Label-Free Imaging of Membrane Potentials by Intramembrane Field Modulation, Assessed by Second Harmonic Generation Microscopy.

Optical interrogation of cellular electrical activity has proven itself essential for understanding cellular function and communication in complex networks. Voltage-sensitive dyes are important tools for assessing excitability but these highly lipophilic sensors may affect cellular function. Label-free techniques offer a major advantage as they eliminate the need for these external probes. In this work, it is shown that endogenous second-harmonic generation (SHG) from live cells is highly sensitive to changes in transmembrane potential (TMP). Simultaneous electrophysiological control of a living human embryonic kidney (HEK293T) cell, through a whole-cell voltage-clamp reveals a linear relation between the SHG intensity and membrane voltage. The results suggest that due to the high ionic strengths and fast optical response of biofluids, membrane hydration is not the main contributor to the observed field sensitivity. A conceptual framework is further provided that indicates that the SHG voltage sensitivity reflects the electric field within the biological asymmetric lipid bilayer owing to a nonzero χeff(2) tensor. Changing the TMP without surface modifications such as electrolyte screening offers high optical sensitivity to membrane voltage (≈40% per 100 mV), indicating the power of SHG for label-free read-out. These results hold promise for the design of a non-invasive label-free read-out tool for electrogenic cells.

Enhancement of Nonlinear Optical Scattering by Gold Nanoparticles through Aggregation-Induced Plasmon Coupling in the Near-Infrared.

Gold nanoparticles (AuNPs) are regarded as promising building blocks in functional nanomaterials for sensing, drug delivery and catalysis. One remarkable property of these particles is the localized surface plasmon resonance (LSPR), which gives rise to augmented optical properties through local field enhancement. LSPR also influences the nonlinear optical properties of metal NPs (MNPs) making them potentially interesting candidates for fast, high resolution nonlinear optical imaging. In this work we characterize and discuss the wavelength dependence of the hyper-Rayleigh scattering (HRS) behavior of spherical gold nanoparticles (GNP) and gold nanorods (GNR) in solution, from 850 nm up to 1300 nm, covering the near-infrared (NIR) window relevant for deep tissue imaging. The high-resolution spectral data allows discriminating between HRS and two photon photoluminescence contributions. Upon particle aggregation, we measured very large enhancements (ca. 104 ) of the HRS intensity in the NIR, which is explained by considering aggregation-induced plasmon coupling effects and local field enhancement. These results indicate that purposely designed coupled nanostructures could prove advantageous for nonlinear optical imaging and biosensing applications.

Resonance Enhancement of Nonlinear Optical Scattering in Monolayer-Protected Gold Clusters.

Monolayer-protected metal clusters (MPCs) have recently gained significant research interest, since they are promising candidates for various applications in bioimaging and catalysis. Besides this, MPCs promise to aid in understanding the evolution of the metallic state from bottom-up principles. MPCs can be prepared with atomic precision, and their nonscalable properties (indicating molecule-like behavior) have been studied with a variety of techniques both theoretically and experimentally. Here, we present spectrally resolved second-order nonlinear optical scattering experiments on thiolate-protected gold clusters (Au130(SR)50, Au144(SR)60, and Au500(SR)120). The three clusters share common resonance enhancement around 490 nm, which is ascribed to an interband transition. This indicates emerging metal-like properties, and we tentatively assign the onset of metal-like behavior somewhere between 102 and 130 gold atoms.

Third-Harmonic Scattering for Fast and Sensitive Screening of the Second Hyperpolarizability in Solution.

Organic materials are promising candidates for integration in optical network components allowing fast communication. Ultimate speeds can be obtained by exploiting third-order nonlinear optical light-matter interactions that ultimately rely on the molecular second hyperpolarizability (γ). The exploration of molecular structure-property relations is crucial to optimize γ but requires state of the art measurement techniques which are both sensitive and efficient. Unfortunately, present-day methods for probing the performance of third-order nonlinear optical (NLO) materials fail to meet at least one of those requirements. We have developed third-harmonic scattering (THS) as an alternative method to measure γ in solution, featuring a simple experimental setup and straightforward data analysis. Since the signal strength relies on |γ|2, the method proves to be very sensitive and allows rapid screening of organic molecules in dilute solutions for potential use in third-order NLO applications. In this manuscript, we demonstrate the experimental procedure and calibration of THS and have determined the second hyperpolarizability |γ| of commonly used solvents, which can be used as an internal calibration standard. As a proof of concept we determined γ of trans-stilbene and found it to be in excellent agreement with values obtained by other techniques.

Conformational Changes of a Surface-Tethered Polymer during Radical Growth Probed with Second-Harmonic Generation.

