The degradation of chlorophyll pigments in dairy silage: the timeline of anaerobic fermentation.

BACKGROUND: Detection of chlorophyll metabolites in milk has recently been suggested to be an indicator of a grass-fed diet fed for cattle. Such a means of detection, however, is complicated when the grazing season is over because cattle can be fed fermented silage ingredients, such as alfalfa and corn silage. During fermentation, chlorophyll compounds and other pigments undergo degradation due to the accumulation of lactic acid and the resultant decline in pH. RESULTS: We monitored degradation of chlorophyll compounds by measuring the fluorescence and absorption spectra of silage extracts. The spectroscopic evidence supports the hypothesis that chlorophylls are converted into fluorescent products, such as pheophytin, and further cleaved into pheophorbide. The degradation starts with dechelation and removal of the magnesium ion to produce pheophytin. Further removal of the phytol chain from pheophytin results in the production of pheophorbide. CONCLUSIONS: The fluorescence intensity of these degradation products is reduced compared to that of the parent molecule. These findings are important in understanding the fluorescent signal in milk when cows consume silage rather than fresh pasture grass. © 2020 Society of Chemical Industry.

Fluorescence quenching of the SYBR Green I-dsDNA complex by in situ generated magnetic ionic liquids.

Magnetic ionic liquids (MILs) with metal-containing cations are promising extraction solvents that provide fast and high efficiency extraction of DNA. Hydrophobic MILs can be generated in situ in a methodology called in situ dispersive liquid-liquid microextraction. To consolidate the sample preparation workflow, it is desirable to directly use the DNA-enriched MIL microdroplet in the subsequent analytical detection technique. Fluorescence-based techniques employed for DNA detection often utilize SYBR Green I, a DNA binding dye that exhibits optimal fluorescence when bound to double-stranded DNA. However, the MIL may hinder the fluorescence signal of the SYBR Green I-dsDNA complex due to quenching. In this study, MILs with metal-containing cations were selected and their fluorescence quenching effects evaluated using FÓ§rster Resonance Energy Transfer and quantified using Stern-Volmer models. The MILs were based on N-substituted imidazole ligands (with butyl- and benzyl- groups as substituents) coordinated to Ni2+ or Co2+ metal centers as cations, and paired with chloride anions. The effects of NiCl2 and CoCl2 salts and of the 1-butyl-3-methylimidazolium chloride ionic liquid on the fluorophore complex were also studied to understand the components of the MIL structure that are responsible for quenching. The metal within the MIL chemical structure was found to be the main component contributing to fluorescence quenching. FÓ§rster critical distances between 11.9 and 18.8 Å were obtained for the MILs, indicating that quenching is likely not due to non-radiative energy transfer but rather to spin-orbit coupling or excited-state electron transfer. The MILs were able to be directly used in qPCR and fluorescence emission measurements using a microplate reader for detection, demonstrating their applicability in fluorescence-based detection methods. Graphical abstract.

Spectral Narrowing Accompanies Enhanced Spatial Resolution in Saturated Coherent Anti-Stokes Raman Scattering (CARS): Comparisons of Experiment and Theory.

We demonstrate theoretically and confirm experimentally the mechanism by which spectral narrowing accompanies enhanced spatial resolution in a saturated coherent anti-Stokes Raman scattering (CARS) signal that is demodulated at the third harmonic (3f) of the pump modulation frequency (f). Under these modulation conditions, theory predicts a narrowing of the full width at half-maximum (FWHM) of the CARS spectrum by a factor of 2.0 with respect to that of the spectrum obtained by demodulation at the fundamental frequency. Theory also predicts an improvement of spatial resolution by a factor of 1.7. Experimentally, narrowing of the FWHM of the CARS spectrum of 1,4-bis((E)-2-methylstyryl) benzene (MSB) crystals by a factor of 2.5 is observed upon saturation. Further experimental confirmation is provided from investigating diamond particles, for which spectral narrowing was enhanced by a factor of 2.8 and spatial resolution was enhanced by a factor of 2. Details of the mechanism and execution of the saturated CARS experiment are elucidated and limits to its applicability are suggested, one of which is the conclusion that the saturation approach is not suitable for extraction of harmonics beyond 3f. In this work, we have developed a more comprehensive understanding of the correlation between the observed experimental results and experimental factors than has been previously reported.

