Analysis Of Fluorescence Emission Research Articles

Synthesis, structure and characterization of two Cd(II) coordination polymers with helical structures

Two coordination polymers, [Cd2Cl2(1,3-BDC)(TTBT)(H2O)] (1) and [CdCl(1,4-HBDC)(TTBT)] (2) (1,3-BDC = 1,3-benzenedicarboxylate, 1,4-BDC = 1,4-benzenedicarboxylate, and TTBT = 10,11,12,13-tetrahydro-4,5,9,14-tetraaza-benzo[b]triphenylene), have been prepared under hydrothermal conditions and characterized by elemental analyses, IR spectra, thermal gravimetry (TG), fluorescence emission and single crystal X-ray diffraction analyses. The crystal structures were determined by X-ray diffraction and refined by full-matrix least-squares methods to R = 0.0621 and wR = 0.1543 using 4937 reflections with I > 2σ(I) for 1; and R = 0.0525 and wR = 0.1427 using 3386 reflections with I > 2σ(I) for 2. Compound 1 possesses a chiral chain structure. Adjacent chiral chains are further linked through π–π interactions between TTBT ligands to give a three-dimensional supramolecular architecture. Compound 2 displays a helical chain structure, with neighboring chains stacked by π–π interactions, generating an unusual three-dimensional supramolecular structure.

Design and Characterization of Optofluidic Photonic Crystal Structures for the Detection of Fluorescent-Labeled Biomolecules

The combination of optics and microfluidics into optofluidic systems has provided a means of creating portable integrated platforms for performing fluorescence emission analyses in fieldable systems. We present here the design of a photonic crystal transducer for highly sensitive and selective fluorescence-based biomolecule detection using a reduced-size lattice nanocavity tuned for resonance at emission wavelengths. This novel approach to biosensing is based upon photonic crystal defect structures that are engineered to act as waveguides, optical resonant cavities, and nanofluidic flow channels. This paper will discuss modeling, design, and nanofabrication of optofluidic photonic crystal (PhC) transducers in silicon and GaN materials. First-order results of PhC optical property characterization will be provided. Preliminary simulations indicate that fluorescence emission intensity inside of a PhC resonant defect nanocavity is enhanced to a level 26 times greater than emission outside of a defect due to resonant optical confinement and the Purcell effect.

Control of Peripheral Light-Harvesting Complex Synthesis by a Bacteriophytochrome in the Aerobic Photosynthetic Bacterium Bradyrhizobium Strain BTAi1

The recent sequence analysis of the photosynthetic and plant-symbiotic Bradyrhizobium sp. strain BTAi1 revealed the unexpected presence of a pucBA operon encoding the apoproteins of peripheral light-harvesting (LH) complexes. This pucBA operon is found close to a bacteriophytochrome gene (BphP3(B BTAi1)) and a two-component transcriptional regulator gene (TF(BTAi1) gene). In this study, we show that BphP3(B BTAi1) acts as a bona fide bacteriophytochrome and controls, according to light conditions, the expression of the pucBA operon found in its vicinity. This light regulatory pathway is very similar to the one previously described for chromo-BphP4(Rp) in Rhodopseudomonas palustris and conducts the synthesis of a peripheral LH complex. This LH complex presents a single absorption band at low temperature, centered at 803 nm. Fluorescence emission analysis of intact cells indicates that this peripheral LH complex does not act as an efficient light antenna. One putative function of this LH complex could be to evacuate excess light energy in order to protect Bradyrhizobium strain BTAi1, an aerobic anoxygenic photosynthetic bacterium, against photooxidative damage during photosynthesis.

