Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water–glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.     

Lanthanide Complex-Based Fluorescence Label for Time-Resolved Fluorescence Bioassay

Different from organic fluorescence dyes, fluorescent lanthanide complexes have the fluorescence properties of long fluorescence lifetime, large Stokes shift and sharp emission profile, which makes them favorable be used as the fluorescent labeling reagents for microsecond time-resolved fluorescence bioassay. Lanthanide complex-based fluorescence labels have been successfully used for highly sensitive time-resolved fluorescence immunoassay, DNA hybridization assay, cell activity assay, and bioimaging microscopy assay. Since the technique allows easy distinction of the specific fluorescence signal of the long-lived label from short-lived background noises associated with biological samples, scattering lights (Tyndall, Rayleigh and Raman scatterings) and the optical components (cuvettes, filters and lenses), the sensitivity of fluorescence bioassay has been remarkably improved. This paper summarized the recent developments of lanthanide complex-based fluorescence labels and their applications in time-resolved fluorescence bioassays mainly based on the authors’ researches and relative publications.     

Dipyrrylmetheneboron Difluorides as Labels in Two-Photon Excited Fluorometry. Part II–Nucleic Acid Hybridization Assays

Five two-photon excitable dipyrrylmetheneboron difluoride labels (dipyrrylmethene-BF₂ labels) with fluorescence emission maximum between 530 and 590 nm, and a frequently used rhodamine label, TAMRA, were conjugated to aminomodified oligonucleotides. The performance of the labeled oligonucleotides was studied in a separation-free nucleic acid hybridization assay using ArcDia^™ TPX bioaffinity assay technology. The results show that oligonucleotide conjugates of dipyrrylmethene-BF₂ labels provide higher two-photon excited fluorescence yield and better assay sensitivity than corresponding TAMRA conjugate. The effect of conjugation on photophysical properties of the labels and performance of the labeled oligonucleotides in separation-free hybridization assay is discussed.     

Fluorescence fluctuation spectroscopy and its artifacts: simulations and tests

Fluorescence fluctuation spectroscopy (FFS)[1,2] is a kind of fluorescence detection techniques suitable for monitoring interaction and transportation processes in a small detection zone of biological systems. FFS detects and analyzes the fluorescence signal of the fluorescent molecules in the detection zone to retrieve the dynamical parameters of the system including concentration, diffusion coefficient and interaction constants. FFS utilizes various statistical methods to analyze the data,…     

Absorption and fluorescence of 8-azasteroids in the gas phase

Extensive studies of the spectral-luminescent characteristics of four 8-azasteroids and a model compound 2-(3,3-dimethyl-1,2,3,4-tetrahydro-1-isoquinolidene)-5,5-dimethyl-1,3-cyclohexanedion in the gas phase have been made. From the analysis of the dependences of the absorption spectra on the vapor pressure (T_low) and the fluorescence spectra on T_low and the exciting radiation wavelength (_exc) a conclusion on the presence in the vapors of the investigated 8-azasteroids of three absorption and fluorescence centers (S-, M-, and L-centers) has been drawn. The absorption spectra of these centers strongly overlap. Their long-wave absorption boundaries have been determined. The dependence of the fluorescence spectra of all three centers on _exc, which is inherent in rarefied gases of individual organic molecules, is observed. The S-centers are the molecules of the initial steroids, and the M- and L-centers are the molecules of thermo- and phototransformations of the initial steroids. The model compound in the gas phase is characterized by the same dependences of the fluorescence spectra on T_low and _exc as those inherent in 8-azasteroids. Taking into account the additional data obtained as a result of investigation of the absorption and fluorescence spectra of solutions of the substances extracted from vacuum cells after the investigation of 8-azasteroids and the model compound in the gas phase, conclusions on the nature of the M- and L-centers have been drawn.__________Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 72, No. 1, pp. 48–57, January–February, 2005.     

Heteroannihilation of the excited states of associates and monomers of fluorescein dyes on the silica surface at low temperatures

The processes of homo-and hetero-triplet—triplet annihilation (TTA) of Bengali rose on the spongy silicon surface have been investigated. It has been found that upon pulse photoexcitation on the silica surface photophysical processes involving the triplet states of monomers and their associates can develop. It has been shown that the processes of hetero-TTA are inhibited due to the decomposition of associates under the influence of hexane molecules in the molecular clusters of the dye adsorbates. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 72, No. 6, pp. 734–737, November–December, 2005.