Masahito Yamanaka

Introduction to super-resolution microscopy

In this review, we introduce the principles of spatial resolution improvement in super-resolution microscopies that were recently developed. These super-resolution techniques utilize the interaction of light and fluorescent probes in order to break the diffraction barrier that limits spatial resolution. The imaging property of each super-resolution technique is also compared with the corresponding conventional one. Typical applications of the super-resolution techniques in biological research are also introduced.

Beyond the diffraction-limit biological imaging by saturated excitation microscopy

We demonstrate high-resolution fluorescence imaging in biological samples by saturated excitation (SAX) microscopy. In this technique, we saturate the population of fluorescence molecules at the excited state with high excitation intensity to induce strong nonlinear fluorescence responses in the center of laser focus, which contributes the improvement of the spatial resolution in three dimensions. Using SAX microscopy, we observed stained microtubules in HeLa cells with improved spatial resolution. We also measured the relation of the fluorescence and excitation intensity with several kinds of fluorescence dyes and, in the results, confirmed that SAX microscopy has the potential to observe any kind of fluorescence samples in current usage.

A fast- and positively photoswitchable fluorescent protein for ultralow-laser-power RESOLFT nanoscopy

Fluorescence nanoscopy has revolutionized our ability to visualize biological structures not resolvable by conventional microscopy. However, photodamage induced by intense light exposure has limited its use in live specimens. Here we describe Kohinoor, a fast-switching, positively photoswitchable fluorescent protein, and show that it has high photostability over many switching repeats. With Kohinoor, we achieved super-resolution imaging of live HeLa cells using biocompatible, ultralow laser intensity (0.004 J/cm2) in reversible saturable optical fluorescence transition (RESOLFT) nanoscopy.

SAX microscopy with fluorescent nanodiamond probes for high-resolution fluorescence imaging

We report the use of fluorescent nanodiamonds (FNDs) as a photostable fluorescent probe for high resolution saturated excitation (SAX) microscopy. We confirmed that FNDs show a nonlinear fluorescence response under saturated excitation conditions generated by intense excitation light. Using FNDs, we quantified the spatial resolution improvement inherent in SAX microscopy, and experimentally demonstrated the scalability of the spatial resolution of SAX microscopy. The photostability of the FNDs allowed us to perform nanoparticle imaging of a multicolor-stained macrophage cell with a spatial resolution beyond the diffraction limit.

Determination of the Expanded Optical Transfer Function in Saturated Excitation Imaging and High Harmonic Demodulation

Most limits of a microscope can be understood in terms of its spatial frequency mapping ability, known as the optical transfer function (OTF). Here, we demonstrate the expansion of the confocal fluorescence microscopy OTF under conditions where the population of excitation states of the sample is saturated. We show how the OTF of saturated excitation can be determined by estimating fluorescence signals using a five-level molecular electronic state model. We then show how modulating excitation and demodulating fluorescence signals can extract high frequency components. We provide experimental validation by demonstrating that harmonic demodulation can extract high frequency OTF components.

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 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.

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

Abstract. 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 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.

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

Abstract. 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.

High-speed ultrahigh-resolution spectral domain optical coherence tomography using high-power supercontinuum at 0.8 µm wavelength

We demonstrated high-speed ultrahigh-resolution (UHR) optical coherence tomography (OCT) in the 800 nm wavelength region. A high-power coherent supercontinuum (SC) and a high-speed line scan camera were used to construct a spectral domain OCT. The axial resolution was 3.1 µm in air and 2.3 µm in tissue. The dependence of sensitivity on the SC power and A-scan rate was examined. For the A-scan rate of 70 kHz, the sensitivity of 104 dB was achieved for the SC power higher than 60 mW. High-speed in vivo UHR-OCT imaging was demonstrated for zebrafish embryo and swimming medaka.