Search

We previously developed a fully automated spherical aberration compensation microscope system, Deep-C, to obtain spherical aberration-free images, but the contrast-based algorithm (Peak-C) may limit applications for low signal-to-noise ratio images. Herein we propose a new spatial frequency-based algorithm called Peak-F and compared its performance to Peak-C. Unlike Peak-C, Peak-F is robust to any noise level since it is independent of the dynamic range of the images, and it does not suffer from image saturation. Finally, Peak-F was implemented in a two-photon microscope to observe living aged and young mouse brains. Consequently, the average refractive index of brain tissue was higher in old mice than in young mice. The Peak-F algorithm determines high-resolution microscopic images stably and robustly.

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.

Nonlinear optical interaction is crucial to all-optical signal processing. In metallic nanostructures, both linear and nonlinear optical interactions can be greatly enhanced by surface plasmon resonance (SPR). In the last few decades, saturation and reverse saturation of absorption in plasmonic materials have been unraveled. It is known that scattering is one of the fundamental light–matter interactions and is particularly strong in metallic nanoparticles due to SPR. However, previous methods measure response from ensemble of nanoparticles and did not characterize scattering on a single particle basis. Here we report that backscattering from an isolated gold nanoparticle exhibits not only saturation, but also reverse saturation. Wavelength-dependent and intensity-dependent studies reveal that nonlinear scattering is dominated by SPR and shares a similar physical origin with nonlinear absorption. The reversibility and repeatability of saturable scattering (SS) and reverse saturable scattering (RSS) are val...

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.

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.

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.

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.

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.

Recently, we discovered, for the first time, reverse saturable scattering in a single gold nanoparticle. When incident intensity increases, the scattering intensity dependence of 80-nm gold nanoparticles evolves from linear, to saturation, and to reverse saturation sequentially. The intensity dependence in reverse saturable scattering region is significantly steeper than that in the linear region. With the aid of a confocal microscope, the full width half maximum of the single-particle point spread function can be reduced down to 80 nm, which is beyond the diffraction limit. Our finding shows great potential for superresolution imaging application without bleaching.

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.

We show that scattering from a single gold nanoparticle is saturable for the first time. Wavelength-dependent study reveals that the saturation behavior is governed by depletion of surface plasmon resonance, not the thermal effect. We observed interesting flattening of the point spread function of scattering from a single nanoparticle due to saturation. By extracting the saturated part of scattering via temporal modulation, we achieve λ/8 point spread function in far-field imaging with unambiguous separation of adjacent particles.

Recently, we reported that the localized surface plasmon resonance (LSPR) could strongly affect to the nonlinear light scattering from a single gold nanoparticle. The saturation and reverse satiation effect of light scattering were observed under the laser irradiation at the resonance [1, 2], but not under the off-resonance conditions.

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.

We observed the saturation of plasmonic light scattering on gold nanoparticles. Extracting the nonlinear signals due to the saturation, we demonstrated super-resolution imaging of two nearby gold nanoparticles.

Recently, several superresolution methods have been demonstrated to revolutionize the way we access the nano-world. However, these techniques are based on switching or saturation of fluorescence, and consequently are limited by switching reversibility and photobleaching. Here we show first superresolution far-field imaging based on scattering, which is a universal phenomenon without bleaching. Our principal finding is that scattering from plasmonics particles is saturable. By extracting the saturated part, sub-80-nm resolution is achieved. This work not only expands the horizon of superresolution imaging from fluorescence to scattering, but also points out the possibility of resolution enhancement for all imaging modalities that utilize plasmon properties.

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.

Nowadays, nonlinear optics plays an unprecedented role in research not only because of its intriguing physical mechanisms but the wide range of photonic functionalities, such as frequency generation, ultrafast signal processing, microscopy, etc. [1]. However, optical nonlinearities are usually weak. One of the most promising candidates is the nonlinearity of localized surface plasmon resonance (LSPR) in noble metal nanostructure. The strongly enhanced nonlinearity is usually attributed to the strong surface field enhancement due to the nanometer scale and the interband transitions near visible light. Many examples had been discovered, such as Kerr effect [2], saturable and reverse-saturable absorption (SA and RSA) [3].

In this work, we investigated, both theoretically and experimentally, the saturable scattering in a single gold nanoparticle for the first time. In theoretical part, we used different models of the nonlinear properties to explain the nonlinear responses in gold material. In experimental part, multi-color confocal microscopy was used to observe the scattering of a single gold nanoparticle. As a result, by a resonant excitation, saturable scattering was observed with moderate excitation intensity (~10 7 W/cm 2 ); with even higher excitation intensity (>10 9 W/cm 2 ), reverse saturable scattering was observed, indicating the existence of higher order nonlinear properties. To completely comprehend the mechanism of this saturable scattering, we applied three kinds of excitation wavelengths (405nm, 532nm and 671nm) and four kinds of gold nanoparticle with different diameters (40nm, 50nm, 80nm and 100nm) to demonstrate the wavelength dependence and size dependence. Since the scattering of gold nanoparticles is significantly enhanced by localized surface plasmon resonance, we compared these dependencies with the spectral properties induced by LSPR and found that they match the spectra, revealing that the saturation is dominated by plasmon resonance. Besides, by fitting the dependencies, linear and nonlinear hyperpolarizability of a single gold nanoparticle were also deduced.

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.