Yasuo Yonemaru

Ultrasmall all-optical plasmonic switch and its application to superresolution imaging

Because of their exceptional local-field enhancement and ultrasmall mode volume, plasmonic components can integrate photonics and electronics at nanoscale, and active control of plasmons is the key. However, all-optical modulation of plasmonic response with nanometer mode volume and unity modulation depth is still lacking. Here we show that scattering from a plasmonic nanoparticle, whose volume is smaller than 0.001 μm3, can be optically switched off with less than 100 μW power. Over 80% modulation depth is observed, and shows no degradation after repetitive switching. The spectral bandwidth approaches 100 nm. The underlying mechanism is suggested to be photothermal effects, and the effective single-particle nonlinearity reaches nearly 10−9 m2/W, which is to our knowledge the largest record of metallic materials to date. As a novel application, the non-bleaching and unlimitedly switchable scattering is used to enhance optical resolution to λ/5 (λ/9 after deconvolution), with 100-fold less intensity requirement compared to similar superresolution techniques. Our work not only opens up a new field of ultrasmall all-optical control based on scattering from a single nanoparticle, but also facilitates superresolution imaging for long-term observation.

Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters.

Abstract. Saturated excitation (SAX) microscopy offers high-depth discrimination predominantly due to nonlinearity in the fluorescence response induced by the SAX. Calculation of the optical transfer functions and the edge responses for SAX microscopy revealed the contrast improvement of high-spatial frequency components in the sample structure and the effective reduction of background signals from the out-of-focus planes. Experimental observations of the edge response and x-z cross-sectional images of stained HeLa cells agreed well with theoretical investigations. We applied SAX microscopy to the imaging of three-dimensional cultured cell clusters and confirmed the resolution improvement at a depth of 40 μm. This study shows the potential of SAX microscopy for super-resolution imaging of deep parts of biological specimens.

Saturated Excitation Microscopy with Optimized Excitation Modulation

Saturated excitation (SAX) microscopy utilizes the nonlinear relation between fluorescence emission and excitation under saturated excitation to improve the spatial resolution of confocal microscopy. In this study, we theoretically and experimentally investigate the saturation of fluorescence excitation under modulated excitation to optimize the excitation conditions for SAX microscopy. Calculation of the relationships between fluorescence and excitation intensity with different modulation frequencies reveals that the lifetime of the triplet state of the fluorescent probe strongly affects the strength of the demodulated fluorescence signals. We also find that photobleaching shows little dependence on the modulation frequency. These investigations allow us to determine the optimum excitation conditions, that is, the conditions providing sufficient fluorescence saturation without strong photobleaching. For a sample stained with ATTO Rho6G phalloidin, we estimate the optimal excitation conditions, which are produced with 50 kHz excitation modulation and a 50 μsec pixel dwell time, and successfully perform three-dimensional imaging with sub-diffraction resolution.

Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging

Abstract. The simultaneous observation of multiple fluorescent proteins (FPs) by optical microscopy is revealing mechanisms by which proteins and organelles control a variety of cellular functions. Here we show the use of visible-light based two-photon excitation for simultaneously imaging multiple FPs. We demonstrated that multiple fluorescent targets can be concurrently excited by the absorption of two photons from the visible wavelength range and can be applied in multicolor fluorescence imaging. The technique also allows simultaneous single-photon excitation to offer simultaneous excitation of FPs across the entire range of visible wavelengths from a single excitation source. The calculation of point spread functions shows that the visible-wavelength two-photon excitation provides the fundamental improvement of spatial resolution compared to conventional confocal microscopy.

Point spread function analysis with saturable and reverse saturable scattering

Nonlinear plasmonics has attracted a lot of interests due to its wide applications. Recently, we demonstrated saturation and reverse saturation of scattering from a single plasmonic nanoparticle, which exhibits extremely narrow side lobes and central peaks in scattering images [ACS Photonics 1(1), 32 (2014)]. It is desirable to extract the reversed saturated part to further enhance optical resolution. However, such separation is not possible with conventional confocal microscope. Here we combine reverse saturable scattering and saturated excitation (SAX) microscopy. With quantitative analyses of amplitude and phase of SAX signals, unexpectedly high-order nonlinearities are revealed. Our result provides greatly reduced width in point spread function of scattering-based optical microscopy. It will find applications in not only nonlinear material analysis, but also high-resolution biomedical microscopy.

Measurement of Scattering Nonlinearities from a Single Plasmonic Nanoparticle.

