measurement-scattering-nonlinearities-from-single-plasmonic

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 ...

Osaka University

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 for sub-diffraction-limited imaging of cell clusters.

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.

Measurement of a saturated emission of optical radiation from gold nanoparticles: application to an ultrahigh resolution microscope.

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.

Saturated excitation microscopy with optimized excitation modulation.

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.

Point spread function analysis with saturable and reverse saturable scattering.

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.

Article	Year
Saturated excitation microscopy for sub-diffraction-limited imaging of cell clusters. Journal of biomedical optics\| PubMed ID: 24296998	2013
Measurement of a saturated emission of optical radiation from gold nanoparticles: application to an ultrahigh resolution microscope. Physical review letters\| PubMed ID: 24483931	2014
Saturated excitation microscopy with optimized excitation modulation. Chemphyschem : a European journal of chemical physics and physical chemistry\| PubMed ID: 24488765	2014
Point spread function analysis with saturable and reverse saturable scattering. Optics express\| PubMed ID: 25401635	2014
Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging. Journal of biomedical optics\| PubMed ID: 26238663	2015

Yasuo Yonemaru

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