Unveiling the Photo- and Thermal-Stability of Cesium Lead Halide Perovskite Nanocrystals.

Lead halide perovskites possess unique characteristics that are well-suited for optoelectronic and energy capture devices, however, concerns about their long-term stability remain. Limited stability is often linked to the methylammonium cation, and all-inorganic CsPbX3 (X=Cl, Br, I) perovskite nanocrystals have been reported with improved stability. In this work, the photostability and thermal stability properties of CsPbX3 (X=Cl, Br, I) nanocrystals were investigated by means of electron microscopy, X-ray diffraction, thermogravimetric analysis coupled with FTIR (TGA-FTIR), ensemble and single particle spectral characterization. CsPbBr3 was found to be stable under 1-sun illumination for 16 h in ambient conditions, although single crystal luminescence analysis after illumination using a solar simulator indicates that the luminescence states are changing over time. CsPbBr3 was also stable to heating to 250 °C. Large CsPbI3 crystals (34±5 nm) were shown to be the least stable composition under the same conditions as both XRD reflections and Raman bands diminish under irradiation; and with heating the γ (black) phase reverts to the non-luminescent δ phase. Smaller CsPbI3 nanocrystals (14±2 nm) purified by a different washing strategy exhibited improved photostability with no evidence of crystal growth but were still thermally unstable. Both CsPbCl3 and CsPbBr3 show crystal growth under irradiation or heat, likely with a preferential orientation based on XRD patterns. TGA-FTIR revealed nanocrystal mass loss was only from liberation and subsequent degradation of surface ligands. Encapsulation or other protective strategies should be employed for long-term stability of these materials under conditions of high irradiance or temperature.

Photoinduced Trans-to-cis Phase Transition of Polycrystalline Azobenzene at Low Irradiance Occurs in the Solid State.

The ability to produce large-scale, reversible structural changes in a variety of materials by photoexcitation of a wide variety of azobenzene derivatives has been recognized for almost two decades. Because photoexcitation of trans-azobenzene produces the cis-isomer in solution, it has generally been inferred that the macroscopic structural changes occurring in materials are also initiated by a similar large-amplitude trans-to-cis isomerization. This work provides the first demonstration that a trans-to-cis photoisomerization occurs in polycrystalline azobenzene, and is consistent with the previously hypothesized nature of the trigger in the photoactuated mechanisms of the materials in question. It is also demonstrated that under low irradiance, trans-to-cis isomerization occurs in the solid (not via a pre-melted phase); and the presence of the cis-isomer thus lowers the melting point of the sample, providing a liquid phase. A variety of experimental techniques were employed, including X-ray diffraction measurements of polycrystalline azobenzene during exposure to laser irradiation and fluorescence measurements of the solid sample. A practical consequence of this work is that it establishes trans-azobenzene as an easily obtainable and well-defined control for monitoring photoinduced structural changes in X-ray diffraction experiments, using easily accessible laser wavelengths.

Photon Counting Data Analysis: Application of the Maximum Likelihood and Related Methods for the Determination of Lifetimes in Mixtures of Rose Bengal and Rhodamine B.

