Masayasu Taki

Fluorescence Imaging of Intracellular Cadmium Using a Dual-Excitation Ratiometric Chemosensor

We described here a coumarin-based dual-excitation ratiometric probe for cadmium, CadMQ. This fluorescence sensor has high quantum yields of 0.59 and 0.70 in the metal-free and Cd2+-bound forms, respectively, and has a dissociation constant of 0.16 nM for Cd2+. CadMQ is cell permeable and locates within the acidic compartments of the cells. We further show that CadMQ is a useful tool to ratiometrically probe the change in the intracellular Cd2+ levels with the use of two excited wavelengths.

Development of Highly Sensitive Fluorescent Probes for Detection of Intracellular Copper(I) in Living Systems

We describe here the new copper-selective fluorescent probes FluTPA1 and FluTPA2, in which the tetradentate ligand tris[(2-pyridyl)methyl]amine (TPA) is connected to a reduced form of a fluorescein platform through a benzyl ether linkage. These probes selectively react with copper ions in the presence of submillimolar glutathione and emit intense green fluorescence from the reaction product, O-methylfluorescein. Confocal images of live cells further show that FluTPA2 is membrane-permeable and allows visualization of Cu(I) present in living cells.

Rosamine-Based Fluorescent Chemosensor for Selective Detection of Silver(I) in an Aqueous Solution

The synthesis and photophysical properties of a rosamine-based fluorescent chemosensor, RosAg, for detecting Ag ion in an aqueous solution are described. This fluorescent sensor has a negligible quantum yield (<0.005) in the absence of Ag(+), whereas a significant increase in fluorescence is observed upon complexation with Ag(+) under physiological conditions. The crystal structure of the silver complex with the chelator moiety of RosAg reveals a trigonal-planar coordination geometry in which three S atoms occupy the metal center. Although a strong coordinative interaction of Ag-N is not observed in the crystal structure, the (1)H NMR experiments suggest that aniline nitrogen is likely to be associated with the Ag(+) center in the solution state. This may inhibit the photoinduced electron transfer process and result in the enhancement of fluorescence.

Environment-Sensitive Fluorescent Probe: A Benzophosphole Oxide with an Electron-Donating Substituent†

Electron-donating aryl groups were attached to electron-accepting benzophosphole skeletons. Among several derivatives thus prepared, one benzophosphole oxide was particularly interesting, as it retained high fluorescence quantum yields even in polar and protic solvents. This phosphole-based compound exhibited a drastic color change of its fluorescence spectrum as a function of the solvent polarity, while the absorption spectra remained virtually unchanged. Capitalizing on these features, this phosphole-based compound was used to stain adipocytes, in which the polarity of subcellular compartments could then be discriminated on the basis of the color change of the fluorescence emission.

Rosamine-Based Fluorescent Sensor with Femtomolar Affinity for the Reversible Detection of a Mercury Ion

A Hg(2+)-selective fluorescent sensor, RosHg, has been developed based on a rosamine platform. RosHg exhibited a ∼20-fold increase in fluorescence emission upon binding with Hg(2+), and the enhanced fluorescence was immediately decreased when glutathione was added to a solution of the Hg-RosHg complex. The dissociation constant for the Hg(2+) complex was determined to be 0.10 fM by using a set of Hg(2+)/Mg(2+)/ethylenediaminetetraacetic acid buffer solutions. Confocal microscopy experiments demonstrated that this sensor can monitor changes of the Hg(2+) level in the mitochondria of living cells.

Development of a cholesterol-conjugated fluorescent sensor for site-specific detection of zinc ion at the plasma membrane.

A cholesterol-conjugated fluorescence Zn(2+) sensor based on the fluorescein platform was designed and synthesized. The cholesterol moiety is essential for localizing the Zn(2+) sensor to the cell membrane, allowing the sensor to probe changes in the Zn(2+) concentration in a localized area of the cell.