Plasmonics, which are based on the collective oscillation of electrons due to light excitation, involve strongly enhanced local electric fields and thus have potential applications in nonlinear optics, which requires extraordinary optical intensity. One of the most studied nonlinearities in plasmonics is nonlinear absorption, including saturation and reverse saturation behaviors. Although scattering and absorption in nanoparticles are closely correlated by the Mie theory, there has been no report of nonlinearities in plasmonic scattering until very recently. Last year, not only saturation, but also reverse saturation of scattering in an isolated plasmonic particle was demonstrated for the first time. The results showed that saturable scattering exhibits clear wavelength dependence, which seems to be directly linked to the localized surface plasmon resonance (LSPR). Combined with the intensity-dependent measurements, the results suggest the possibility of a common mechanism underlying the nonlinear behaviors of scattering and absorption. These nonlinearities of scattering from a single gold nanosphere (GNS) are widely applicable, including in super-resolution microscopy and optical switches. In this paper, it is described in detail how to measure nonlinearity of scattering in a single GNP and how to employ the super-resolution technique to enhance the optical imaging resolution based on saturable scattering. This discovery features the first super-resolution microscopy based on nonlinear scattering, which is a novel non-bleaching contrast method that can achieve a resolution as low as l/8 and will potentially be useful in biomedicine and material studies.

Plasmon saturation induced super-resolution imaging

Conventionally, super-resolution imaging is achieved by manipulating the on/off switching of fluorophores, or by saturation of fluorescence emission. To prevent the photobleaching of fluorophores, we demonstrate novel superresolution imaging based on saturation of scattering from plasmonic particles, for the first time. With spectral studies, we have confirmed the saturation is directly linked to surface plasmon resonance effect. With the aid of saturation excitation microscopy, plus field concentration due to nonlinear plasmon resonance, we have achieved optical resolution below 80-nm based on scattering. Our study will open up a completely new paradigm for super-resolution microscopy.

Saturated excitation microscopy using differential excitation for efficient detection of nonlinear fluorescence signals

We report a method to increase the efficiency of detecting nonlinear fluorescence signals in saturated excitation (SAX) microscopy. With this method, we compare fluorescence signals obtained under different degrees of saturated excitation to extract the nonlinear fluorescent signal induced by saturated excitation. Compared to conventional SAX microscopy using the harmonic demodulation technique, the detection efficiency of the fluorescence signal can be increased up to 8 and 32 times in imaging using the second-order and the third-order nonlinear fluorescence signals, respectively. We combined this approach with pulsed excitation, which is effective to reduce photobleaching effects, and achieved super-resolution imaging using third-order nonlinear fluorescence signals induced by saturated excitation of an organic dye. The resolution improvement was confirmed in the observations of fluorescent beads, actin-filaments in HeLa cells, and a spine in mouse brain tissue.We report a method to increase the efficiency of detecting nonlinear fluorescence signals in saturated excitation (SAX) microscopy. With this method, we compare fluorescence signals obtained under different degrees of saturated excitation to extract the nonlinear fluorescent signal induced by saturated excitation. Compared to conventional SAX microscopy using the harmonic demodulation technique, the detection efficiency of the fluorescence signal can be increased up to 8 and 32 times in imaging using the second-order and the third-order nonlinear fluorescence signals, respectively. We combined this approach with pulsed excitation, which is effective to reduce photobleaching effects, and achieved super-resolution imaging using third-order nonlinear fluorescence signals induced by saturated excitation of an organic dye. The resolution improvement was confirmed in the observations of fluorescent beads, actin-filaments in HeLa cells, and a spine in mouse brain tissue.

Superresolution imaging based on nonlinearities of plasmonic scattering

We demonstrated novel non-bleaching contrast for super-resolution imaging based on saturation and on/off switching of scattering from plasmonic particles, for the first time. Our study opens up new paradigms for both plasmonics and super-resolution microscopy.

Size and wavelength dependency of saturable scattering by a single gold nanosphere embedded in dielectric material

The wavelength and size dependencies of nonlinear scattering by a single gold nanosphere immersed in oil are presented. We show that the wavelength dependency fits well with the scattering spectrum by Mie solution, reflecting that the nonlinear scattering is dominated by the field enhancement from plasmonic effects. The tendency for different sizes is consistent with the results of degenerate four-wave mixing in the literature, showing that the saturation behavior is governed by the Kerr nonlinearity resonantly enhanced via intraband transition. Thus we conclude that the saturable scattering in our case is attributed to intraband χ(3), with nonlinear behavior enhanced by LSPR.