It is often convenient to know the minimum amount of data needed to obtain a result of desired accuracy and precision. It is a necessity in the case of subdiffraction-limited microscopies, such as stimulated emission depletion (STED) microscopy, owing to the limited sample volumes and the extreme sensitivity of the samples to photobleaching and photodamage. We present a detailed comparison of probability-based techniques (the maximum likelihood method and methods based on the binomial and the Poisson distributions) with residual minimization-based techniques for retrieving the fluorescence decay parameters for various two-fluorophore mixtures, as a function of the total number of photon counts, in time-correlated, single-photon counting experiments. The probability-based techniques proved to be the most robust (insensitive to initial values) in retrieving the target parameters and, in fact, performed equivalently to 2-3 significant figures. This is to be expected, as we demonstrate that the three methods are fundamentally related. Furthermore, methods based on the Poisson and binomial distributions have the desirable feature of providing a bin-by-bin analysis of a single fluorescence decay trace, which thus permits statistics to be acquired using only the one trace not only for the mean and median values of the fluorescence decay parameters but also for the associated standard deviations. These probability-based methods lend themselves well to the analysis of the sparse data sets that are encountered in subdiffraction-limited microscopies.

Plant hemoglobins may be maintained in functional form by reduced flavins in the nuclei, and confer differential tolerance to nitro-oxidative stress.

The heme of bacteria, plant and animal hemoglobins (Hbs) must be in the ferrous state to bind O(2) and other physiological ligands. Here we have characterized the full set of non-symbiotic (class 1 and 2) and 'truncated' (class 3) Hbs of Lotus japonicus. Class 1 Hbs are hexacoordinate, but class 2 and 3 Hbs are pentacoordinate. Three of the globins, Glb1-1, Glb2 and Glb3-1, are nodule-enhanced proteins. The O(2) affinity of Glb1-1 (50 pm) was the highest known for any Hb, and the protein may function as an O(2) scavenger. The five globins were reduced by free flavins, which transfer electrons from NAD(P)H to the heme iron under aerobic and anaerobic conditions. Class 1 Hbs were reduced at very fast rates by FAD, class 2 Hbs at slower rates by both FMN and FAD, and class 3 Hbs at intermediate rates by FMN. The members of the three globin classes were immunolocalized predominantly in the nuclei. Flavins were quantified in legume nodules and nuclei, and their concentrations were sufficient to maintain Hbs in their functional state. All Hbs, except Glb1-1, were expressed in a flavohemoglobin-deficient yeast mutant and found to confer tolerance to oxidative stress induced by methyl viologen, copper or low temperature, indicating an anti-oxidative role for the hemes. However, only Glb1-2 and Glb2 afforded protection against nitrosative stress induced by S-nitrosoglutathione. Because this compound is specifically involved in transnitrosylation reactions with thiol groups, our results suggest a contribution of the single cysteine residues of both proteins in the stress response.

Enhanced stability and activity of cellulase in an ionic liquid and the effect of pretreatment on cellulose hydrolysis.

We discuss the hydrolysis of cellulose using a pure cellulase: endo-1,4-ß-D-glucanase (EG) from the fungus, Aspergillus niger, in buffer, the pure ionic liquid (IL), tris-(2-hydroxyethyl)-methylammonium methylsulfate (HEMA), and various mixtures of the two at different temperatures. Steady-state fluorescence and absorbance studies were performed to monitor the stability and activity of EG using cellulose azure as the substrate. EG attains its highest activity at 45°C in buffer and denatures at ∼55°C. On the other hand, HEMA imparts substantial stability to the enzyme, permitting the activity to peak at 75°C. The relative roles of temperature, viscosity, pH, polarity, and the constituent ions of the ILs on the hydrolysis reaction are examined. It is demonstrated that pretreatment of cellulose with ILs such as BMIM Cl, MIM Cl, and HEMA results in more rapid conversion to glucose than hydrolysis with cellulose that is not pretreated. The percent conversion to glucose from pretreated cellulose is increased when the temperature is increased from 45 to 60°C. Two different ILs are used to increase the efficiency of cellulose conversion to glucose. Cellulose is pretreated with BMIM Cl. Subsequent hydrolysis of the pretreated cellulose in 10-20% solutions of HEMA in buffer provides higher yields of glucose at 60°C. Finally, to our knowledge, this is the first study dealing with a pure endoglucanase from commercial A. niger. This enzyme not only shows higher tolerance to ILs, such as HEMA, but also has enhanced thermostability in the presence of the IL.