Optical Detection of Triggered Atherosclerotic Plaque Disruption by Fluorescence Emission Analysis¶

Optical Detection of Triggered Atherosclerotic Plaque Disruption by Fluorescence Emission Analysis¶

Smart anionic polyelectrolytes based on natural polymer for complexation of cationic surfactant

AbstractThe thermosensitive polyelectrolytes were obtained by grafting 2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid (AMPS) onto hydroxypropylcellulose (HPC), a biodegradable polysaccharide. The interactions of the polymers with dodecyltrimethylammonium chloride (DTAC), a model cationic surfactant, were studied. It was found by the measurements of the surface tension and the analysis of fluorescence emission of pyrene used as a fluorescent probe, that the HPC–AMPS graft polymers strongly interact with DTAC with the formation of polymer–surfactant complexes. The critical aggregation concentrations of these polymer–surfactant systems were found to be of the order of 10−5 mol/dm3. The polymers were found to be potentially useful in the purification of water from cationic surfactants. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 2401–2407, 2006

Effects of flunitrazepam on the L α-H II phase transition of phosphatidylethanolamine using merocyanine 540 as a fluorescent indicator

Previous studies of our group demonstrated that flunitrazepam is a lipophilic drug capable of interacting with membranes through a partition equilibrium phenomenon. Its localization at the phospholipid polar head region could explain the decrease in the size of dipalmitoylphosphatidylcholine (dpPC) vesicles, through a mechanism that involves the increment in the relative volume of this region with a subsequent increase in the vesicle’s surface curvature. In the present work, we investigated if flunitrazepam can affect the L α-H II phase transition of phosphatidylethanolamine through a similar mechanism. This study was approached by using merocyanine 540, a dye sensitive to the molecular packing of membrane lipids. A detailed analysis of merocyanine absorption and fluorescence emission and excitation spectra was performed. The results indicated that the fluorescence emitted came mainly from the monomeric form of merocyanine and that it resulted a good indicator of this phase transition, as was previously described. Flunitrazepam did not affect significantly the onset of the phase transition but showed a tendency to diminish the dye fluorescence emission intensity, which could involve a lower partition of merocyanine in the vesicles. Moreover, the results suggest that this drug produced a delay in the completeness of the phase transition and a decrement in the cooperativity of this phenomenon.

Theoretical analysis of phase-resolved fluorescence emission from fingerprint samples

Basic theory for the detection and imaging of latent fingerprints using phase-resolved optical method is described. In this work, extraction of single fluorescence emission from fingerprint samples deposited on non-fluorescing substrate is described. Also, the significance of incorporating the heterodyne concept in the signal-processing scheme in the formulated theoretical model is explained.

A flexible and accurate quantification algorithm for electron probe X-ray microanalysis based on thin-film element yields

Quantitative analysis by means of electron probe X-ray microanalysis (EPXMA) of low Z materials such as silicate glasses can be hampered by the fact that ice or other contaminants build up on the Si(Li) detector beryllium window or (in the case of a windowless detector) on the Si(Li) crystal itself. These layers act as an additional absorber in front of the detector crystal, decreasing the detection efficiency at low energies (<5 keV). Since the layer thickness gradually changes with time, also the detector efficiency in the low energy region is not constant. Using the normal ZAF approach to quantification of EPXMA data is cumbersome in these conditions, because spectra from reference materials and from unknown samples must be acquired within a fairly short period of time in order to avoid the effect of the change in efficiency. To avoid this problem, an alternative approach to quantification of EPXMA data is proposed, following a philosophy often employed in quantitative analysis of X-ray fluorescence (XRF) and proton-induced X-ray emission (PIXE) data. This approach is based on the (experimental) determination of thin-film element yields, rather than starting from infinitely thick and single element calibration standards. These thin-film sensitivity coefficients can also be interpolated to allow quantification of elements for which no suitable standards are available. The change in detector efficiency can be monitored by collecting an X-ray spectrum of one multi-element glass standard. This information is used to adapt the previously determined thin-film sensitivity coefficients to the actual detector efficiency conditions valid on the day that the experiments were carried out. The main advantage of this method is that spectra collected from the standards and from the unknown samples should not be acquired within a short period of time. This new approach is evaluated for glass and metal matrices and is compared with a standard ZAF method.

