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PubMed2001

Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues.

In our previous paper (Reshetnyak, Ya. K., and E. A. Burstein. 2001. Biophys. J. 81:1710-1734) we confirmed the existence of five statistically discrete classes of emitting tryptophan fluorophores in proteins. The differences in fluorescence properties of tryptophan residues of these five classes reflect differences in interactions of excited states of tryptophan fluorophores with their microenvironment in proteins. Here we present a system of describing physical and structural parameters of microenvironments of tryptophan residues based on analysis of atomic crystal structures of proteins. The application of multidimensional statistical methods of cluster and discriminant analyses for the set of microenvironment parameters of 137 tryptophan residues of 48 proteins with known three-dimensional structures allowed us to 1) demonstrate the discrete nature of ensembles of structural parameters of tryptophan residues in proteins; 2) assign spectral components obtained after decomposition of tryptophan fluorescence spectra to individual tryptophan residues; 3) find a correlation between spectroscopic and physico-structural features of the microenvironment; and 4) reveal differences in structural and physical parameters of the microenvironment of tryptophan residues belonging to various spectral classes.

Analysis of Variance↗
PubMed1999

Imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast.

We show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (GFP) can be monitored by imaging microscopy in living yeast. This work is based on the constitutive expression of a GFP-containing fusion protein and the inducible expression of the tobacco etch virus (TEV) protease. In the fusion protein, the P4.3 GFP mutant is linked to the YS65T GFP mutant by a spacer bearing the TEV protease-specific cleavage site.

Endopeptidases↗
PubMed2001

Binding and recognition of GATATC target sequences by the EcoRV restriction endonuclease: a study using fluorescent oligonucleotides and fluorescence polarization.

Oligonucleotides labeled with hexachlorofluorescein (hex) have enabled the interaction of the restriction endonuclease EcoRV with DNA to be evaluated using fluorescence anisotropy. The sensitivity of hex allowed measurements at oligonucleotide concentrations as low as 1 nM, enabling K(D) values in the low nanomolar range to be measured. Both direct titration, i.e., addition of increasing amounts of the endonuclease to hex-labeled oligonucleotides, and displacement titration, i.e., addition of unlabeled oligonucleotide to preformed hex-oligonucleotide/EcoRV endonuclease complexes, have been used for K(D) determination. Displacement titration is the method of choice; artifacts due to any direct interaction of the enzyme with the dye are eliminated, and higher fluorescent-labeled oligonucleotide concentrations may be used, improving signal-to-noise ratio. Using this approach (with three different oligonucleotides) we found that the EcoRV restriction endonuclease showed a preference of between 1.5 and 6.5 for its GATATC target sequence at pH 7.5 and 100 mM NaCl, when the divalent cation Ca2+ is absent. As expected, both the presence of Ca2+ and a decrease in pH value stimulated the binding of specific sequences but had much less effect on nonspecific ones.

Binding, Competitive↗
PubMed1998

Fluorescence correlation microscopy (FCM)-fluorescence correlation spectroscopy (FCS) taken into the cell.

Confocal fluorescence correlation spectroscopy (FCS) and other confocal spectroscopic techniques are ideally suited for the analysis of molecular interactions at the subcellular level. However, one requires exact positioning in three dimensions within the cell. Our instrument integrates FCS with high sensitivity digital imaging microscopy and high precision positioning. We present first measurements of intracellular FCS, with specification of the instrumental requirements and methods of data analysis. We propose the term fluorescence correlation microscopy (FCM) for this extended modality of FCS.

Dextrans↗
PubMed1994

Fluorescence array detector for large-field quantitative fluorescence cytometry.

A prototypic fluorescence array detector (FAD) has been designed and constructed which is capable of quantifying single-cell fluorescence emissions from a statistically significant population of cell-sized objects (over 10(3)) on a solid substrate. The system is comprised of a cryogenically cooled CCD, 50 mW air-cooled argon ion laser, and optics that image a large (1 x 1 cm) field at 1:1 (no magnification). The CCD is effectively treated as a two-dimensional array of 2.7 x 10(5) independent 20 x 20 microns photodetectors, with each cell-sized object imaged across only a few CCD pixels. Algorithms have been developed for focusing, image segmentation, shading correction, and noise rejection; performance data for the FAD with fluorescent calibration beads are presented. The FAD is a simple alternative to microscope-based imaging cytometry, allowing large-field imaging without a scanning stage.

