StayGold photostability under different illumination modes.

StayGold is a bright fluorescent protein (FP) that is over one order of magnitude more photostable than any of the currently available FPs across the full range of illumination intensities used in widefield microscopy and structured illumination microscopy, the latter of which is a widefield illumination-based technique. To compare the photostability of StayGold under other illumination modes with that of three other green-emitting FPs, namely EGFP, mClover3, and mNeonGreen, we expressed all four FPs as fusions to histone 2B in HeLa cells. Unlike the case of widefield microscopy, the photobleaching behavior of these FPs in laser scanning confocal microscopy (LSCM) is complicated. The outstanding photostability of StayGold observed in multi-beam LSCM was variably attenuated in single-beam LSCM, which produces intermittent and instantaneously strong illumination. We systematically examined the effects of different single-beam LSCM beam-scanning patterns on the photostability of the FPs in living HeLa cells. This study offers relevant guidelines for researchers who aim to achieve sustainable live cell imaging by resolving problems related to FP photostability. We also provide evidence for measurable sensitivity of the photostability of StayGold to chemical fixation.

Spatial frequency-based correction of the spherical aberration in living brain imaging.

Optical errors, including spherical aberrations, hinder high-resolution imaging of biological samples due to biochemical components and physical properties. We developed the Deep-C microscope system to achieve aberration-free images, employing a motorized correction collar and contrast-based calculations. However, current contrast-maximization techniques, such as the Brenner gradient method, inadequately assess specific frequency bands. The Peak-C method addresses this issue, but its arbitrary neighbor selection and susceptibility to the noise limit its effectiveness. In this paper, we emphasize the importance of a broad spatial frequency range for accurate spherical aberration correction and propose Peak-F. This spatial frequency-based system utilizes a fast Fourier transform as a bandpass filter. This approach overcomes Peak-C's limitations and comprehensively covers the low-frequency domain of image spatial frequencies.

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 µm(3), 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) m(2)/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.

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.

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

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.

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.

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

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.

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

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.