Temperature-Dependent Constrained Diffusion of Micro-Confined Alkylimidazolium Chloride Ionic Liquids.

Alkylimidazolium chloride ionic liquids (ILs) have many uses in a variety of separation systems, including micro-confined separation systems. To understand the separation mechanism in these systems, the diffusion properties of analytes in ILs under relevant operating conditions, including micro-confinement dimension and temperature, should be known. For example, separation efficiencies for various IL-based microextraction techniques are dependent on the sample volume and temperature. Temperature-dependent (20-100 °C) fluorescence recovery after photobleaching (FRAP) was utilized to determine the diffusion properties of a zwitterionic, hydrophilic dye, ATTO 647, in alkylimidazolium chloride ILs in micro-confined geometries. These micro-confined geometries were generated by sandwiching the IL between glass substrates that were separated by ∼1 to 100 µm. From the measured temperature-dependent FRAP data, we note alkyl chain length-, thickness-, and temperature-dependent diffusion coefficients, with values ranging from 0.021 to 46 µm2/s. Deviations from Brownian diffusion are observed at lower temperatures and increasingly less so at elevated temperatures; the differences are attributed to alterations in intermolecular interactions that reduce temperature-dependent nanoscale structural heterogeneities. The temperature- and thickness-dependent data provide a useful foundation for efficient design of micro-confined IL separation systems.

Comparison of the dielectric response obtained from fluorescence upconversion measurements and molecular dynamics simulations for coumarin 153-apomyoglobin complexes and structural analysis of the complexes by NMR and fluorescence methods.

We present a comparison of the dielectric response obtained from fluorescence upconversion experiments and from molecular dynamics simulations of the complexes of coumarin 153 with five apomyoglobins (apoMbs): wild-type horse heart (HH-WT) and those of wild-type sperm whale (SW-WT); its two triple mutants, L29F/H64Q/V68F and H64L/V68F/P88A; and its double mutant, L29F/V68L. Comparisons between experimental and simulated solvation relaxation functions, C(t)s, for the wild-type proteins range from very good to excellent. For the three mutants we investigated, however, agreement between experiment and simulation was considerably inferior. Thus, an NMR study of the complex of the HH-WT complex apoMb, and fluorescence energy transfer and anisotropy studies of the five complexes, were performed to investigate the structures upon which the simulations were based. The NMR measurements confirm our earlier conclusions that the C153 lies in the heme pocket of the HH-WT apoMb. For the wild-type complexes, fluorescence energy transfer measurements provide two rise times, suggesting a definite spatial relationship between the two Trp donors and the C153 acceptor. These results confirm the structural integrity of the wild-type complexes and validate the initial structures used for the molecular dynamics simulations. On the other hand, the three mutants provided single exponential rise times for energy transfer, suggesting that the position of the C153 used in the simulations may have been in error or that the C153 is mobile on the time scale of the energy transfer experiment. Fluorescence anisotropy studies also suggest that the double mutant was not structurally intact. Furthermore, examination of these systems demonstrates the sensitivity of C153 to its environment and permits the observation of differences in the heme pockets. These results point to the importance of structural characterization of modified proteins used in studies of the dielectric response and suggest strategies for performing molecular dynamics simulations of modified proteins.

Localization of Nonblinking Point Sources Using Higher-Order-Mode Detection and Optical Heterodyning: Developing a Strategy for Extending the Scope of Molecular, Super-resolution Imaging.