Using chlorophyll fluorescence to determine stress inEucalyptus grandisseedlings

Synopsis Since several environmental factors affect the functioning of the photosynthetic system, the status of the photosynthetic apparatus is a good indicator of the plant in terms of stress and stress adaptation. The measurement of chlorophyll fluorescence with a portable fluorimeter has been shown to be a useful non-destructive method for the investigation of the structure and function of the photosynthetic apparatus. A quantitative analysis of fluorescence emission, called the JIP-test, has also been introduced by which the behaviour of the photosynthetic system in response to environmental changes can be monitored. A pilot trial was conducted to determine the effect of light, water and nutrient stress on the fluorescence emission of Eucalyptus grandis seedlings. Flourescencc measurements were made on potted plants with a Hansatech Plant Efficiency Analyser, everyone to two days from stress initiation until trial termination. The results indicate that water and light stress in E. grandis seedlings ca...

Membrane potential changes after infection of monocytes by Toxoplasma gondii

Membrane potential changes in host cell plasma membrane were analyzed and the parasitophorous vacuole membrane (PVM) potential was characterized after infection by Toxoplasma gondii. Human monocytes infested by T. gondii were stained with two membrane potential sensitive dyes, DiOC 6(3) carbocyanine and DiSBAC 2(3) bis-oxonol, before fluorescence emission analysis by confocal laser scanning microscopy. After 24 and 48 h of infection, 34 and 39%, respectively, of monocytes showed several parasites (from two to six) per cell. At these infection times, significant decreases in cytoplasmic emissions were observed for both DiOC 6(3) and DiSBAC 2(3). Thus, hyperpolarisation of the host plasma membrane would occur consecutively to infection. Inside the parasitophorous vacuole, the fluorescence intensity of DiOC 6(3) and DiSBAC 2(3) increased significantly from 6 to 24 h after infection and the PVM became less polarised. Involvement of different ATPases in the membrane potential of infected monocytes was evaluated with ouabain, DCCD, omeprazole and sodium orthovanadate, ATPase inhibitors. All inhibitors induced a depolarisation of the plasma membrane. In the parasitophorous vacuole compartment, DCCD, omeprazole and sodium orthovanadate but not ouabain caused a significant depolarisation of the PVM, suggesting that H +, H +/K + and P-type ATPases were at the origin of the PVM potential. This is the first report showing the presence of ion transporters in the T. gondii PVM and the existence of at least two members of the P-type family of ion pumps: an electrogenic H +ATPase and an electroneutral H +/K + ATPase.

A new detection system with polycapillary conic collimator for high-localized analysis of X-ray fluorescence emission

In this paper, we present a new detection system conceived for the X-ray fluorescence (XRF) analysis of the radiation emitted by very small areas on the sample. The system is based on the use of a polycapillary conic collimator which captures the XRF emitted only from a local area of the sample independently on how extended is the X-ray excitation. The use of a Peltier-cooled silicon drift detector allows to achieve high energy resolution with a very compact system configuration. In this first prototype, a spatial resolution of about 150 /spl mu/m has been verified in the analysis of thin metal strips. Moreover, the results of a first X-Y scanning for elemental mapping on a sample surface are also reported.

Hydrophobic Clustering in Acid-Denatured IL-2 and Fluorescence of a Trp NH···π H-bond

The single tryptophan at position 121 of human interleukin-2 (IL-2) can form an NH···π hydrogen bond with Phe 117 involving the indole nitrogen and the benzene aromatic ring. At pH 5.5, this type of aromatic interaction results in a fluorescence quantum yield three-fold lower than that of a fully solvent exposed tryptophan. At pH 2.1, IL-2 forms a compact denatured state with twice the emission intensity of the native protein. Global analysis of time-resolved fluorescence emission at multiple emission wavelengths shows that native and acid-denatured IL-2 can be described by four decay components. The fractional amplitudes of the shortest sub-nanosecond lifetimes are higher in the native state, suggesting rapid quenching due to the NH···π hydrogen bond. In the denatured state, longer lifetimes have greater fractional amplitudes, indicating a smaller population of hydrogen-bonded species. Electrostatic-dipolar relaxation of the tryptophan microenvironment upon excitation is greater in the native-state of IL-2 than the acid-denatured state. This suggests that acid-denaturation sequesters Trp 121 from polar residues, while maintaining an interaction with Phe 117. This is consistent with the model of secondary structure preservation and hydrophobic clustering in molten-globule intermediates.

