Neurons form complex networks and communicate through synaptic connections. The molecular dynamics of cell surface molecules at synaptic terminals are essential for elucidating synaptic transmission and plasticity in biological neural networks. To achieve artificial control of synaptic transmission in neural networks at the single-synapse level, we propose and demonstrate the application of optical trapping for laser-induced perturbation to cellular molecules on neurons. In this study, we investigated the effects of optical forces on the dynamics of cell molecules in an optical trap on neurons. The diffusion properties of the cell surface molecules under optical trapping were evaluated using fluorescence analysis with single-particle tracking and fluorescence correlation spectroscopy. Molecular diffusion at the cell surface of neurons was compared to that of lipid molecules in artificial bilayers. Moreover, the molecular dynamics in an optical trap without fluorescent labeling under live cell conditions was evaluated using Raman spectroscopy.
Neuronal stimulation is essential to understand information processing in brain systems. Spatiotemporal patterns of neuronal activity can be modified by external stimuli. Recent studies have shown that neurons can be stimulated by short-pulse laser processing of the cell membrane. An optical vortex with a helical wavefront possesses an orbital angular momentum (OAM) enables the inward twisting of ablated materials, thereby processing further precisely cells beyond a conventional Gaussian beam. We herein study the mechanisms of neuronal stimulation with a focused nanosecond optical vortex. The focused nanosecond optical vortex on the cell membrane of rat hippocampal neurons induces extracellular Ca2+ influx and neuronal activity elicitation. Morphological changes of the neuronal cell membrane due to nanosecond optical vortex irradiation is also evaluated with fluorescence recovery after photobleaching. After the deposition of a single pulse of nanosecond optical vortex on the cell membrane of neurons, the fluorescence intensity of membrane probe at the focal region significantly decreases, however, it recovers within 5 seconds. Such dynamics suggests that the transient disruption occurs at the cell membrane based on laser ablation and recovers due to lateral diffusion of membrane molecules. The diffusion coefficients of membrane molecules after optical vortex irradiation are larger than those of Gaussian beam irradiation, and the disrupted membrane areas are smaller than the expected ones as the optical vortex focal region. These differences are attributed to the fact that the disruption of cell membrane owing to laser ablation and subsequent membrane diffusion are assisted by OAM transfer effects.
Neurons in the brain communicate by releasing and receiving neurotransmitters at synapse. Synaptic vesicles (SVs) that encapsulate neurotransmitters play an important role for neuronal communication. We demonstrate that optical trapping of synaptic vesicles in cultured rat hippocampal neurons regulates the neuronal network activity. The neuronal electrical activity was evaluated by extracellular potential measurement using microelectrodes arrays (MEAs). When a near-infrared trapping laser was focused on synaptic vesicles labeled with FM1-43 dye, fluorescence caused by two-photon absorption was observed at the focal spot. The fluorescence intensity gradually increased during the laser irradiation time at the laser power of 500 mW, indicating that optical trapping forces cause the assembly of SVs at the focal spot. In the extracellular potential measurement of neuronal electrical activity, spike number of spontaneous neuronal activity increased under optical trapping of SVs. The synchronicity of neuronal network activity by cross-correlation analysis increased after the laser irradiation under higher laser power conditions. These results suggest that neuronal electrical activity can be manipulated by optical trapping of synaptic vesicles.
For the purpose of precise manipulation of single nanoparticles by optical trapping, we demonstrated optical trapping of nanoparticles enhanced depending on the wavelength of excitation laser. The optical trapping dynamics of quantum dot (QD) nanoparticles at the focal spot was evaluated by fluorescence correlation spectroscopy (FCS). The simultaneous irradiation with excitation and near-infrared lasers increased the average transit time of QDs at the focal spot, which depended on the laser power and the wavelength of the excitation laser. This suggests that the particle motion of QD nanoparticles is constrained at the laser focus due to enhancement of optical trapping based on the resonant optical response.
DNA changes its conformation by combining a transcription factor or transcription factor complex on specific base sequences. We investigated the conformation changes by using local surface plasmon resonance of two gold nanoparticles linked to each other via the DNA, which compose a nano-dimer. Gap distance of the nano-dimer is reduced due to the DNA conformation change or bending, then the plasmon resonance shifts to longer wavelength. By measuring the plasmon resonant wavelength, gap distance is determined with a calibration curve prepared beforehand. Hence, conformation change of DNA bound with transcription factors is evaluated at nanoscale or sub-nanoscale. For example, a bending angle was determined to be 61.3º when SOX2, one of transcription factors, was bound on a double-stranded DNA having DC5 sequence and the DNA changes conformation. Binding SOX2 and PAX6 together on DC5 sequence, bending angles were evaluated to be 61.3º at SOX2 side and 5.7º at PAX6 side, respectively. When we used DNA having a DC5-con sequence which is a little different from DC5 sequence, bending angles were evaluated to be 61.1º at SOX2 side and 2.3º at PAX6 side. Such small difference in DNA conformations can be distinguished by using the local surface plasmon resonance. We also observed DNA conformation change by binding SOX2 on DC5 in real time and duration for conformation change was determined to be less than 100 msec. Such binding of DNA and transcription factors has possibility for a driving component for nano-machines.