While the stochastic, "blinking" nature of fluorescent systems has enabled the super-resolution of their localization by the fitting of their point-spread functions (PSFs), this strategy cannot be exploited for similar resolution of "nonblinking" systems, such as those that might be encountered in a coherent Raman experiment. An alternative method for subdiffraction-limited imaging lies in the exploitation of optical heterodyning. For example, if a Gaussian PSF (a TEM00 mode) of a point emitter is displaced with respect to the origin of the optical system, photons in the higher-order TEM modes carry information about that displacement. Information concerning the displacement can be extracted from photons in these higher-order modes. These photons can be collected by optical heterodyning, which exploits the large gain in a detector's response to an optical signal from an emitter coupled to a local oscillator, which is prepared in the TEM of interest, e.g., TEM10. We have generalized and developed the heterodyning technique to localize point emitters via the detection of higher-order spatial modes. We have developed a theoretical approach to find a practical estimation limit of the localization parameters using a realistic model that accounts for shot noise, background noise, and Gaussian noise. To demonstrate the applicability of the method, we designed experiments in which a laser is a surrogate for one and two point emitters. Using the Fisher information and its accompanying Cramér-Rao lower bound, we demonstrate super-resolution localization in these cases: we show that objects can be localized to roughly 2-3 orders of magnitude of their point-spread function's size for a given optical system. Finally and most importantly, it is suggested that the results will ultimately be generalizable to multiple emitters and, most importantly, to "nonblinking" molecular systems, which will be essential for broadening the scope of super-resolution measurements beyond the limits of fluorescence-based techniques.

Fast and non-destructive determination of water content in ionic liquids at varying temperatures by Raman spectroscopy and multivariate regression analysis.

Imidazolium acetate ionic liquids (ILs) have been utilized as promising solvents in many applications that involve varying water content and temperature. These experimental variables affect the anion-cation intermolecular interactions, which in turn influence the performance of the ILs in these applications. This paper shows Raman spectroscopy can be used as an operando method to measure water content in IL solvents when simultaneous temperature changes may occur. The Raman spectra of 1-alkyl-3-methylimidazolium acetate ILs (alkyl chain length n = 2, 4, 6, 8) with varying water content (from 0.028 to 0.899 water mole fraction) and temperature (from 78.1 K to 423.1 K) were measured. Increasing the water content or decreasing the temperature of the tested ILs weakens the anion-cation intermolecular interactions. The water content of these ILs can be quantified even in conditions when the temperature is changing using Raman spectroscopy combined with multivariate regression analysis, including principal component regression (PCR), partial-least-squares regression (PLSR), and artificial neural networks (ANNs). The ANN model combined with partial-least-squares (PLS) achieved the highest prediction accuracy of water content in ILs at varying temperatures (RMSECV = 0.017, R2CV = 99.1%, RMSEP = 0.019, R2P = 98.8%, RPD = 8.93). Raman spectroscopy provides a potential fast non-destructive operando method to monitor the water content of ILs even in applications when the temperature may be simultaneously altered; this information can lead to the optimized use of these ILs in many applications.

Fluorescence spectroscopy of the retina for diagnosis of transmissible spongiform encephalopathies.

The feasibility of exploiting fluorescence spectra of the eye for diagnosis of transmissible spongiform encephalopathies (TSEs) was examined. Retinas from scrapie-positive sheep were compared with scrapie-negative sheep using fluorescence spectroscopy, and distinct differences in the fluorescence intensity and spectroscopic signatures were observed. The characteristic fluorescent signatures are thought to be the result of an accumulation of lipofuscin in the retina. It appears that the eye, in particular the retina, is a useful tissue for noninvasive examination of some neurological pathologies such as scrapie. The development of procedures based on examinations of the eye that permit the detection of neurological disorders in animals holds great promise.

Solvation dynamics of the fluorescent probe PRODAN in heterogeneous environments: contributions from the locally excited and charge-transferred states.