Polarization Measurements on Single Pigment-Protein Complexes

Individual antenna complexes from different photosynthetic units have been investigated by single molecule spectroscopy. In such energy transfer systems the polarization of the fluorescence emission gives valuable information about the nature of the emitting state, which is not readily available with other methods like fluorescence excitation or emission spectroscopy. The peripheral antenna light harvesting complex from purple bacterium Rhodopseudomonas Acidophila shows predominantly linear polarized fluorescence emission at low temperature, whereas at room temperature the fluorescence is randomly polarized. This is attributed to the fact, that at low temperature in the fluorescence emitting state the excitation energy is localized mainly on 4-5 chromophores. Analysis of the fluorescence emission of single peripheral antenna complexes of green plants indicate that for trimers of this species more than one Chlorophyll is responsible for the final fluorescence emission, which points towards a weak intermonomer coupling in the complex.

Optical detection of triggered atherosclerotic plaque disruption by fluorescence emission analysis.

Fluorescence emission analysis (FEA) has proven to be very sensitive for the detection of elastin, collagen and lipids, which are recognized as the major sources of autofluorescence in vascular tissues. FEA has also been reported to detect venous thromboemboli. In this paper we have tested the hypothesis that FEA can reproducibly detect in vivo and in vitro triggered plaque disruption and thrombosis in a rabbit model. Fluorescence emission (FE) spectra, recorded in vivo, detected Russell's viper venom (RVV)-induced transformation of atherosclerotic plaque. FE intensity at 410-490 nm 4 weeks after angioplasty was significantly lower (P < 0.0033 by analysis of variance) in RVV-treated rabbits when compared to control animals with stable plaque. FE spectral profile analyses also demonstrated a significant change in curve shape as demonstrated by polynomial regression analysis (R2 from 0.980 to 0.997). We have also demonstrated an excellent correlation between changes in FE intensity and the structural characteristics detected at different stages of "unstable atherosclerotic plaque" development using multiple regression analysis (R2 = 0.989). Thus, FEA applied in vivo is a sensitive and highly informative diagnostic technique for detection of triggered atherosclerotic plaque disruption and related structural changes, associated with plaque transformation, in a rabbit model.

A Fluorescence Investigation of the Active Site ofPseudomonas aeruginosa Exotoxin A

Single tryptophan mutant proteins of a catalytically active domain III recombinant protein (PE24) from Pseudomonas aeruginosa exotoxin A were prepared by site-directed mutagenesis. The binding of the dinucleotide substrate, NAD+, to the PE24 active site was studied by exploiting intrinsic tryptophan fluorescence for the wild-type, single Trp, and tryptophan-deficient mutant proteins. Various approaches were used to study the substrate binding process, including dynamic quenching, CD spectroscopy, steady-state fluorescence emission analysis, NAD+-glycohydrolase activity, NAD+ binding analysis, protein denaturation experiments, fluorescence lifetime analysis, steady-state anisotropy measurement, stopped flow fluorescence spectroscopy, and quantum yield determination. It was found that the conservative replacement of tryptophan residues with phenylalanine had little or no effect on the folded stability and enzyme activity of the PE24 protein. Dynamic quenching experiments indicated that when bound to the active site of the enzyme, the NAD+ substrate protected Trp-558 from solvent to a large extent but had no effect on the degree of solvent exposure for tryptophans 417 and 466. Also, upon substrate binding, the anisotropy of the Trp-417(W466F/W558F) protein showed the largest increase, followed by Trp-466(W417F/W558F), and there was no effect on Trp-558(W417F/W466F). Furthermore, the intrinsic tryptophan fluorescence exhibited the highest degree of substrate-induced quenching for the wild-type protein, followed in decreasing order by Trp-417(W466F/W558F), Trp-558(W417F/W466F), and Trp-466(W417F/W558F). These data provide evidence for a structural rearrangement in the enzyme domain near Trp-417 invoked by the binding of the NAD+ substrate.

Site-directed mutagenesis of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase.