The surface-induced polymerization of a chromophore-functionalized monomer was probed in situ for the first time using a nonlinear optical technique, second-harmonic generation. During the first hours of the polymerization reaction, dramatic changes in the tilt angle of the chromophore-functionalized side groups were observed. Following evaluation of the nonlinear optical data with those obtained from atomic force microscopy and ultraviolet-visible, we conclude that second-harmonic generation efficiently probes the polymerization reaction and the conformational changes of the surface-grafted polymer. With polymerization time, the conformation of the surface-tethered polymer changes from a conformation with the polymer backbone and its side groups flat on the surface, i.e., a "pancake" conformation, to a conformation where the polymer backbone is stretched away combined with tilted side groups or an enlarged tilt angle distribution, i.e., a "brush-type" conformation.

Chiral Side Groups Trigger Second Harmonic Generation Activity in 3D Octupolar Bipyrimidine-Based Organic Liquid Crystals.

The design of efficient noncentrosymmetric materials remains the ultimate goal in the field of organic second-order nonlinear optics. Unlike inorganic crystals currently used in second-order nonlinear optical applications, organic materials are an attractive alternative owing to their fast electro-optical response and processability, but their alignment into noncentrosymmetric film remains challenging. Here, symmetry breaking by judicious functionalization of 3D organic octupoles allows the emergence of multifunctional liquid crystalline chromophores which can easily be processed into large, flexible, thin, and self-oriented films with second harmonic generation responses competitive to the prototypical inorganic KH2 PO4 crystals. The liquid-crystalline nature of these chiral organic films also permits the modulation of the nonlinear optical properties owing to the sensitivity of the supramolecular organization to temperature, leading to the development of tunable macroscopic materials.

Ultrasmall Superparamagnetic Iron Oxide Nanoparticles with Europium(III) DO3A as a Bimodal Imaging Probe.

A new prototype consisting of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles decorated with europium(III) ions encapsulated in a DO3A organic scaffold was designed as a platform for further development of bimodal contrast agents for MRI and optical imaging. The USPIO nanoparticles act as negative MRI contrast agents, whereas the europium(III) ion is a luminophore that is suitable for use in optical imaging detection. The functionalized USPIO nanoparticles were characterized by TEM, DLS, XRD, FTIR, and TXRF analysis, and a full investigation of the relaxometric and optical properties was conducted. The typical luminescence emission of europium(III) was observed and the main red emission wavelength was found at 614 nm. The relaxometric study of these ultrasmall nanoparticles showed r2 values of 114.8 mM(-1) Fes(-1) at 60 MHz, which is nearly double the r2 relaxivity of Sinerem(®).

Chiral phase transfer and enantioenrichment of thiolate-protected Au102 clusters.

The Au102(p-MBA)44 cluster (p-MBA: para-mercaptobenzoic acid) is observed as a chiral compound comprised of achiral components in its single-crystal structure. So far the enantiomers observed in the crystal structure are not isolated, nor is the circular dichroism spectrum known. A chiral phase transfer method is presented which allows partial resolution of the enantiomers by the use of a chiral ammonium bromide, (-)-1R,2S-N-dodecyl-N-methylephedrinium bromide ((-)-DMEBr). At sufficiently low concentration of (-)-DMEBr, the phase transfer from water to chloroform is incomplete. Both the aqueous and organic phases show optical activity of near mirror image relationship. Differences in the spectra are ascribed to the formation of diastereomeric salts. At high concentrations of (-)-DMEBr, full phase transfer is observed. The organic phase, however, still displays optical activity. We assume that one of the diastereomers has very strong optical activity, which overrules the cancelation of the spectra with opposite sign. Comparison with computations further corroborates the experimental data and allows a provisional assignment of handedness of each fraction.

Orientational changes of supported chiral 2,2'-dihydroxy-1,1'binaphthyl molecules.

Well defined thin molecular films of 2,2'-dihydroxy-1,1'binaphthyl (binol) molecules at coverages between 5 × 10(15) molecules per cm(2) and 10(17) molecules per cm(2) on thin glass (BK7) substrates were investigated under ultra-high-vacuum (UHV) conditions. Second-Harmonic-Generation Optical-Rotatory-Dispersion measurements (SHG-ORD) were performed using a dedicated spectroscopic setup which allows for the determination of the rotation angle of the SH-signal of two enantiomers. Rotation angles of up to 38 degrees were measured. The chirality of the two enantiomers has been studied at 674 nm (337 nm resonance wavelength) in the transmission mode. Coverage dependent orientation evolution of binol molecular films was revealed by precise monitoring of the surface coverage while performing SHG-ORD experiments. We show that the molecules reach their final orientation at a surface coverage of 5 × 10(16) molecules per cm(2). From the obtained experimental data the ratio of chiral and achiral susceptibility components could be calculated and was observed to change with coverage.