The coexistence of different excited states with different properties of the same chromophores could have significant consequences for the accurate characterization of solvation dynamics in a heterogeneous environment, such as a protein. The purpose of this work is to study the contributions of the locally excited (LE) and charge-transferred (CT) states of the fluorescent probe molecule 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) to its solvation dynamics in the heterogeneous environment provided by reverse micelles formed by sodium 1,4-bis-(2-ethylhexyl) sulfosuccinate (AOT)/n-heptane/water. We have found that the LE and CT states of PRODAN solvate on different time scales in reverse micelles (2 and approximately 0.4 ns, respectively), consistent with results suggested in the literature, and have concluded that PRODAN's use as a probe of heterogeneous environments must be used with caution and that, more importantly, the same caution must be exercised with any chromophore capable of emitting from different excited states.

Excited-state intramolecular hydrogen atom transfer and solvation dynamics of the medicinal pigment curcumin.

The potential use of the naturally occurring yellow-orange pigment curcumin as a photodynamic therapy agent is one of the most exciting applications of this medicinal compound. Although subnanosecond spectroscopy has been used to investigate the photophysical processes of curcumin, the time resolution is insufficient to detect important and faster photoinduced processes, including solvation and excited-state intramolecular hydrogen atom transfer (ESIHT). In this study, the excited-state photophysics of curcumin is studied by means of ultrafast fluorescence upconversion spectroscopy. The results show two decay components in the excited-state kinetics with time scales of 12-20 ps and approximately 100 ps in methanol and ethylene glycol. The resulting prominent isotope effect in the long component upon deuteration indicates that curcumin undergoes ESIHT in these solvents. The short component (12-20 ps) is insensitive to deuteration, and multiwavelength fluorescence upconversion results show that this decay component is due to solvation of excited-state curcumin.

Considerations for the construction of the solvation correlation function and implications for the interpretation of dielectric relaxation in proteins.

The dielectric response of proteins is conveniently measured by monitoring the time-dependent Stokes shift of an associated chromophore. The interpretation of these experiments depends critically upon the construction of the solvation correlation function, C(t), which describes the time-dependence of the Stokes shift and hence the dielectric response of the medium to a change in charge distribution. We provide an analysis of various methods of constructing this function and review selected examples from the literature. The naturally occurring amino acid, tryptophan, has been frequently used as a probe of solvation dynamics in proteins. Its nonexponential fluorescence decay has stimulated the generation of an alternative method of constructing C(t). In order to evaluate this method, we have studied a system mimicking tryptophan. The system is comprised of two coumarins (C153 and C152) having different fluorescence lifetimes but similar solvation times. The coumarins are combined in different proportions in methanol to make binary probe mixtures. We use fluorescence upconversion spectroscopy to obtain wavelength-resolved kinetics of the individual coumarins in methanol as well as the binary mixtures of 75:25, 50:50, and 25:75 of C153:C152. The solvation correlation functions are constructed for these systems using different methods and are compared.

Influence of chiral ionic liquids on stereoselective fluorescence quenching by photoinduced electron transfer in a naproxen dyad.

In a previous study of a naproxen dyad in a pair of N-methylimidazoliummethyl menthylether-NTf(2) chiral ionic liquids (J. Phys. Chem. B 2008, 112, 7555), we observed that though intramolecular electron transfer was impeded, a consistent small stereodifferentiation in the fluorescence lifetime of the dyad was obtained. We proposed that this discrimination was purely electronic in nature and did not arise from geometrical effects, which can influence nonradiative rate processes, such as intramolecular electron transfer. In our present work, we have studied the interaction of the same chiral naproxen dyad molecule in both the previously studied menthyl-based NTf(2) ionic liquids and also in bis(tertrabutylphosphonium) (TBP) d-,l-tartrate ionic liquids. Unlike in the menthyl-based IL pair, the amount of quenching is different in the bis(TBP) tartrate enantiomeric liquids and the tartrate enantiomers have a different temperature dependence on the nonradiative rate of the dyad. This chiral discrimination most likely arises from the steric effects of the different conformations of the chiral molecules. We have shown that the viscosity and polarity of the solvents can influence the rate of electron transfer. On the other hand, no such electron transfer quenching is observed in the menthyl-based NTf(2) IL solvents. To our knowledge, this is the first example of chiral ionic liquids inducing a stereoselective fluorescence quenching by photoinduced, intramolecular electron transfer.