Flow Cytometry↗
PubMed1990

Steady-state fluorescence and time-resolved fluorescence monitor changes in tryptophan environment in arginase from Saccharomyces cerevisiae upon removal of catalytic and structural metal ions.

Yeast arginase is a trimeric protein of identical subunits, each containing three tryptophans. Time-resolved fluorescence and steady-state fluorescence were employed to monitor the effects of removing the weakly bound catalytic Mn2+ as well as the tightly bound structural Zn2+/Mn2+. Resolution of the total native emission spectrum into decay-associated spectra (DAS) yielded components with lifetimes of 0.1, 1.2, and 4.0 ns. Upon removal of the catalytic metal, the intensities increased approximately 20% while the lifetimes increased less than 10%, and the DAS were unchanged except in intensity. The two major components are well resolved, but the 0.1-ns term is small and dominated by scattered excitation. In contrast, removal of the structural metal increased decay times to 0.2, 1.8, and 5.3 ns. More important, both native DAS red-shifted and became indistinguishable. These data suggest that removal of the catalytic metal does little to change the microenvironments of the individual tryptophans while removal of the structural metal causes partial unfolding of the protein. The excitation spectra for the active and inactive trimers were resolved into their excitation DAS (IEDAS), suggesting ground-state heterogeneity of the fluorescent species. In contrast, the excitation spectra of arginase without the structural metal could not be resolved due to the indistinguishable DAS. The tryptophans are quenched by acrylamide but not by cesium or iodide. Global analysis of the acrylamide quenching data resulted in two quenching decay-associated spectra (QDAS) which correlated well with the DAS. Since the apoenzyme does not exhibit tryptophan accessibility to either positive or negative ionic quenchers, one must assume that the "unfolded" monomeric protein retains considerable tertiary structure.

Apoenzymes↗
PubMed2003

Thermally reversible fluorescent polymorphs of alkoxy-cyano-substituted diphenylbutadienes: role of crystal packing in solid state fluorescence.

Correlation of fluorescence and crystal packing in thermally interconvertible polymorphic states of octyloxy-cyano-substituted diphenylbutadiene possessing visually distinguishable fluorescence reveals that solid state fluorescence of this class of derivatives depends on their monomer-J-aggregate ratio, controlled by variations in their molecular packing.

Journal Article↗
PubMed2001

Morphology, fluorescence properties, and their photothermal changes of poly(substituted thiophene) films revealed by near-field fluorescence microspectroscopy.

The morphology and fluorescence spectrum of poly(3-[2-(N-dodecylcarbamoyloxy)ethyl]thiophene-2,5-diyl) film were examined with spatial resolution of 100 nm using near-field fluorescence microspectroscopy. Fluorescence spectra observed at protruding domains were blue-shifted compared with flat areas, and further blue-shift was observed there more appreciably by long-time irradiation via a near-field scanning optical microscope probe. It is considered that the polymer chains at the protruding domains take disordered conformations, in which conjugated lengths are shorter and further disordering can be induced more easily by irradiation compared with those in the flat areas.

Journal Article↗
PubMed2004

Self-diffusion of rodlike and spherical particles in a matrix of charged colloidal spheres: a comparison between fluorescence recovery after photobleaching and fluorescence correlation spectroscopy.