A Phosphole Oxide Based Fluorescent Dye with Exceptional Resistance to Photobleaching: A Practical Tool for Continuous Imaging in STED Microscopy

The development of stimulated emission depletion (STED) microscopy represented a major breakthrough in cellular and molecular biology. However, the intense laser beams required for both excitation and STED usually provoke rapid photobleaching of fluorescent molecular probes, which significantly limits the performance and practical utility of STED microscopy. We herein developed a photoresistant fluorescent dye C-Naphox as a practical tool for STED imaging. With excitation using either a λ=405 or 488 nm laser in protic solvents, C-Naphox exhibited an intense red/orange fluorescence (quantum yield ΦF >0.7) with a large Stokes shift (circa 5900 cm(-1) ). Even after irradiation with a Xe lamp (300 W, λex =460 nm, full width at half maximum (FWHM)=11 nm) for 12 hours, 99.5 % of C-Naphox remained intact. The high photoresistance of C-Naphox allowed repeated STED imaging of HeLa cells. Even after recording 50 STED images, 83 % of the initial fluorescence intensity persisted.

Water‐Soluble N‐Heterocyclic Carbene‐Protected Gold Nanoparticles: Size‐Controlled Synthesis, Stability, and Optical Properties

NHC-AuI complexes were used to prepare stable, water-soluble, NHC-protected gold nanoparticles. The water-soluble, charged nature of the nanoparticles permitted analysis by polyacrylamide gel electrophoresis (PAGE), which showed that the nanoparticles were highly monodisperse, with tunable core diameters between 2.0 and 3.3 nm depending on the synthesis conditions. Temporal, thermal, and chemical stability of the nanoparticles were determined to be high. Treatment with thiols caused etching of the particles after 24 h; however larger plasmonic particles showed greater resistance to thiol treatment. These water-soluble, bio-compatible nanoparticles are promising candidates for use in photoacoustic imaging, with even the smallest nanoparticles giving reliable photoacoustic signals.

Human Spire Interacts with the Barbed End of the Actin Filament

Spire is an actin nucleator that initiates actin polymerization at a specific place in the cell. Similar to the Arp2/3 complex, spire was initially considered to bind to the pointed end of the actin filament when it generates a new actin filament. Subsequently, spire was reported to be associated with the barbed end (B-end); thus, there is still no consensus regarding the end with which spire interacts. Here, we report direct evidence that spire binds to the B-end of the actin filament, under conditions where spire accelerates actin polymerization. Using electron microscopy, we visualized the location of spire bound to the filament by gold nanoparticle labeling of the histidine-tagged spire, and the polarity of the actin filament was determined by image analysis. In addition, our results suggest that multiple spires, linked through one gold nanoparticle, enhance the acceleration of actin polymerization. The B-end binding of spire provides the basis for understanding its functional mechanism in the cell.

Super-Photostable Phosphole-Based Dye for Multiple-Acquisition Stimulated Emission Depletion Imaging

As stimulated emission depletion (STED) microscopy can provide structural details of cells with an optical resolution beyond the diffraction limit, it has become an indispensable tool in cell biology. However, the intense STED laser beam usually causes rapid photobleaching of the employed fluorescent dyes, which significantly limits the utility of STED microscopy from a practical perspective. Herein we report a new design of super-photostable dye, PhoxBright 430 (PB430), comprising a fully ring-fused π-conjugated skeleton with an electron-accepting phosphole P-oxide unit. We previously developed a super-photostable dye C-Naphox by combining the phosphole unit with an electron-donating triphenylamine moiety. In PB430, removal of the amino group alters the transition type from intramolecular charge transfer character to π-π* transition character, which gives rise to intense fluorescence insensitive to molecular environment in terms of fluorescence colors and intensity, and bright fluorescence even in aqueous media. PB430 also furnishes high solubility in water, and is capable of labeling proteins with maintaining high fluorescence quantum yields. This dye exhibits outstanding resistance to photoirradiation even under the STED conditions and allows continuous acquisition of STED images. Indeed, using a PB430-conjugated antibody, we succeed in attaining a 3-D reconstruction of super-resolution STED images as well as photostability-based multicolor STED imaging of fluorescently labeled cytoskeletal structures.