Uptake quantification of gold nanoparticles inside of cancer cells using high order image correlation spectroscopy.

The application of gold nanoparticles (AuNPs) in cancer therapeutics and diagnostics has recently reached a clinical level. Functional use of the AuNP in theranostics first requires effective uptake into the cells, but accurate quantification of AuNPs cellular uptake in real-time is still a challenge due to the destructive nature of existing characterization methods. The optical imaging-based quantification method is highly desirable. Here, we propose the use of high-order image correlation spectroscopy (HICS) as an optical imaging-based nanoparticle quantification technique. Coupled with dark field microscopy (DFM), a non-destructive and easy quantification method could be achieved. We demonstrate HICS analysis on 80 nm AuNPs coated with cetyltrimethylammonium bromide (CTAB) uptake in HeLa cells to calculate the percentage of aggregate species (dimer) in the total uptake and their relative scattering quantum yield inside the cells, the details of which are not available with other quantification techniques. The total particle uptake kinetics measured were in a reasonable agreement with the literature.

Ablation in Externally Applied Electric and Magnetic Fields.

To harness light-matter interactions at the nano-/micro-scale, better tools for control must be developed. Here, it is shown that by applying an external electric and/or magnetic field, ablation of Si and glass under ultra-short (sub-1 ps) laser pulse irradiation can be controlled via the Lorentz force F = e E + e [ v × B ] , where v is velocity of charge e, E is the applied electrical bias and B is the magnetic flux density. The external electric E-field was applied during laser ablation using suspended micro-electrodes above a glass substrate with an air gap for the incident laser beam. The counter-facing Al-electrodes on Si surface were used to study debris formation patterns on Si. Debris was deposited preferentially towards the negative electrode in the case of glass and Si ablation. Also, an external magnetic field was applied during laser ablation of Si in different geometries and is shown to affect ripple formation.

Direct Chemical Synthesis of Plasmonic Black Colloidal Gold Superparticles with Broadband Absorption Properties.

Self-assembly of plasmonic metal nanoparticles can provide an opportunity of creating colloidal superparticles with fascinating optical properties arising from interparticle plasmonic coupling, but typically requires multiple steps involving solvent and/or ligand exchange. We developed a direct, one-step chemical synthesis of plasmonic black colloidal Au superparticles with broadband absorption in visible and near-infrared regions. During the synthesis, the Au superparticles were formed through self-assembly of in-situ-formed Au nanoparticles driven by solvophobic interactions between nanoparticles and solvent. These superparticles could be solution-processed to fabricate a thin film, which exhibited near-perfect absorption over a broad range from 400 nm to 2.5 µm as well as the excellent antireflective property. Thanks to their broadband absorption property, the Au superparticles showed good performances for near-infrared surface-enhanced Raman spectroscopy and light-to-heat conversion.

The Effect of Nanoparticles on the Cluster Size Distributions of Activated EGFR Measured with Photobleaching Image Correlation Spectroscopy.

The epidermal growth factor receptor (EGFR) is an important cell surface receptor in normal physiology and disease. Recent work has shown that EGF-gold nanoparticle conjugates can influence cell behaviour, but the underlying mechanism at the receptor quaternary structural level remains poorly understood.In the present work, the cluster density and cluster size of activated (phosphorylated) EGFR clusters in HeLa cells were determined with photobleaching image correlation spectroscopy. EGFR activation was probed via immunofluorescence-detected phosphorylation of tyrosines (pY-mAb) located in the kinase domain of EGFR (Y845) and at the EGFR cytoplasmic tail (Y1173). Cell activation was probed via nuclear extracellular-regulated kinase (ERK) phosphorylation. The cluster size of activated EGFR was 1.3-2.4 pY-mAb/cluster in unstimulated HeLa cells. EGF or nanorod treatment led to an increase in EGFR oligomers containing multiple phosphotyrosines (>2 phosphotyrosines per EGFR oligomer, average cluster size range = 3-5 pY-mAb/cluster) which paralleled increases in nuclear p-ERK. In contrast, EGF-nanorods decreased the contribution from higher-order phospho-clusters and decreased nuclear p-ERK relative to the nanorod control. These studies provide direct evidence that targeted nanotechnology can manipulate receptor organization and lead to changes in receptor activation and subsequent signalling processes.

