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.

Surface plasmon resonance (SPR) is widely used as a label-free method for monitoring molecular interactions such as antigen-antibody interactions by measuring the shift in the SPR angle due to changes in the refractive index of the sensing surface. Recently, SPR sensors based on Fano resonance have been proposed to improve the sensitivity of SPR sensors [1]. In our previous report, SF11/Ag/SiO2/TiO2 multilayer system for Fano resonant SPR was developed and applied to the detection of glucose concentration, successfully improving the sensitivity by about one order of magnitude [2].

Azo-polymer thin film, containing azo-benzene molecules, forms unique surface reliefs due to photoisomerization reaction under light irradiation [1]. It has been considered to be a promising material for various optical application. In addition, the formation of complex reliefs by optical vortex and skyrmion has recently attracted much attention in topological and quantum optics [2]. In previous, the formation process of azo-polymer has been studied by observing distinct deformed structures at different irradiation times using atomic force microscopy (AFM). To reveal the formation mechanisms, real-time nanoscale visualization of the azo-polymer deformation process is highly desirable.

We have developed an optical microscope based on surface plasmon resonance (SPR) to observe the dynamics of potassium ion (K+) concentrations. We formed a polyvinyl chloride (PVC) film containing ionophores that trap K+ on top of a gold film that excites surface plasmon. By measuring the attenuated total reflection spectra when injecting pure water or K+ solution on the fabricated substrate, we confirmed that the ionophore chelation reaction changed the PVC refractive index. Subsequently, we constructed a custom-made optical system for SPR imaging, mounted the substrate, and imaged the two-dimensional diffusion of K+ concentration changes when high concentrations of K+ solution were injected onto the substrate. In addition, we formed supported lipid bilayers on the substrate, mimicking cell membranes. Compared with the movie without lipid bilayer formation, that with supported lipid bilayers was confirmed to have taken longer time to increase the concentration due to its shielding effect of K+. Finally, we added K+ channels into the lipid bilayer membrane and visualized a K+ diffusion using the same procedure as that in the previous imaging experiment. We succeeded in observing the dynamics of K+ concentration change through the K+ channels inserted in the lipid bilayer. The addition of K+ channels weakened the shielding effect and shortened the time required for the K+ concentration to increase.

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.

We investigated the distribution changes of cytochrome c in mitochondria of neurons by Raman imaging. Cytochrome c gradually spread to neurites, implying that mitochondria increased in areas of high energy demand, including synaptic site.

The incident angle dependence of Raman scattering intensity of Rhodamine 6G enhanced by propagating surface plasmon resonance was quantitatively evaluated, and the dependence was found to be in good agreement with electromagnetic field analysis.

We have developed a non-invasive and high-resolution imaging method for the two-dimensional dynamics of metal ions in the vicinity of cells based on Surface Plasmon Resonance Microscope.

The determination of optical constants (i.e., real and imaginary parts of the complex refractive index (n(c)) and thickness (d)) of ultrathin films is often required in photonics. It may be done by using, for example, surface plasmon resonance (SPR) spectroscopy combined with either profilometry or atomic force microscopy (AFM). SPR yields the optical thickness (i.e., the product of n(c) and d) of the film, while profilometry and AFM yield its thickness, thereby allowing for the separate determination of n(c) and d. In this paper, we use SPR and profilometry to determine the complex refractive index of very thin (i.e., 58 nm) films of dye-doped polymers at different dye/polymer concentrations (a feature which constitutes the originality of this work), and we compare the SPR results with those obtained by using spectroscopic ellipsometry measurements performed on the same samples. To determine the optical properties of our film samples by ellipsometry, we used, for the theoretical fits to experimental data, Bruggeman's effective medium model for the dye/polymer, assumed as a composite material, and the Lorentz model for dye absorption. We found an excellent agreement between the results obtained by SPR and ellipsometry, confirming that SPR is appropriate for measuring the optical properties of very thin coatings at a single light frequency, given that it is simpler in operation and data analysis than spectroscopic ellipsometry.

The incident angle dependence of Raman scattering for five vibrational modes enhanced by propagating surface plasmon resonance was investigated by measuring the Raman scattering intensity of Rhodamine 6G on a silver thin film. Owing to the wavelength dependence of the resonance angle, the Raman scattering intensity for each Raman band was maximized at a different angle. The angular dependence of the enhanced Raman scattered light was the product of the surface plasmon's angular dependence on the excitation light and Raman scattered light. This is in good agreement with electromagnetic field analysis, including the resonant Raman effect.

The excitatory synaptic transmission is mediated by glutamate (GLU) in neuronal networks of the mammalian brain. In addition to the synaptic GLU, extra-synaptic GLU is known to modulate the neuronal activity. In neuronal networks, GLU uptake is an important role of neurons and glial cells for lowering the concentration of extracellular GLU and to avoid the excitotoxicity. Monitoring the spatial distribution of intracellular GLU is important to study the uptake of GLU, but the approach has been hampered by the absence of appropriate GLU analogs that report the localization of GLU. 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 microelectrode 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 GLU with equivalent effects for facilitating the neuronal activity. We anticipate GLU-D developing as a promising analog of GLU for studying the dynamics of glutamate during neuronal activity.

Surface enhanced absorption is a plasmonic effect parenting to surface enhanced fluorescence and Raman scattering, and it was clearly reported to occur in the infrared region of the spectrum of light. In this paper, we unambiguously show that it also occurs in the visible region of the spectrum by using a dye; i.e. an azo-dye, which exhibits a good light absorption in that region, and gold nanoparticles, which act as plasmonic nanoantennas that capture and re-radiate light, when the azo-dyes and the nanoparticles are incorporated in the bulk of solid films of polymer. In such a configuration, it is possible to use a dye concentration much larger than that of the nanoparticles and absorption path lengths much larger than those of the molecularly thin layers used in surface enhanced effects studies. In addition, the dye undergoes shape and orientation change; i.e. isomerization and reorientation, upon polarized light absorption; and the observation of surface enhanced visible absorption is done by two separate experiments; i.e. UV-visible absorption spectroscopy and photo-induced birefringence, since the signals detected from both experiments are directly proportional to the extinction coefficient of the dye. Both the dye's absorption and photoorientation are enhanced by the presence of the nanoparticles.

Two-color light was irradiated to azo-polymer films to investigate the mechanism of light-induced polymer movement. A collimated green light was used for inducing softening of the polymer, while a focused red light was used for inducing optical gradient force. We found from the topology change of the film that polymer movement was induced only when both green and red light were simultaneously irradiated. This finding indicated that the polymer movement was induced by optical gradient force generated by the red light under the condition that the polymer was softened by repeatable photoisomerization by the green light.