Non-Linear Optical Activity of Chiral Bipyrimidine-Based Thin Films.

An original series of bipyrimidine-based chromophores featuring alkoxystyryl donor groups bearing short chiral (S)-2-methylbutyl chains in positions 4, 3,4 and 3,5, connected to electron-accepting 2,2-bipyrimidine rings, has been developed. Their linear and non-linear optical properties were studied using a variety of techniques, including one- and two-photon absorption spectroscopy, fluorescence measurements, as well as Hyper-Rayleigh scattering to determine the first hyperpolarizabilities. Their electronic and geometrical properties were rationalized by TD-DFT calculations. The thermal properties of the compounds were also investigated by a combination of polarized light optical microscopy, differential scanning calorimetry measurements and small-angle X-ray scattering experiments. The derivatives were found not to have mesomorphic properties, but to exhibit melting temperatures or cold crystallization behavior that enabled the isolation of well-organized thin films. The nonlinear optical properties of amorphous or crystalline thin films were studied by wide-field second harmonic generation and multiphoton fluorescence imaging, confirming that non-centrosymmetric crystal organization enables strong second and third harmonic generation. This new series confirms that our strategy of functionalizing 3D organic octupoles with short chiral chains to generate non-centrosymmetric organized thin films enables the development of highly second order nonlinear optical active materials without the use of corona-poling or tedious deposition techniques.

Nanostripe length dependence of plasmon-induced material deformations.

Following the impact of a single femtosecond light pulse on nickel nanostripes, material deformations-or "nanobumps"-are created. We have studied the dependence of these nanobumps on the length of nanostripes and verified the link with plasmons. More specifically, local electric currents can melt the nanostructures in the hotspots, where hydrodynamic processes give rise to nanobumps. This process is further confirmed by independently simulating local magnetic fields, since these are produced by the same local electric currents.

Chiral thin films of metal oxide.

In this paper, we describe for the first time the synthesis of new chiral nanosized metal oxide surfaces based on chiral self-assembled monolayers (SAMs) coated with metal oxide (TiO2) nanolayers. In this new type of nanosize chiral surface, the metal oxide nanolayers enable the protection of the chiral self-assembled monolayers while preserving their enantioselective nature. The chiral nature of the SAM/TiO2 films was characterized by variety of unique techniques, such as second-harmonic generation circular dichroism (SHG-CD), quartz crystal microbalance, and chiral adsorption measurements with circular dichroism spectroscopy. The chiral resolution abilities of the SAMs coated with metal oxide (TiO2) nanolayers were investigated in the crystallization of a racemic mixture of threonine and glutamic acid. Our proposed methodology for the preparation of nanoscale chiral surfaces described in this article could open up opportunities in other fields of chemistry, such as chiral catalysis.

Plasmon enhanced fluorescence from meticulously positioned gold nanoparticles, deposited by ultra sonic spray coating on organic light emitting diodes.

Enhancement of the spontaneous emission of fluorophores aided by plasmonic nanoparticles (PNPs) prompts the growth of plasmonic organic light emitting diodes (OLEDs). Together with the spatial dependence of the fluorophore and PNPs on enhanced fluorescence, the surface coverage of the PNPs controls the charge transport in OLEDs. Hence, here, the spatial and surface coverage reliance of plasmonic gold nanoparticles is controlled by a roll-to-roll compatible ultrasonic spray coating technique. A 2-fold enhancement in the multi photon fluorescence is seen by two-photon fluorescence microscopy for a polystyrene sulfonate (PSS) stabilized gold nanoparticle located 10 nm away from the super yellow fluorophore. Fluorescence enhancement combined with ∼2% surface coverage of PNPs, provides a 33%, 20% and ∼40% increase in the electroluminescence, luminous efficacy and external quantum efficiency, respectively.

Point group symmetry determination via observables revealed by polarized second-harmonic generation microscopy: (2) applications.

In this work, the theory presented in part 1 (van der Veen, M. A.; Vermoortele, F.; De Vos, D. E.; Verbiest, T. Anal. Chem. 2012, DOI: 10.1021/ac300936q) for determination of the point groups symmetry based on easily distinguishable observables present in simple polarization dependent tests in second harmonic generation microscopy is tested. It is shown experimentally that the methodology can be applied for point group symmetry determination for a variety of structures among which molecular crystals and host/guest systems where the symmetry of the guest molecules cannot be inferred from conventional diffraction methods. Uniquely, this second-harmonic generation based method can discriminate between chiral and achiral structures regardless of their orientation. The method allows for in situ and in vivo studies with spatial resolution.

Point group symmetry determination via observables revealed by polarized second-harmonic generation microscopy: (1) theory.

