In Situ Real-Time Observation of Photoinduced Nanoscale Azo-Polymer Motions Using High-Speed Atomic Force Microscopy Combined with an Inverted Optical Microscope.

High-speed atomic force microscopy (HS-AFM) is an indispensable technique in the field of biology owing to its imaging capability with high spatiotemporal resolution. Furthermore, recent developments established tip-scan stand-alone HS-AFM combined with an optical microscope, drastically improving its versatility. It has considerable potential to contribute to not only biology but also various research fields. A great candidate is a photoactive material, such as an azo-polymer, which is important for optical applications because of its unique nanoscale motion under light irradiation. Here, we demonstrate the in situ observation of nanoscale azo-polymer motion by combining tip-scan HS-AFM with an optical system, allowing HS-AFM observations precisely aligned with a focused laser position. We observed the dynamic evolution of unique morphologies in azo-polymer films. Moreover, real-time topographic line profile analyses facilitated precise investigations of the morphological changes. This important demonstration would pave the way for the application of HS-AFM in a wide range of research fields.

Plasmonic coupled modes in metal-dielectric multilayer structures: Fano resonance and giant field enhancement.

We provide an overview of Fano resonance and plasmon induced transparency (PIT) as well as on plasmons coupling in planar structures, and we discuss their application in sensing and enhanced spectroscopy. Metal-insulator-metal (MIM) structures, which are known to support symmetric and anti-symmetric surface plasmon polaritons (SPPs) arising from the coupling between two SPPs at the metal-insulator interfaces, exhibit anticrossing behavior of the dispersion relations arising from the coupling of the symmetric SPP and the metal/air SPP. Multilayer structures, formed by a metal film and a high-index dielectric waveguide (WG), separated by a low-index dielectric spacer layer, give narrow resonances of PIT and Fano line shapes. An optimized Fano structure shows a giant field intensity enhancement value of 106 in air at the surface of the high-index dielectric WG. The calculated field enhancement factor and the figure of merit for the sensitivity of the Fano structure in air can be 104 times as large as those of the conventional surface plasmon resonance and WG sensors.

Preparation of Pt nanoclusters with different emission wavelengths and their application in Co2+ detection.

Differently colored fluorescent Pt nanoclusters (NCs) are easily prepared using polyethyleneimine as the stabilizing ligand and environmentally friendly L-ascorbic acid as the reducing agent. The quantum yields of the blue- (1:5), green- (1:20), and yellow-emitting (1:25) NCs are 4 %, 11 %, and 7 %, respectively. The fluorescent Pt NCs show excellent photostability and are sensitive to Co(2+) ions (with a detection limit of 500 nM).

Detection of Glutamate Encapsulated in Liposomes by Optical Trapping Raman Spectroscopy.

The transmission of neuronal information is propagated through synapses by neurotransmitters released from presynapses to postsynapses. Neurotransmitters released from the presynaptic vesicles activate receptors on the postsynaptic membrane. Glutamate acts as a major excitatory neurotransmitter for synaptic vesicles in the central nervous system. Determining the concentration of glutamate in single synaptic vesicles is essential for understanding the mechanisms of neuronal activation by glutamate in normal brain functions as well as in neurological diseases. However, it is difficult to detect and quantitatively measure the concentration of glutamate in single synaptic vesicles owing to their small size, i.e., ∼40 nm. In this study, to quantitatively evaluate the concentrations of the contents in small membrane-bound vesicles, we developed an optical trapping Raman spectroscopic system that analyzes the Raman spectra of small objects captured using optical trapping. Using artificial liposomes encapsulating glutamate that mimic synaptic vesicles, we investigated whether spontaneous Raman scattered light of glutamate can be detected from vesicles trapped at the focus using optical forces. A 575 nm laser beam was used to simultaneously perform the optical trapping of liposomes and the detection of the spontaneous Raman scattered light. The intensity of Raman scattered light that corresponds to lipid bilayers increased with time. This observation suggested that the number of liposomes increased at the focal point. The number of glutamate molecules in the trapped liposomes was estimated from the calibration curve of the Raman spectra of glutamate solutions with known concentration. This method can be used to measure the number of glutamate molecules encapsulated in synaptic vesicles in situ.

Deuterated Glutamate-Mediated Neuronal Activity on Micro-Electrode Arrays.