Monitoring the accumulation of lipofuscin in aging murine eyes by fluorescence spectroscopy.

The integrated fluorescence of murine eyes is collected as a function of age. This fluorescence is attributed to pigments generally referred to as lipofuscin and is observed to increase with age. No difference in fluorescence intensity is observed between the eyes of males or females. This work provides a benchmark for further studies that are planned in order to use such signatures as markers of central nervous system (CNS) tissue or even of diseased CNS tissue and provides a basis for determining the age of a healthy animal.

A Bayesian Approach for Extracting Fluorescence Lifetimes from Sparse Data Sets and Its Significance for Imaging Experiments.

The measurement of fluorescence lifetimes, especially in small sample volumes, presents the dual challenge of probing a small number of fluorophores and fitting the concomitant sparse data set to the appropriate excited-state decay function. A common method of analysis, such as the maximum likelihood (ML) technique, assumes a uniform probability distribution of the parameters describing the fluorescence decay function. An improvement is thus suggested by implementing a suitable nonuniform distribution, as is provided by a Bayesian framework, where the distribution of parameters is obtained from both their prior knowledge and the evidence-based likelihood of an event for a given set of parameters. We have also considered the Dirichlet prior distribution, whose mathematical form enables analytical solutions of the fitting parameters to be rapidly obtained. If Gaussian and exponential prior distributions are judiciously chosen, they reproduce the experimental target lifetime to within 20% with as few as 20 total photon counts for the data set, as does the Dirichlet prior distribution. But because of the analytical solutions afforded by the Dirichlet prior distribution, it is proposed to employ a Dirichlet prior to search parameter space rapidly to provide, if necessary, appropriate parameters for subsequent employment of a Gaussian or exponential prior distribution.

Characterization of the Photophysical Behavior of DFHBI Derivatives: Fluorogenic Molecules that Illuminate the Spinach RNA Aptamer.

( Z)-5-(3,5-Difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4 H-imidazol-4-one (DFHBI) and its analogues are fluorogenic molecules that bind the Spinach aptamer (a small RNA molecule), which was selected for imaging RNA. They are extremely weakly fluorescent in liquid solvents. It had been hypothesized that photoisomerization is a very efficient nonradiative process of deactivation. We show, consistent with the results of other studies, that if the isomerization is impeded, the fluorescence signal is enhanced significantly. In addition, we provide a thorough characterization of the photophysical behavior of DFHBI and its derivatives, notably that of ( Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-3-((perfluorophenyl)methyl)-3,5-dihydro-4 H-imidazol-4-one (PFP-DFHBI) in various solvent environments. Solvent-dependent studies were performed with various mixtures of solvents. The results suggest that hydrogen bonding or strong interactions of the solvents with the phenolic-OH group change the absorption band near 420-460 nm and the nature of emission near 430 and 500 nm through various degrees of stabilization and the transformation between the neutral and the anionic species at both ground and excited states. These observations are confirmed by using a methoxy-substituted molecule (( Z)-5-(4-methoxybenzylidene)-2,3-dimethyl-3,5-dihydro-4 H-imidazol-4-one), where the 420-460 nm band is absent in the presence of methanol and the spectra are similar to those of PFP-DFHBI in noninteracting solvents, such as acetonitrile and dichloromethane. Thus, in addition to the major role of photoisomerization as a nonradiative process of deactivation of the excited state, the fluorescence of DFHBI-type molecules is very sensitively dependent upon the pH of the medium as well as upon solvent-specific interactions, such as hydrogen-bonding ability and polarity.