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.
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.
KEYWORDS: Microscopy, Confocal microscopy, Luminescence, Spatial resolution, Signal detection, 3D image processing, Point spread functions, Glasses, Optical transfer functions, Demodulation
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.
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.
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 (~107 W/cm2); with even higher excitation intensity (>109 W/cm2), 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.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.