Atsushi Miyawaki

Control of Calcium Signal Propagation to the Mitochondria by Inositol 1,4,5-Trisphosphate-binding Proteins

Cytosolic Ca2+ ([Ca2+]c) signals triggered by many agonists are established through the inositol 1,4,5-trisphosphate (IP3) messenger pathway. This pathway is believed to use Ca2+-dependent local interactions among IP3 receptors (IP3R) and other Ca2+ channels leading to coordinated Ca2+ release from the endoplasmic reticulum throughout the cell and coupling Ca2+ entry and mitochondrial Ca2+ uptake to Ca2+ release. To evaluate the role of IP3 in the local control mechanisms that support the propagation of [Ca2+]c waves, store-operated Ca2+ entry, and mitochondrial Ca2+ uptake, we used two IP3-binding proteins (IP3BP): 1) the PH domain of the phospholipase C-like protein, p130 (p130PH); and 2) the ligand-binding domain of the human type-I IP3R (IP3R224–605). As expected, p130PH-GFP and GFP-IP3R224–605 behave as effective mobile cytosolic IP3 buffers. In COS-7 cells, the expression of IP3BPs had no effect on store-operated Ca2+ entry. However, the IP3-linked [Ca2+]c signal appeared as a regenerative wave and IP3BPs slowed down the wave propagation. Most importantly, IP3BPs largely inhibited the mitochondrial [Ca2+] signal and decreased the relationship between the [Ca2+]c and mitochondrial [Ca2+] signals, indicating disconnection of the mitochondria from the [Ca2+]c signal. These data suggest that IP3 elevations are important to regulate the local interactions among IP3Rs during propagation of [Ca2+]c waves and that the IP3-dependent synchronization of Ca2+ release events is crucial for the coupling between Ca2+ release and mitochondrial Ca2+ uptake.

Nanoaquarium: integrated microchips fabricated by ultrafast laser for understanding phenomena and functions of microorganisms

We demonstrate to fabricate microfluidic chips integrated with some functional microcomponents such as optical attenuators and optical waveguides by femtosecond laser direct writing for understanding phenomena and functions of microorganisms. Femtosecond laser irradiation followed by annealing and wet etching in dilute hydrofluoric acid solution resulted in fabrication of three-dimensional microfludic structures embedded in a photosensitive glass. The embedded microfludic structures enabled us to easily and efficiently observe Phormidium gliding to the seedling root, which accelerates growth of the vegetable. In addition, integration of optical attenuators and optical waveguides into the microfluidic structures clarified the mechanism of the gliding movement of Phormidium. We termed such integrated microchips nanoaquariums, realizing the highly efficient and functional observation and analysis of various microorganisms.

3D microstructuring inside glass by ultrafast laser

We demonstrate three-dimensional (3D) microstructuring inside glass by ultrafast laser to fabricate microfluidic chips integrated with some functional microcomponents such as optical attenuators and optical waveguides. The fabricated microchips are applied to understand phenomena and functions of microorganisms and cyanobacteria. Ultrafast laser irradiation followed by thermal treatment and wet etching in dilute hydrofluoric acid solution resulted in fabrication of 3D microfludic structures embedded in a photosensitive glass. The embedded microfludic structures enabled us to easily and efficiently observe Phormidium gliding to the seedling root, which accelerates growth of the vegetable. In addition, integration of optical attenuators and optical waveguides into the microfluidic structures clarified the mechanism of the gliding movement of Phormidium. We termed such integrated microchips nanoaquariums, realizing the highly efficient and functional observation and analysis of various microorganisms.

Nanoaquarium fabricated by femtosecond laser 3D micromachining: Investigation on phormidium assemblage

Internal modification of transparent materials such as glass can be carried out using multiphoton absorption induced by a femtosecond (fs) laser. The fs-laser modification followed by thermal treatment and successive chemical wet etching in a hydrofluoric (HF) acid solution forms three-dimensional (3D) hollow microstructures embedded in photosensitive glass. This technique is a powerful method for directly fabricating 3D microfludic structures integrated with some functional microcomponents inside a photosensitive glass microchip. We used the fabricated microchips, referred to as a nanoaquarium, for dynamic observations of living microorganisms. In this paper, among some examples, we focus on exploring mechanism of Phormidium assemblage to seedling root for growth promotion of vegetable using the nanoaquarium.Internal modification of transparent materials such as glass can be carried out using multiphoton absorption induced by a femtosecond (fs) laser. The fs-laser modification followed by thermal treatment and successive chemical wet etching in a hydrofluoric (HF) acid solution forms three-dimensional (3D) hollow microstructures embedded in photosensitive glass. This technique is a powerful method for directly fabricating 3D microfludic structures integrated with some functional microcomponents inside a photosensitive glass microchip. We used the fabricated microchips, referred to as a nanoaquarium, for dynamic observations of living microorganisms. In this paper, among some examples, we focus on exploring mechanism of Phormidium assemblage to seedling root for growth promotion of vegetable using the nanoaquarium.

Selective excitation in nonlinear microscopy by using an ultra-broadband pulse

We show that the selective excitation of vibrational mode is achieved by a single broadband pulse to focus its bandwidth into a narrow spectral region. The spectral focusing is performed by controlling the spectral phase.

Multifarious Two‐Photon Fluorescence Microscopy Employing Ultrabroadband Femtosecond Laser Pulses

We demonstrate two‐photon excited fluorescence (TPEF) microscopy employing the novel spectral phase modulation, which permits not only the selective excitation but also the simultaneous excitation together with controlling TPEF intensities from various fluorophores freely. The selective excitation is applied to the sequential dual‐color imaging without emission crosstalk and FRET imaging of a HeLa cell. By the simultaneous excitation, dual‐color imaging with various signal ratios including equal signal ratio is successfully achieved.