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Considerable efforts have been devoted to the development of spectroscopic probes that are sensitive to water and can be used to monitor, for example, biological and chemical processes involving dehydration or hydration. Continuing this line of research, herein we show that 7-cyanoindole can serve as a sensitive fluorescence probe of hydration as its fluorescence properties, including intensity, peak wavelength and lifetime, depend on the amount of water in nine water-organic solvent mixtures. Our results indicate that 7-cyanoindole is not only able to reveal the underlying microheterogeneity of these binary solvent systems, but also offers distinct advantages. These include: (1) its fluorescence intensity increases more than ten times upon going from a hydrated to a dehydrated environment; (2) its peak wavelength shifts as much as 35 nm upon dehydration; (3) its single-exponential fluorescence decay lifetime increases from 2.0 ns in water to 8-16 ns in water-organic binary mixtures, making it viable to distinguish between differently hydrated environments via fluorescence lifetime measurements; and (4) its absorption spectrum is significantly red-shifted from that of indole, making selective excitation of its fluorescence possible in the presence of naturally occurring amino-acid fluorophores. Moreover, we find that for seven binary mixtures the fluorescence lifetimes of 7-cyanoindole measured at solvent compositions where maximum microheterogeneity occurs correlate linearly with the peak wavenumbers of its fluorescence spectra obtained in the respective pure organic solvents. This suggests that the microheterogeneities of these binary mixtures bear certain similarity, a phenomenon that warrants further investigation.

Several cyanotryptophans have been shown to be useful biological fluorophores. However, how their fluorescence lifetimes vary with solvent has not been examined. In this regard, herein we measure the fluorescence decay kinetics as well as the absorption and emission spectra of six cyanoindoles in different solvents. In particular, we find, among other results, that only 4-cyanoindole affords a long fluorescence lifetime and hence high quantum yield in H2O. Therefore, our measurements provide not only a guide for choosing which cyanotryptophan to use in practice but also data for computational modeling of the substitution effect on the electronic transitions of indole.

Tryptophan (Trp) fluorescence has been widely used to interrogate the structure, dynamics, and function of proteins. In particular, it provides a convenient and site-specific means to probe a protein's hydration status and dynamics. Herein, we show that a tryptophan analogue, 5-cyanotryptophan (TrpCN), can also be used for this purpose, but with the benefit of enhanced sensitivity to hydration. This conclusion is reached based on measurements of the static and time-resolved fluorescence properties of 5-cyanoindole, TrpCN, and TrpCN-containing peptides in different solvents, which indicate that upon dehydration the fluorescence quantum yield (QY) and lifetime (τF) of TrpCN undergo a much greater change in comparison to those of Trp. For example, in H2O the QY of TrpCN is less than 0.01, which increases to 0.11 in 1,4-dioxane. Consistently, the fluorescence decay kinetics of TrpCN in H2O are dominated by a 0.4 ns component, whereas in 1,4-dioxane the kinetics are dominated by a 6.0 ns component. The versatile utility of TrpCN as a sensitive fluorescence reporter is further demonstrated in three applications, where we used it (1) to probe the solvent property of a binary mixture consisting of dimethyl sulfoxide and H2O, (2) to monitor the binding interaction of an antimicrobial peptide with lipid membranes, and (3) to differentiate two differently hydrated environments in a folded protein.

The C≡N stretching frequency and fluorescence quantum yield of p-cyanophenylalanine are sensitive to environment. As such, this unnatural amino acid has found broad applications, ranging from studying how proteins fold to determining the local electric field of membranes. Herein, we demonstrate that the fluorescence of p-cyanophenylalanine can be quenched by selenomethionine through an electron transfer process occurring at short distances, thus further expanding its spectroscopic utility. Using this fluorophore-quencher pair, we are able to show that short polyproline peptides (1–4 prolines) are not rigid; instead, they sample a bimodal conformational distribution.

