KEYWORDS: Microscopy, Luminescence, Spatial resolution, 3D image processing, Confocal microscopy, Two photon excitation microscopy, Stereoscopy, Point spread functions, Objectives, Time lapse microscopy
<p>Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.</p>
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(<sup>3)</sup>, with nonlinear behavior enhanced by LSPR.