Terahertz thermometry of gold nanospheres in water.

The photo-thermal effects of plasmonic nanoparticles are promising for cancer therapies. These treatments would greatly benefit from real-time, multi-scale temperature mapping by non-invasive means. Here we show that intense terahertz time domain spectroscopy can be used as a non-contact and high-resolution thermometer of water solutions. Using this technique, we measure the temperature change, triggered by femtosecond amplified laser pulses, of a solution of gold nanospheres in water. Extensions of this ultra-fast and non-invasive technique could open the door to real-time micro-thermometry of single cells without fluorescent labels.

Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells.

Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

Inhibiting EGFR clustering and cell proliferation with gold nanoparticles.

Gold nanoparticles are functionalized with epidermal growth factor (EGF) molecules and incubated with HeLa cells. These new complexes mechanically interfere with the activation of EGF receptors in a length-dependent manner. Protein-functionalized gold nanoparticles hold great potential for unveiling the fundamental characteristics of cell receptors and for future pharmacological studies on receptor targeting.

Below melting point photothermal reshaping of single gold nanorods driven by surface diffusion.

Plasmonic gold nanorod instability and reshaping behavior below melting points are important for many future applications but are yet to be fully understood, with existing nanoparticle melting theories unable to explain the observations. Here, we have systematically studied the photothermal reshaping behavior of gold nanorods irradiated with femtosecond laser pulses to report that the instability is driven by curvature-induced surface diffusion rather than a threshold melting process, and that the stability dramatically decreases with increasing aspect ratio. We successfully utilized the surface diffusion model to explain the observations and found that the activation energy for surface diffusion was dependent on the aspect ratio of the rods, from 0.6 eV for aspect ratio of 5 to 1.5 eV for aspect ratio less than 3. This result indicates that the surface atoms are much easier to diffuse around in larger aspect ratio rods than in shorter rods and can induce reshaping at any given temperature. Current plasmonics and nanorod applications with the sharp geometric features used for greater field enhancement will therefore need to consider surface diffusion driven shape change even at low temperatures.

Electron-beam lithography of plasmonic nanorod arrays for multilayered optical storage.

In this paper we demonstrate multilayer fabrication of plasmonic gold nanorod arrays using electron-beam lithography (EBL), and show that this structure could be used for multilayered optical storage media capable of continuous-wave (cw) laser readout. The gold nanorods fabricated using the EBL method are aligned perfectly and homogeneous in size and shape, allowing the polarization response of surface plasmon resonance (SPR) to be observed through ensemble array. This property in turn permits polarization detuned SPR readout possible and other manipulations such as progressively twisted arrays through the multilayers to make cw readout possible through deeper layers without too much extinction loss. The layered gold nanorod arrays are separated by thick spacer layer to enable the optical resolving of individual layers. Using this method, we demonstrated four-fold reduction in extinction loss for cw readout in three-layer structure. The current technique of multilayer fabrication and readout can be useful in 3-dimensional fabrication of plasmonic circuits and structures.

Ultra-pure, water-dispersed Au nanoparticles produced by femtosecond laser ablation and fragmentation.

Aqueous solutions of ultra-pure gold nanoparticles have been prepared by methods of femtosecond laser ablation from a solid target and fragmentation from already formed colloids. Despite the absence of protecting ligands, the solutions could be (1) fairly stable and poly size-dispersed; or (2) very stable and monodispersed, for the two fabrication modalities, respectively. Fluorescence quenching behavior and its intricacies were revealed by fluorescence lifetime imaging microscopy in rhodamine 6G water solution. We show that surface-enhanced Raman scattering of rhodamine 6G on gold nanoparticles can be detected with high fidelity down to micromolar concentrations using the nanoparticles. Application potential of pure gold nanoparticles with polydispersed and nearly monodispersed size distributions are discussed.

Aggregation distributions on cells determined by photobleaching image correlation spectroscopy.