The excitatory synaptic transmission is mediated by glutamate in neuronal networks of the mammalian brain. In addition to the synaptic glutamate, extra-synaptic glutamate is known to modulate the neuronal activity. In neuronal networks, glutamate uptake is an important role of neurons and glial cells for lowering the concentration of extracellular glutamate and to avoid the excitotoxicity by glutamate. Monitoring the spatial distribution of intracellular glutamate is important to study the uptake of glutamate, but the approach has been hampered by the absence of appropriate glutamate analogs that report the localization of glutamate. Deuterium-labeled glutamate (GLU-D) is a promising tracer for monitoring the intracellular concentration of glutamate, but physiological properties of GLU-D have not been studied. Here we study the effects of extracellular GLU-D for the neuronal activity by using primary cultured rat hippocampal neurons that form neuronal networks on microelectrodes array. The frequency of firing in the spontaneous activity of neurons increased with the increasing concentration of extracellular GLU-D. The frequency of synchronized burst activity in neurons increased similarly as we observed in the spontaneous activity. These changes of the neuronal activity with extracellular GLU-D were suppressed by antagonists of glutamate receptors. These results suggest that GLU-D can be used as an analog of glutamate with equivalent effects for facilitating the neuronal activity. We anticipate GLU-D developing as a promising analog of glutamate for studying the dynamics of glutamate during neuronal activity.

Time-resolved observation of surface-enhanced Raman scattering from gold nanoparticles during transport through a living cell.

We perform time-resolved observation of living cells with gold nanoparticles using surface-enhanced Raman scattering (SERS). The position and SERS spectra of 50-nm gold nanoparticles are simultaneously observed by slit-scanning Raman microscopy with high spatial and temporal resolution. From the SERS observation, we confirm the attachment of the particles on the cell surface and the entry into the cell with the subsequent generation of SERS signals from nearby molecules. We also confirm that the strong dependence of SERS spectra on the position of the particle during the transportation of the particle through the cell. The obtained SERS spectra and its temporal fluctuation indicate that the molecular signals observable by this technique are given only from within a limited volume in close proximity to the nanoparticles. This confirms the high spatial selectivity and resolution of SERS imaging for observation of biomolecules involved in cellular events in situ.

Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells.

The early detection of HER2 (human epidermal growth factor receptor 2) status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs) have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs) using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC). As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs) with fluorescence quantum yields of 0.23∼0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line) was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission) on the fluorescence image of KPL-4 cells.

Raman microscopy for dynamic molecular imaging of living cells.

We demonstrate dynamic imaging of molecular distribution in unstained living cells using Raman scattering. By combining slit-scanning detection and optimizing the excitation wavelength, we imaged the dynamic molecular distributions of cytochrome c, protein beta sheets, and lipids in unstained HeLa cells with a temporal resolution of 3 minutes. We found that 532-nm excitation can be used to generate strong Raman scattering signals and to suppress autofluorescence that typically obscures Raman signals. With this technique, we reveal time-resolved distributions of cytochrome c and other biomolecules in living cells in the process of cytokinesis without the need for fluorescent labels or markers.

DFT vibrational calculations of rhodamine 6G adsorbed on silver: analysis of tip-enhanced Raman spectroscopy.

The tip-enhanced near-field Raman (TERS) bands of Rhodamine 6G (R6G), that we reported earlier [Chem. Phys. Lett. 2001, 335, 369.], are assigned on the basis of density-functional theory (DFT) calculations at the 6-311++G(d,p) level. The Raman and infrared intensities as well as frequencies of the vibrational modes are used for band assignments. These vibrational modes, in combination with characterization of resonant electronic transitions using time-dependent DFT calculations, predict spectral changes in resonant Raman and surface-enhanced resonant Raman scatterings of R6G. Moreover, the TERS spectra of R6G are analyzed in detail, where interactions between the tip and R6G molecules and their enhancement mechanisms are discussed. Finally, we propose a novel Raman spectroscopy technique capable of detecting molecular vibrations at sub-nanometer scale.

Near-field optics and spectroscopy for molecular nano-imaging.

Application of near-field optical microscopy with a sharp metallic probe to Raman spectroscopy brings microanalysis of materials to their nano-identification and imaging. The local plasmon polariton excitation on the probe tip results in the localization and amplification of the optical field at the vicinity of the tip. The tip-enhanced near-field Raman spectroscopy has analyzed DNA base molecules and single-walled carbon nanotubes (SWNTs) with the nanometric spatial resolution and sufficient sensitivity. Combined with tip pressurization and nonlinear effects, the tip-enhanced near-field Raman spectroscopy gives additional spectral information or improves the spatial resolution and sensitivity. This article introduces the recent progresses on the tip-enhanced near-field Raman spectroscopy and imaging.