The fluorescence recovery after photobleaching (FRAP) method and the fluorescence correlation spectroscopy (FCS) have been applied on suspensions of highly charged colloidal spheres with a small content of rod-shaped tobacco mosaic virus (TMV) particles. Since these methods only determine the self-diffusion coefficient of the fluorescently labeled species, D(S) of the rods and the spheres could independently be measured. The ionic strength of the dispersion medium has been varied to measure self-diffusion of rods and spheres in dependence on the degree of order of the matrix spheres. In contrast to FRAP, which allows the determination of the long-time self-diffusion coefficient D(S) (L), FCS measures self-diffusion on a shorter time scale. Thus a comparison of the results that were obtained by FCS and FRAP, in combination with Brownian Dynamics simulations, gives insight into the time dependence of the self-diffusion coefficient of an interacting colloidal system. As the mean interparticle distance of the matrix is of the same order of magnitude as the length of a TMV rod, the rotational motion is influenced by the assembly of spheres around a TMV particle. Since FCS is sensitive both to translational and rotational motion, whereas FRAP, which probes the diffusion at much larger length scales, is only sensitive to the translational motion of TMV, the comparison of diffusion coefficients measured employing FRAP and FCS can give some insights in the rotational diffusion: the experimental data indicate a slowing down of the rotational motion of a TMV rod with increasing structural order of the matrix spheres.

Journal Article↗
PubMed1985

Combined demonstration of an intraneuronal fluorescent dye and formaldehyde-induced catecholamine fluorescence in the guinea pig superior cervical ganglion.

A method is introduced for combined demonstration of an intracellular fluorescent dye (Lucifer Yellow-CH) and of formaldehyde-induced catecholamine fluorescence using as a model individual postganglionic neurons projecting to small intensely fluorescent cells in the guinea pig superior cervical ganglion. The application of this method to the peripheral and central nervous system offers the opportunity of studying potential sites of communication between monoamine containing cells and selected neurons prior to detailed ultrastructural studies.

Animals↗
PubMed1998

Flow cytometric characterization and classification of multiple dual-color fluorescent microspheres using fluorescence lifetime.

FlowMetrix (Luminex, Austin, TX) microspheres were recently introduced as a platform for bead-based assays involving antibodies, enzymes, toxins, and nucleic acids. The procedure involves classification of the microspheres by their orange and red fluorescence and quantitation of the BODIPY-tagged biological probes by their green fluorescence. In an attempt to increase the number of fluorochromes available for the biological probes, we explored the possibility of using excited singlet state lifetime as an alternative to one of the fluorochromes. For a set of 20 dual-color microspheres the excited singlet state lifetimes were measured using the total emissions (>515 nm), the orange emissions (515-600 nm), and the red emissions (>665 nm). The microspheres could not all be resolved in bivariates of fluorescence intensity versus excited singlet state lifetime. However, 13 of the microspheres could be resolved using the total emissions and lifetime. Although this result required both fluorochromes, the merits and limitations of this approach to other systems are briefly discussed.

Flow Cytometry↗
PubMed1999

Time-resolved fluorescence: 1996-1998

Luminescence continues to provide comprehensive literature surveys which will be published in most issues. These are a continuation of the literature surveys begun in 1986 in the Journal of Bioluminescence and Chemiluminescence which, up until 1998, encompassed more than 6000 references cited by year or specialized topic. With this newly named journal these searches are expanding to reflect the journal's wider scope. In future we will cover all fundamental and applied aspects of biological and chemical luminescence and include not only bioluminescence and chemiluminescence but also fluorescence, time resolved fluorescence, electrochemiluminescence, phosphorescence, sonoluminescence, lyoluminescence and triboluminescence. The compilers would be pleased to receive any comments from the readership. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗
PubMed2005

Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water.

To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence-based live/dead staining method using ViaGram Red+ Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probe (referred as LDS-FISH). The proposed LDS-FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single-cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4 degrees C, 13 degrees C, 18 degrees C, and 24 degrees C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS-FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24 degrees C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24 degrees C. The proposed LDS-FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters.

Apoptosis↗
PubMed2005

Online identification of the fluorescent whitening agent 4,4-bis(2-sulfostyrol)biphenyl using a sweeping technique combined with capillary electrophoresis/77 K fluorescence spectroscopy.

