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Green-to-red photoconvertible fluorescent proteins (PCFPs) are key players in advanced microscopy schemes such as photoactivated localization microscopy (PALM). Whereas photoconversion and red-state blinking in PCFPs have been studied intensively, their green-state photophysical behavior has received less attention. Yet dark states in green PCFPs can become strongly populated in PALM schemes and exert an indirect but considerable influence on the quality of data recorded in the red channel. Furthermore, green-state photoswitching in PCFPs can be used directly for PALM and has been engineered to design highly efficient reversibly switchable fluorescent proteins (RSFPs) amenable to various nanoscopy schemes. Here, we demonstrate that green mEos4b efficiently switches to a long-lived dark state through cis-trans isomerization of its chromophore, as do most RSFPs. However, by combining kinetic crystallography, molecular dynamics simulations, and Raman spectroscopy, we find that the dark state in green mEos4b is much more dynamic than that seen in switched-off green IrisFP, a biphotochromic PCFP engineered from the common EosFP parent. Our data suggest that H-bonding patterns maintained by the chromophore in green PCFPs and RSFPs in both their on- and off-states collectively control photoswitching quantum yields. The reduced number of H-bonds maintained by the dynamic dark chromophore in green mEos4b thus largely accounts for the observed lower switching contrast as compared to that of IrisFP. We also compare the long-lived dark states reached from green and red mEos4b, on the basis of their X-ray structures and Raman signatures. Altogether, these data provide a unifying picture of the complex photophysics of PCFPs and RSFPs. ispartof: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY vol:142 issue:25 pages:10978-10988 ispartof: location:United States status: published

Six new thiophene oligomers, here designated as tripodal-like oligothienyl imidazoles, were synthesized and have been investigated in ethanol solution at room and low temperature. The oligomers bear a common core where two or more thiophenes are linked to one or more imidazole unit which further links through its alpha position to a different number of incremental thiophene units. The study involves a comprehensive spectral and photophysical investigation where the properties of the singlet and triplet states have been investigated regarding absorption, fluorescence and phosphorescence, transient triplet-triplet absorption together with all relevant quantum yields (fluorescence, internal conversion, intersystem crossing and singlet oxygen) and lifetimes. In addition, DFT quantum chemical calculations were performed to gain a detailed understanding of the molecular geometry and optical properties of the investigated oligomers. From the overall data, the radiative (kF) and radiationless (kNR, kIC and kISC) rate constants have been determined and it is shown that, in contrast with the parent oligothiophenes, the radiative competes with the radiationless deactivation channels. The results show that, by comparison with the oligothiophene counterparts, there is an augment of the relative contributions of the internal conversion and fluorescence processes relative to the S1~~T1 intersystem crossing. Phosphosphorescence emission was found for the simplest member of the investigated compounds, with a low quantum yield and a lifetime of 8 picos. The data also show that the introduction of a 4,5-dithienyl-imidazole moiety in a bi- or terthiophene oligomer results in, respectively, a 20-fold and a 3-fold increase of the fluorescence quantum yield relative to their oligothiophene counterparts., Fundação para a Ciência e a Tecnologia (FCT)

A new optical-fiber-based spectrofluorometer for in vivo or in vitro detection of delayed fluorescence is presented and characterized. This compact setup is designed so that it can be readily adapted for future clinical use. Optical excitation is done with a nitrogen laser-pumped, tunable dye laser, emitting in the UV-vis part of the spectrum. Excitation and luminescence signals are carried to and from the biological tissues under investigation, located out of the setup enclosure, by a single optical fiber. These measurements, as well as measurements performed without a fiber on in vitro samples in a thermostable quartz cell, in a controlled-atmosphere enclosure, are possible due to the efficient collection of the laser-induced luminescence light which is collected and focused on the detector with a high aperture parabolic mirror. The detection is based on a gated photomultiplier which allows for time-resolved measurements of the delayed fluorescence intensity. Thus, relevant luminescence lifetimes, typically in the sub-microsecond-to-millisecond range, can be measured with near total rejection of the sample's prompt fluorescence. The instrument spectral and temporal resolution, as well as its sensitivity, is characterized and measurement examples are presented. The primary application foreseen for this setup is the monitoring and adjustment of the light dose delivered during photodynamic therapy. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3558846]

The emission properties of some aza-stilbenes (2-, 3- and 4-styrylpyridine) and thio-stilbenes [2- and 3-styrylthiophene and 1,2-di-(3-thienyl)ethene] have been investigated after inclusion in commercial (NaY) and cation-exchanged (TlY) faujasite zeolites to get information on the triplet properties through population of the T 1 state induced by the heavy atom effect. The fluorescence properties in NaY and TlY were compared with those reported in solution. The phosphorescence spectra, observed in TlY at liquid nitrogen temperature, allowed the energy levels of the T 1 states to be obtained. Phosphorescence lifetimes were also measured. Their comparison with the lifetime known for stilbene showed that the radiative decay is little affected by the heteroatoms.

The steady state and time resolved fluorescence and the triplet-triplet absorption of p-dimethylaminobenzonitrile (DMABN) in presence of alpha- and beta-cyclodestrin (CD) were investigated at various host and guest concentrations and temperatures. The formation of 1:1 and 1:2 DMABN:alpha-CD and 1:1 and 2:2 complexes DMABN:beta-CD complexes was ascertained by applying global analysis methods. The "pure" fluorescence spectra as well as the emission quantum yields and lifetimes and the triplet properties of the various associates were determined. The role of environmental features in the radiative and non-radiative deactivation of the LE and ICT excited states of the complexed DMABN was elucidated.