The organization of molecules into macromolecular (nanometer scale), supramolecular complexes (submicron-to-micron scale), and within subcellular domains, is an important architectural principle of cellular biology and biochemistry. Determining the precise nature and distribution of complexes within the cellular milieu is a challenging biophysical problem. Time-series analysis of laser scanning confocal microscopy images by image correlation spectroscopy (ICS) or fluctuation moments methods provides information on aggregation, flow, and dynamics of fluorescently tagged macromolecules. All the methods to date require a brightness standard to relate the experimental data to absolute aggregation. In this article, we show that ICS as a function of gradual photobleaching is a sensitive indicator of aggregation distribution on the submicron scale. Specifically, in photobleaching ICS, the extent of nonlinearity of the apparent cluster density as a function of bleaching is related to the size of clusters. The analysis is tested using computer simulations on model aggregate systems and then applied to an experimental determination of Aß peptide aggregation on nerve cells. The analysis reveals time-dependent increases in Aß1-42 peptide aggregation. Globally, the datasets could be described by a monomer-dimer-tetramer-hexamer or a monomer-dimer-trimer-pentamer model. The results demonstrate the utility of photobleaching with ICS for determining aggregation states on the supramolecular scale in intact cells without the requirement for a brightness standard.

Detuned surface plasmon resonance scattering of gold nanorods for continuous wave multilayered optical recording and readout.

In a multilayered structure of absorptive optical recording media, continuous-wave laser operation is highly disadvantageous due to heavy beam extinction. For a gold nanorod based recording medium, the narrow surface plasmon resonance (SPR) profile of gold nanorods enables the variation of extinction through mulilayers by a simple detuning of the readout wavelength from the SPR peak. The level of signal extinction through the layers can then be greatly reduced, resulting more efficient readout at deeper layers. The scattering signal strength may be decreased at the detuned wavelength, but balancing these two factors results an optimal scattering peak wavelength that is specific to each layer. In this paper, we propose to use detuned SPR scattering from gold nanorods as a new mechanism for continuous-wave readout scheme on gold nanorod based multilayered optical storage. Using this detuned scattering method, readout using continuous-wave laser is demonstrated on a 16 layer optical recording medium doped with heavily distributed, randomly oriented gold nanorods. Compared to SPR on-resonant readout, this method reduced the required readout power more than one order of magnitude, with only 60 nm detuning from SPR peak. The proposed method will be highly beneficial to multilayered optical storage applications as well as applications using a continuous medium doped heavily with plasmonic nanoparticles.

Enhanced degree of temporal coherence through temporal and spatial phase coupling within a focused supercontinuum.

In the diffraction of a supercontinuum source, a redistribution of amplitude and phase at the focal region is incurred by the coupling between the supercontinuum and the spatial phase caused by the lens diffraction, making it extremely difficult to predict the focal behaviour. We show that the coupling between the temporal phase of a SC source and the spatial phase from the diffraction by a low numerical aperture (NA) lens causes dramatic alterations in the spectra and the temporal coherence near the focal region, and that this effect is maximized in points of singularity. Furthermore, we show that such an enhancement in temporal coherence can be controlled by the pulse evolution through the photonic crystal fiber, in which nonlinear and disperive effects such as the soliton fission process provides the key phase evolution necessary for dramatically changing the coherence time of the focused electromagnetic wave.

White light scattering spectroscopy and electron microscopy of laser induced melting in single gold nanorods.

We present the first measurements of laser induced melting and reshaping of single gold nanorods. Using a combination of white light scattering spectroscopy and electron microscopy we find a melting energy of 260 fJ for nanorods with an average size of 92 x 30 nm. Contrary to previous reports on ensembles of nanorods, this melting energy corresponds well to the theoretical prediction of 225 fJ. We observe a gradual shape change from a long and thin rod to a shorter and wider rod, which eventually collapses into a sphere when enough laser energy is deposited. We also observe that higher aspect ratio particles are thermodynamically less stable, leading to a greater reduction of the aspect ratio at lower laser pulse energy densities.

Five-dimensional optical recording mediated by surface plasmons in gold nanorods.