When the size of metallic nanoparticles becomes smaller than 1 nm, of which nanostructures are composed of several tens of atoms, the plasmonic effect disappears and the electronic energy levels of the nanoparticles called as nanoclusters are quantized. Then, the nanoclusters can emit fluorescence of which wavelength depends on their size. We investigated synthetic method of Platinum nanoclusters (Pt NCs) that exhibit blue to yellow photoluminescence by a facile one-pot reduction method. They were synthesized from the mixture of H2PtCl6, hyper-branched polyethylenimine (PEI), and L-ascorbic acid, resulting in the formation stabilized with the amino groups in the cavities formed by coiled PEI ligands. The chain conformation of cationic polymer PEI depends on pH of solution. By controlling pH of the synthesis solution, the size of Pt NCs@PEI changes and their fluorescent wavelength can be tuned. Pt NCs@PEI were applied to the labeling of Chemokine receptors of the membrane of cancer HeLa cells and Glutamate receptors of the membrane of neural cells by binding them to an antibody via a conjugate protein for bio-imaging. They showed lower cell cytotoxicity than other nanoparticles such as Q-dots@COOH, indicating that they have better cell viability and great potential for biological applications.
Modern farming relies highly on pesticides to protect agricultural food items from insects for high yield and better
quality. Increasing use of pesticide has raised concern about its harmful effects on human health and hence it has become
very important to detect even small amount of pesticide residues. Raman spectroscopy is a suitable nondestructive
method for pesticide detection, however, it is not very effective for low concentration of pesticide molecules. Here, we
report an approach based on plasmonic enhancement, namely, particle enhanced Raman spectroscopy (PERS), which is
rapid, nondestructive and sensitive. In this technique, Raman signals are enhanced via the resonance excitation of
localized plasmons in metallic nanoparticles. Gold nanostructures are promising materials that have ability to tune
surface plasmon resonance frequency in visible to near-IR, which depends on shape and size of nanostructures. We
synthesized gold nanorods (GNRs) with desired shape and size by seed mediated growth method, and successfully
detected very tiny amount of pesticide present on food items. We also conformed that the detection of pesticide was not
possible by usual Raman spectroscopy.
Two photon polymerization (TPP) lithography has been established as a powerful tool to develop 3D fine structures of polymer materials, opening up a wide range applications such as micro-electromechanical systems (MEMS). TPP lithography is also promising for 3D micro fabrication of nanocomposites embedded with nanomaterials such as metal nanoparticles. Here, we make use of TPP lithography to fabricate 3D micro structural single wall carbon nanotube (SWCNT)/polymer composites. SWCNTs exhibit remarkable mechanical, electrical, thermal and optical properties, which leads to enhance performances of polymers by loading SWCNTs. SWCNTs were uniformly dispersed in an acrylate UV-curable monomer including a few amounts of photo-initiator and photo-sensitizer. A femtosecond pulsed laser emitting at 780 nm was focused onto the resin, resulting in the photo-polymerization of a nanometric volume of the resin through TPP. By scanning the focus spot three dimensionally, arbitrary 3D structures were created. The spatial resolution of the fabrication was sub-micrometer, and SWCNTs were embedded in the sub-micro sized structures. The fabrication technique enables one to fabricate 3D micro structural SWCNT/polymer composites into desired shapes, and thus the technique should open up the further applications of SWCNT/polymer composites such as micro sized photomechanical actuators.
We present a fabrication method of gold nanorod/ polymer composite microstructures by means of a femtosecond
near-infrared laser light. The mechanism of this method is based on a cooperation of two optical reactions;
two-photon polymerization (TPP) reaction only at the surface of gold nanorods, and optical accumulation of gold
nanorods in photo-polymerizable resin. Gold nanorods were mass-produced by seed mediated growth method, and
were mono-dispersed in photo-resin. The wavelength of the laser light was tuned resonant to two-photon
absorption of the photo-resin, and also close to a longitudinal local surface plasmon resonance (LSPR) mode of the
gold nanorods. The laser light excited LSPR onto gold nanorods, resulting in the formation of thin polymer layer
only at their surface through TPP. Concurrently occurring optical accumulation of gold nanorods by continuous
irradiation of laser light, gold nanorods got together into focus spot. The TPP layer at the surface of gold nanorods
worked as a glue to stick one another for forming their aggregated structure in micro/nano scale. By controlling
the intensity and the exposure time of laser light, an optimal condition was found to induce dominant polymerization
without any thermal damages. The scanning of the focus spot makes it possible to create arbitrary micro/nano
structures. This method has a potential to create plasmonic optical materials by controlling the alignment of gold
nanorods.
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