties of an idealized ensemble of fully aligned, identical, and non-interacting AuNRs embedded in a PVA film. The idealized system is provided by FDTD simulations of a single AuNR, which we carried out using the size- and shape-adapted dielectric function of gold and the software RSOFT.