The non-natural amino acid p-cyanophenylalanine (PheCN) has recently emerged as a useful fluorescent probe of proteins; however, its photophysical properties have not been systematically examined. Herein, we measure the fluorescence quantum yield and the fluorescence lifetime of PheCN in a series of solvents. It is found that the fluorescence lifetime of PheCN shows a linear dependence on the Kamlet–Taft parameter α of the protic solvents used, indicating that the solute-solvent hydrogen bonding interactions mediate the non-radiative decay rate. Thus, results of this study provide a basis for quantitative application of PheCN fluorescence in protein conformational studies.

Fluorescence imaging of cells and tissue can be used to evaluate beta-NADH redox and location. At low temperature, beta-NADH fluorescence intensity increases and therefore sensitivity of imaging increases. In this paper, the temperature dependence of fluorescence was evaluated for beta-NADH in glycerol/water solution and in trehalose/sucrose glass. The average fluorescence lifetime for NADH in glycerol/water is 0.66 ns, compared with 5.3 ns in trehalose/ sucrose at 20 degrees C. Emission spectra were recorded from 290 to 12 K. The fluorescence of beta-NADH in glycerol/water increases approximately 16 fold and the emission shifts about 35 nm to the blue as temperature decreases. Much smaller change is seen for fluorescence of beta-NADH in sugar glass. Below 77 K, the beta-NADH spectral features did not change significantly with temperature change, and so no increase in sensitivity is obtained by going to very low temperatures. It is suggested that the sensitivity of beta-NADH fluorescence is related to water relaxation around the excited state molecule. Differences in water in various tissues may contribute to beta-NADH fluorescence changes when cells are altered.

Imaging oxygen in 3D with submicron spatial resolution can be made possible by combining phosphorescence quenching technique with multiphoton laser scanning microscopy. Because Pt and Pd porphyrin-based phosphorescent dyes, traditionally used as phosphors in biological oxygen measurements, exhibit extremely low two-photon absorption (2PA) cross-sections, we designed a nanosensor for oxygen, in which a 2P absorbing antenna is coupled to a metalloporphyrin core via intramolecular energy transfer (ET) with the purpose of amplifying the 2PA induced phosphorescence of the metalloporphyrin. The central component of the device is a polyfunctionalized Pt porphyrin, whose triplet state emission at ambient temperatures is strong, occurs in the near infrared and is sensitive to O2. The 2PA chromophores are chosen in such a way that their absorption is maximal in the near infrared (NIR) window of tissue (e.g., 700−900 nm), while their fluorescence is overlapped with the absorption band(s) of the core metalloporphyrin, ensuring an efficient antenna-core resonance ET. The metalloporphyrin-antenna construct is embedded inside the protecting dendritic jacket, which isolates the core from interactions with biological macromolecules, controls diffusion of oxygen and makes the entire sensor water-soluble. Several Pt porphyrin-coumarin based sensors were synthesized and their photophyics studied to evaluate the proposed design.

Fluorescence excitation spectra of SO 2 Ar generated in a supersonic expansion near the SO 2 C → X , 0 0 0 , 2 0 1 , and 3 0 2 bands have been obtained. Three features red-shifted by 82, 60 and 41 cm −1 from monomer bands are assigned to the SO 2 Ar complex. The rotational constants of SO 2 (C)Ar are determined to be A = 0.2769 cm −1 , B = 0.0520 cm −1 and C = 0.0488 cm −1 . The corresponding complex structure is obtained. Rapid non-radiative decay induced by the Ar atom has been observed for the 0 0 0 level of the C state complex. Vibrational predissociation is identified for the (010) and (002) levels, 377 and 560 cm −1 above the band origin.

Picosecond time-correlated single-photon counting spectroscopy has been used in rotational coherence measurements to determine the excited-state moments of inertia of p-cyclohexylaniline. This work complements earlier experiments involving single-color time-resolved fluorescence depletion spectroscopy, allowing the separation and identification of the rotational constants in the S 0 and S 1 states. Simulations have shown that the persistence of the K-type recurrences over several dozen cycles is consistent with a difference of ≃2% between the excited-state B and C constants

Microscopic oxygen distributions in heterogeneous phantoms and cells were visualized by means of two-photon laser scanning microscopy, using phosphorescent probes with controllable quenching parameters and enhanced two-photon absorption cross-sections.