We present a methodology based on polarization-controlled second-harmonic generation microscopy that allows one to determine the point group symmetry of noncentrosymmetric structures in situ and in vivo in complex systems regardless of the occurrence of periodicity. Small, randomly oriented structures suffice for the analysis, which is based on simple recognition of observables in four tests. These can be performed in any standard SHG-microscope that allows polarization control of the incident and detected light. The method is resilient to birefringence and light dispersion.

Faraday rotation and its dispersion in the visible region for saturated organic liquids.

Faraday rotation and its dispersion have been measured and calculated in the 400-800 nm wavelength range for a set of saturated organic liquids. The resulting Verdet constants are fitted and trends are analyzed. Comparisons are made to both the polarizability and diamagnetic susceptibility. The data are applied to a connectivity index model, allowing prediction of Verdet constants of aliphatic organic liquids from 400 to 800 nm. The observed correlations and connectivity model improve the understanding of Faraday rotation in diamagnetic materials, allowing for future optimization.

Characterization of magnetization-induced second harmonic generation in iron oxide polymer nanocomposites.

We have measured the magnetization-induced second harmonic generation (MSHG) of a nanocomposite consisting of iron oxide nanoparticles in a polymer film. The existing theoretical framework is extended to include DC magnetic fields in order to characterize the MSHG signal and analyze the measurements. Additionally, magnetic hysteresis loops are measured for four principal polarizer-analyzer configurations, revealing the P(IN)-P(OUT) and S(IN)-P(OUT) polarizer-analyzer configurations to be sensitive to the transverse magnetic field. These results demonstrate the use of MSHG and the applied formalism as a tool to study magnetic nanoparticles and their magnetic properties.

Magnetic-plasmonic nanoparticles for the life sciences: calculated optical properties of hybrid structures.

Magnetic-plasmonic nanoparticles, combining magnetic and plasmonic components, are promising structures for use in life sciences. Optical properties of core-shell magnetite-gold nanostructures, such as the wavelength of the plasmon resonance, the extinction cross-section, and the ratio of scattering to absorption at the plasmon wavelength are critical parameters in the search for the most suitable particles for envisioned applications. Using Mie theory and the discrete dipole approximation (DDA), optical spectra as a function of composition, size, and shape of core-shell nanospheres and nanorods were calculated. Calculations were done using simulated aqueous media, used throughout the life sciences. Our results indicate that in the advantageous near-infrared region (NIR), although magnetic-plasmonic nanospheres produced by available chemical methods lack the desirable tunability of optical characteristics, magnetic-plasmonic nanorods can achieve the desired optical properties at chemically attainable dimensions. The presented results can aid in the selection of suitable magnetic-plasmonic structures for applications in life sciences. FROM THE CLINICAL EDITOR: In this basic science study, magnetic-plasmonic nanoparticles are studied for future applications in life sciences. Optical properties of core-shell magnetite-gold nanostructures, such as the wavelength of the plasmon resonance, the extinction cross-section, and the ratio of scattering to absorption at the plasmon wavelength are critical parameters in the search for the most suitable particles for proposed future applications.

Revealing the Wonder of Natural Photonics by Nonlinear Optics.

Nano-optics explores linear and nonlinear phenomena at the nanoscale to advance fundamental knowledge about materials and their interaction with light in the classical and quantum domains in order to develop new photonics-based technologies. In this perspective article, we review recent progress regarding the application of nonlinear optical methods to reveal the links between photonic structures and functions of natural photonic geometries. Furthermore, nonlinear optics offers a way to unveil and exploit the complexity of the natural world for developing new materials and technologies for the generation, detection, manipulation, and storage of light at the nanoscale, as well as sensing, metrology, and communication.

Uncovering Hidden Dynamics of Natural Photonic Structures Using Holographic Imaging.

In this method, the potential of optics and holography to uncover hidden details of a natural system's dynamical response at the nanoscale is exploited. In the first part, the optical and holographic studies of natural photonic structures are presented as well as conditions for the appearance of the photophoretic effect, namely, the displacement or deformation of a nanostructure due to a light-induced thermal gradient, at the nanoscale. This effect is revealed by real-time digital holographic interferometry monitoring the deformation of scales covering the wings of insects induced by temperature. The link between geometry and nanocorrugation that leads to the emergence of the photophoretic effect is experimentally demonstrated and confirmed. In the second part, it is shown how holography can be potentially used to uncover hidden details in the chemical system with nonlinear dynamics, such as the phase transition phenomenon that occurs in complex oscillatory Briggs-Rauscher (BR) reaction. The presented potential of holography at the nanoscale could open enormous possibilities for controlling and molding the photophoretic effect and pattern formation for various applications such as particle trapping and levitation, including the movement of unburnt hydrocarbons in the atmosphere and separation of different aerosols, decomposition of microplastics and fractionation of particles in general, and assessment of temperature and thermal conductivity of micron-size fuel particles.