Nano-analysis of DNA conformation changes induced by transcription factor complex binding using plasmonic nanodimers.

The plasmon resonant wavelength for a pair of gold nanoparticles, or gold nanodimer, increases inversely with the gap distance between the two nanoparticles. Taking advantage of this property, we performed nanoscale measurements of DNA conformation changes induced by transcription factor binding. Gold nanoparticles were bridged by double-stranded DC5 DNA that included binding sequences for the transcription factors SOX2 and PAX6, which interact on the DC5 enhancer sequence and activate transcription. The gold nanodimers bound by SOX2 shifted the plasmon resonant wavelength from 586.8 to 604.1 nm, indicating that SOX2 binding induces DNA bending. When the SOX2 formed a ternary complex with PAX6 on DC5, the plasmon resonant wavelength showed a further shift to 611.6 nm, indicating additional bending in the DC5 sequence. Furthermore, we investigated DNA conformation changes induced by SOX2 and PAX6 on the DC5-con sequence, which is a consensus sequence of DC5 for the PAX6 binding region that strengthens the PAX6 binding but at the same time disrupts SOX2-PAX6-dependent transcriptional activation. When the PAX6 binding sequence in DC5 was altered to DC5-con, the plasmon resonant wavelength shifted much less to 606.5 nm, which is more comparable to the 603.9 nm by SOX2 alone. These results demonstrate that SOX2-PAX6 cobinding induces a large conformation change in DC5 DNA.

Single molecule detection from a large-scale SERS-active Au79Ag21 substrate.

Detecting and identifying single molecules are the ultimate goal of analytic sensitivity. Single molecule detection by surface-enhanced Raman scattering (SM-SERS) depends predominantly on SERS-active metal substrates that are usually colloidal silver fractal clusters. However, the high chemical reactivity of silver and the low reproducibility of its complicated synthesis with fractal clusters have been serious obstacles to practical applications of SERS, particularly for probing single biomolecules in extensive physiological environments. Here we report a large-scale, free standing and chemically stable SERS substrate for both resonant and nonresonant single molecule detection. Our robust substrate is made from wrinkled nanoporous Au79Ag21 films that contain a high number of electromagnetic "hot spots" with a local SERS enhancement larger than 109. This biocompatible gold-based SERS substrate with superior reproducibility, excellent chemical stability and facile synthesis promises to be an ideal candidate for a wide range of applications in life science and environment protection.

Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells.

To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells.

Subnanometric near-field Raman investigation in the vicinity of a metallic nanostructure.

We present a near-field Raman investigation in the subnanometric vicinity of a metallic nanotip, where the tip-sample distance is precisely controlled by our newly developed time-gated illumination technique. Using this scheme on an isolated carbon nanotube, we have profiled the spatial decay of evanescent light. We also investigated extremely short-ranged chemical and mechanical interactions between the metal on the tip apex and the molecules of an adenine sample, which are observable only within the subnanometric vicinity of the tip. The results show a near-field Raman investigation with an accuracy of better than a few angstroms. Further, this shows strong promise for superhigh resolution in optical microscopy based on this technique.

Nanoscale uniaxial pressure effect of a carbon nanotube bundle on tip-enhanced near-field Raman spectra.

In situ measurement of tip-enhanced near-field Raman spectra of an isolated single-wall carbon nanotube (SWNT) bundle has been demonstrated by applying a uniaxial pressure up to approximately 2 GPa to the bundle via a metal-coated atomic force microscope tip. We investigated the pressure dependences of Raman frequencies and the intensity of the radial breathing mode bands, the D-band and the G-band, which were related to deformation of SWNTs caused by the tip pressure.

Tip-enhanced coherent anti-stokes Raman scattering for vibrational nanoimaging.

An electric field enhanced by a metallic nanoprobe has locally induced coherent anti-Stokes Raman scattering (CARS) of adenine molecules in a nanometric DNA network structure. Owing to the third-order nonlinearity, the excitation of the CARS polarization is extremely confined to the end of the tip apex, resulting in a spatial resolution far beyond the diffraction limit of light. Our tip-enhanced CARS microscope visualized the DNA network structure at a specific vibrational frequency (approximately 1337 cm(-1)) corresponding to the ring-breathing mode of diazole of adenine molecules.