Multiplexed optical recording provides an unparalleled approach to increasing the information density beyond 10(12) bits per cm(3) (1 Tbit cm(-3)) by storing multiple, individually addressable patterns within the same recording volume. Although wavelength, polarization and spatial dimensions have all been exploited for multiplexing, these approaches have never been integrated into a single technique that could ultimately increase the information capacity by orders of magnitude. The major hurdle is the lack of a suitable recording medium that is extremely selective in the domains of wavelength and polarization and in the three spatial domains, so as to provide orthogonality in all five dimensions. Here we show true five-dimensional optical recording by exploiting the unique properties of the longitudinal surface plasmon resonance (SPR) of gold nanorods. The longitudinal SPR exhibits an excellent wavelength and polarization sensitivity, whereas the distinct energy threshold required for the photothermal recording mechanism provides the axial selectivity. The recordings were detected using longitudinal SPR-mediated two-photon luminescence, which we demonstrate to possess an enhanced wavelength and angular selectivity compared to conventional linear detection mechanisms. Combined with the high cross-section of two-photon luminescence, this enabled non-destructive, crosstalk-free readout. This technique can be immediately applied to optical patterning, encryption and data storage, where higher data densities are pursued.

Quantum-rod dispersed photopolymers for multi-dimensional photonic applications.

Nanocrystal quantum rods (QRs) have been identified as an important potential key to future photonic devices because of their unique two-photon (2P) excitation, large 2P absorption cross section and polarization sensitivity. 2P excitation in a conventional solid photosensitive medium has driven all-optical devices towards three-dimensional (3D) platform architectures such as 3D photonic crystals, optical circuits and optical memory. The development of a QR-sensitized medium should allow for a polarization-dependent change in refractive index. Such a localized polarization control inside the focus can confine the light not only in 3D but also in additional polarization domain. Here we report on the first 2P absorption excitation of QR-dispersed photopolymers and its application to the fabrication of polarization switched waveguides, multi-dimensional optical patterning and optical memory. This fabrication was achieved by a 2P excited energy transfer process between QRs and azo dyes which facilitated 3D localized polarization sensitivity resulting in the control of light in four dimensions.

Polarization effects in a highly birefringent nonlinear photonic crystal fiber with two-zero dispersion wavelengths.

A theoretical and experimental study is presented on polarized pulsed propagation from a highly birefringent nonlinear photonic crystal fiber with two-zero dispersion wavelengths. Experimental observations show that the input polarization state can maintain its linearity and that the fiber birefringence creates different spectral properties dependent on the input polarization orientation. The most extensive spectra are obtained for a coupling polarization angles aligned with the fast and slow axis, which is created by the high-order dispersion and Kerr nonlinearity.

Confocal reflection readout thresholds in two-photon-induced optical recording.

Confocal reflection readout thresholds in two-photon-induced optical recording in photoisomerization polymer are studied both theoretically and experimentally. A threshold of the axial response from a planar reflector with a refractive-index change of the order of 10(-2) is revealed. However, the threshold is reduced to 0.006 when strong forward scattering caused by the recorded bits leads to multiple reflection between the bit and the rare surface, which enhances the image contrast and reduces the readout threshold. The quality of the reconstructed bit image is strongly dependent on the refractive-index mismatch at the sample rare interface as well as the distance between the recorded position and the rare surface.

Acoustic oscillations and elastic moduli of single gold nanorods.

We present the first acoustic vibration measurements of single gold nanorods with well-characterized dimensions and crystal structure. The nanorods have an average size of 90 nm x 30 nm and display two vibration modes, the breathing mode and the extensional mode. Correlation between the dimensions obtained from electron microscope images and the vibrational frequencies of the same particle allows us to determine the elastic moduli for each individual nanorod. Contrary to previous reports on ensembles of gold nanorods, we find that the single particle elastic moduli agree well with bulk values.

Rewritable polarization-encoded multilayer data storage in 2,5-dimethyl-4-(p-nitrophenylazo)anisole doped polymer.

We report a rewritable polarization-encoded multilayer data storage method with a polymer film doped with the azo dye DMNPAA (2,5-dimethyl-4-(p-nitrophenylazo)anisole). It is found that under two-photon excitation by a linearly polarized femtosecond laser beam at wavelength 780 nm the optical axis of DMNPAA molecules can be oriented to the perpendicular direction of the beam via a trans-cis-trans isomerization process. As a result, multilayer polarization-encoded optical data storage is demonstrated by recording two letters of a bit spacing of 4 microm in the same region of a given layer. It is shown that erasing and rewriting a particular layer is possible.