The feasibility of combining the techniques of online concentration and CE/low-temperature fluorescence spectroscopy in the detection and identification of E,E-4,4'-bis(2-sulfostyryl)biphenyl (DSBP) in synthetic detergents at 77 K is demonstrated. The technique involves the use of sweeping-MEKC, and was used for the initial online concentration and separation, after which a cryogenic molecular fluorescence experiment was performed at 77 K. The proposed method not only permits the separation and detection of E,E-DSBP in a synthetic detergent sample, but also ensures that the online spectrum is readily distinguishable and can be unambiguously assigned at 77 K. The photoconversion and isomer separation of DSBP are also described.

Biphenyl Compounds↗
PubMed2003

Evaluation of fluorescent probe surface intensities as an indicator of transdermal permeant distributions using wide-area two-photon fluorescence microscopy.

The application of high-speed two-photon fluorescence microscopy (HTPM) to examine transdermal transport processes has enabled the noninvasive visualization of permeant spatial distributions over a larger, more clinically relevant wide area of the skin. Earlier studies demonstrated that the transdermal fluorescent probe distribution over a 2 x 2 mm skin area was well represented by a significantly reduced sampling of the 400 microscale skin sites (100 x 100 microm) constituting the wide area. In the present study, the 400 microscale skin sites are considered individually, and the site-to-site variability in permeant distributions is used as a model to reflect the range in experimentally measured skin permeabilities resulting from the inherent stratum corneum structural heterogeneity. The correlation established between the permeant surface intensity and the corresponding permeant intensity gradient at each skin site provides an indication of the potential for screening transdermal permeant distributions solely based on the evaluation of microscale permeant surface intensities. The strong linear correlation between the intensity gradient and the surface intensity for the hydrophilic model permeant, sulforhodamine B, demonstrated that surface intensities provide a robust indicator of the corresponding transdermal probe distributions at the microscale. For the hydrophobic model permeant, rhodamine B hexyl ester, however, weak correlations were observed between these two parameters. This result suggests that the stratum corneum microscale surface intensity does not validly capture the corresponding intensity gradients for the entire range of skin permeabilities typically encountered as a result of the inherent stratum corneum heterogeneity.

Administration, Cutaneous↗
PubMed1994

Assay of DNA denaturation by polymerase chain reaction-driven fluorescent label incorporation and fluorescence resonance energy transfer.

We have developed a simple and sensitive assay method to monitor DNA denaturation. This method is based on (i) incorporation of fluorescent labels onto complementary DNA strands during target-specific amplification by polymerase chain reaction and (ii) detection of DNA denaturation by fluorescence resonance energy transfer. Fluorescein-11-dUTP and rhodamine-4-dUTP were used to label complementary DNA strands as the energy transfer donors and acceptors, respectively. The complementary DNA strands thus labeled were then annealed by incubation at a constant temperature, and the energy transfer efficiency was monitored during DNA denaturation experiments. Both heat denaturation and alkali denaturation could be monitored by this assay. Some factors affecting the DNA denaturation were also investigated.

DNA↗
PubMed2000

The regulative capacity of prespore amoebae as demonstrated by fluorescence-activated cell sorting and green fluorescent protein.

The ability of prespore Dictyostelium discoideum amoebae to undergo redifferentiation so as to reestablish normal spore/stalk proportioning has been demonstrated in various ways over the years, beginning with the classic microdissection work of K. Raper. The discovery of anterior-like cells in the slug posterior, however, cast doubt on that ability, and more recent experiments using a cell-specific toxin suggested that prespore redifferentiation may not in fact occur. To reexamine this question, we performed fluorescence-activated cell sorting (FACS) upon amoebae expressing a mutated green fluorescent protein gene (S65T-GFP) under the control of a prespore-specific (PsA) promoter. FACS produced prespore cell populations with purities, measured by GFP expression, as high as 99. 5%. Sorted GFP(+) cells were developmentally competent and produced normally proportioned fruits, indistinguishable from those of "sham-sorted" (permissively gated, mixed GFP(+) and GFP(-)) amoebae. This result confirms the developmental totipotency of prespore amoebae.

Animals↗