Rational design of photoconvertible and biphotochromic fluorescent proteins for advanced microscopy applications

Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. IrisFP was recently reported as the first fluorescent protein combining these two types of phototransformations. The introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging. However, the spectroscopic properties of IrisFP are far from being optimal and its tetrameric organization complicates its use as a fusion tag. Here, we demonstrate how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins and develop and characterize a new set of such enhanced optical highlighters derived from mEosFP and Dendra2. We present in particular NijiFP, a promising new fluorescent protein with photoconvertible and biphotochromic properties that make it ideal for advanced fluorescence-based imaging applications.     

Thermodynamic quantities of surface formation of aqueous electrolyte solutions X. Aqueous solution of 2:1 valence-type salts

The behaviors of a series of calcium halides and of alkali earth metal chlorides in the air/water surface region were studied in comparison with those of alkali metal halides by measuring the surface tension increments of solutions. The effect of salts with divalent cations on the surface tension increments is more pronounced than that of uni-univalent salts, but there are some similarities between these two types. It seems that the anions cause a marked effect on surface tension which is proportional to the magnitude of hydration in the bulk water. We also observed a decrease in the entropy change of surface formation with increasing concentration. The importance of an electrical double layer at the surface is discussed in relation to these surface tension increments.     

Photochemically triggered reaction of glucose oxidase in a fused vesicle containing Malachite Green leuconitrile derivative

Glucose oxidase (GOD) was encapsulated in vesicles containing a photoionizable Malachite Green leuconitrile derivative (MGL). Subsequent UV irradiation of MGL afforded the fusion of GOD- and glucose-encapsulating vesicles and thus decreased the concentration of glucose in the vesicles. The time dependence of the vesicle fusion was studied using fluorescent probe molecules. This phototriggered fusion could be instrumental in the development of a system for the production of nanometer-sized bioreactors.     

Interaction of Near-Field Light with Ordered Polystyrene Spheres: Experimental Studies

We have experimentally observed the interaction of near-field light excited in a total internal reflection geometry with a periodic two-dimensional array of polystyrene spheres. The angular- and frequencyscanned attenuated-total-reflection (ATR) method was employed to observe the interaction. ATR spectra indicate that there is an easy axis and a difficult axis for the light propagation. When light is incident toward the easy axis, resonant dips appear in the ATR spectra. The experimental results were compared with those obtained by finite-difference time-domain analysis. The numerical results obtained by this method are able to explain a part of the experimental ones. Parts of the dips in the ATR spectra were considered to stem from propagating and whispering-gallery (WG) modes. That is, the propagation of the WG-modes over several spheres may enhance the coupling between the spheres and the incident light, and consequently may induce resonant structures in the ATR spectra.This paper was originally presented at the 5th International Conference on NEAR FIELD OPTICS and RELATED TECHNOLOGIES (NFO-5), which was held on December 6–10, 1998 at Coganoi Bay Hotel, Shirahama, Japan, in cooperation with the Japan Society of Applied Physics and Mombusho Grant-in Aid for Scientific Research on Priority Areas “Nearfield Nano-optics” Project, sponsored by Japan Society for the Promotion of Science.     

β-Trifluoromethyl-α-functionalized-vinyl sulfides as a potential synthetic intermediate

The β-(trifluoromethyl)vinyl sulfides on treatment with n-BuLi/TMEDA at −78 °C were readily lithiated at an α-position of the sulfanyl group, and the generated β-trifluoromethyl-α-sulfanylvinyl anions were reacted with a variety of electrophiles to give the corresponding β-trifluoromethyl-α-functionalized-vinyl sulfides 4aa-4aq in good to excellent yields. The reactivity of some products has been examined. The palladium-catalyzed cross-coupling reaction as well as homo-coupling reaction of 4af provided the corresponding products in good yields, respectively. The Diels-Alder reaction of cyclic dienes and 14 derived from 4ao provided the desired six-membered cyclic products with high endo-trifluoromethyl group selectivity.     

Formation and photochemistry of organic sulfur compound–chlorine atom complexes

Photochemical reactions of organic sulfur compounds (CH₃SCH₃, C₂H₅SCH₃ and C₂H₅SC₂H₅)–chlorine atom complexes have been studied using a combined pulse radiolysis-laser flash photolysis technique. Excitation of all complexes has resulted in photobleaching with a similar quantum yield (0.37±0.07), independent of solvent polarities and concentration of solutes. The results were compared with previous studies of the analogous dimethyl sulfoxide (DMSO)–Cl complexes. It is concluded that the significant change of photobleaching quantum yields of the excited DMSO–Cl complex observed in the DMSO-CCl₄ mixed solvent is mainly due to the specific solvation effect of DMSO for cations.     

Stereoselective olefination of unfunctionalized ketones via ynolates

Ynolates react with ketones at room temperature to afford alpha,beta,beta-trisubstituted acrylates (tetrasubstituted olefins) with 2:1-8:1 geometrical selectivities. This can be regarded as a new olefination reaction of ketones giving tetrasubstituted olefins in good yield, even in the case of sterically hindered substrates. The reaction mechanism involves cycloaddition of ynolates with a carbonyl group and subsequent thermal electrocyclic ring-opening of the resulting beta-lactone enolates. The stereoselectivity is determined in the ring-opening, which is regulated by torquoselectivity. In this paper, we describe the scope and limitations of olefination of ketones via ynolates and discuss the stereocontrol mechanism.     

Ultrasonic plastic welding using fundamental and higher resonance frequencies

Ultrasonic plastic welding using fundamental and higher resonance frequency vibrations simultaneously was studied. Using higher frequency, welding characteristics is improved due to the larger vibration loss of plastic materials. The 26 kHz welding tip vibrates in maximum velocity of over 4.5 m/s (peak-to-zero value) under a fundamental resonance frequency and there are several higher resonance frequencies up to 95 kHz whose vibration velocities are over one-third that of the fundamental frequency. Welding characteristics of 1.0-mm-thick polypropylene sheets are measured in the cases the vibration system are driven under combined driving voltages of fundamental and higher resonance frequencies. Welded area increases as number of driven higher frequencies increases. The welded area by three frequencies is about three to four times that of the case where only the fundamental frequency is driven. The welding characteristics of ultrasonic plastic welding are improved significantly by driving higher resonance frequencies simultaneously.     

Geometrically selective synthesis of functionalized beta,beta-disubstituted vinylic sulfoxides by Cu-catalyzed conjugate addition of organozinc reagents to 1-alkynyl sulfoxides

[reaction: see text] A new synthetic method of chiral beta,beta-disubstituted vinylic sulfoxides bearing various functionalities has been developed by employing Cu-catalyzed conjugate addition of an organozinc reagent to chiral 1-alkynyl sulfoxide. Since the reaction proceeds with very high syn-selectivity, both geometric beta,beta-disubstituted vinylic sulfoxides were stereoselectively synthesized by changing the combination of 1-alkynyl sulfoxide and the organozinc reagent.