Site-specific mutants have been produced in order to investigate the role of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis-proline in the high resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T. maritima PGK, and may therefore have a role in the independent folding of the two domains or in the 'hinge' bending of the molecule during catalysis. The residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe), and the resulting proteins characterised by differential scanning calorimetry (DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic analysis. Although the secondary and tertiary structure of the Pro204His protein is generally similar to that of the wild-type enzyme as assessed by CD, the enzyme is less stable to heat and guanidinium chloride denaturation than the wild-type. In the denaturation experiments two transitions were observed for both the wild-type and the Pro204His mutant, as have been previously reported for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990) Biochemistry 29, 8683-8689]. The first transition is accompanied by an increase in fluorescence intensity leading to a hyperfluorescent state, followed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild-type protein, suggesting that sequence-distant interactions are more rapidly disrupted in this mutant enzyme than in the wild-type enzyme, while sequence-local interactions are disrupted in a similar way. The Michaelis constants (K(m)) for both 3-phospho-D-glycerate and ATP are increased only by three or fourfold, which confirms that, as expected, the substrate binding sites are largely unaffected by the mutation. However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of 'hinge' bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the wild-type and Pro204His enzymes, with considerable loss of both secondary and tertiary structure. It is proposed that the proline residue at 204 in the 'hinge' region of PGK plays a role in the stability and catalytic mechanism of the enzyme.

Roles of Copper Ligands in the Activation and Secretion ofStreptomyces Tyrosinase

The expression of the melanin operon (melC) of Streptomyces antibioticus requires the chaperone-like protein MelC1 for the incorporation of two copper ions (designated as CuA and CuB) and the secretion of the apotyrosinase (MelC2) via a transient binary complex formation between these two proteins. To investigate whether the copper ligand of tyrosinase is involved in this MelC1.MelC2 binary complex function, six single substitution mutations were introduced into the CuA and CuB sites. These mutations led to differential effects on the stability, copper content, and export function of binary complexes but a complete abolishment of tyrosinase activity. The defects in the tyrosinase activity in mutants were not because of the impairment of the formation of MelC1. MelC2 complex but rather the failure of MelC2 to be discharged from the copper-activated binary complex. Moreover, the impairments on the discharge of the mutant MelC2 from all the mutant binary complexes appeared to result from the structural changes in their apoforms or copper-activated forms of the complexes, as evidenced by the fluorescence emission and circular dichroism spectral analysis. Therefore, each of six copper ligands in Streptomyces tyrosinase binuclear copper sites plays a pivotal role in the final maturation and the discharge of tyrosinase from the binary complex but has a less significant role in its secretion.

Effect of Dimethoate on Photosynthesis and Pigment Fluorescence ofSynechocystissp. PCC 6803

The organophosphorus (OP) insecticides are powerful inhibitors of esterases, and their toxic actions are commonly explained in terms of acetylcholinesterase (EC 3.1.1.7) inhibition but their phytotoxic effects remain unexplained. In this study the effects of an OP insecticide, dimethoate, on cyanobacterial photosynthesis and respiration were measured using the cyanobacteriumSynechocystissp. PCC 6803 as test organism. The insecticide caused enhancement of respiratory O2consumption at all tested concentrations (10–300 μM) while photosynthesis was found to be significantly affected at concentrations ≥50 μM.From fluorescence emission analysis, oxygen exchange measurement, and determination of14CO2incorporation, it was found that dimethoate caused inhibition of photosynthetic electron transport, resulting in increase of PS II fluorescence and reduction in photosynthetic carbon fixation. An increase of nonphotochemical quenching was caused by the insecticide through the increase in acidity of the thylakoid lumen. Furthermore, detachment of phycobilisomes (PBS) from the PS II reaction centers was observed in term of increase in PBS fluorescence in treated cultures. This detachment is expected to be caused by membrane fluidity changes. The fluorescence enhancement of PS II was more than that of the PBS.

ACCLIMATION OF THE PHOTOSYNTHETIC APPARATUS OF PALMARIA PALMATA (RHODOPHYTA) TO LIGHT QUALITIES THAT PREFERENTIALLY EXCITE PHOTOSYSTEM I OR PHOTOSYSTEM II1

ABSTRACT Acclimation of the photosynthetic apparatus to light absorbed primarily by phycobilisomes (which transfer energy predominantly to photosystem II) or absorbed by chlorophyll a (mainly present in the antenna of photosystem I) was studied in the macroalga Palmaria palmata L. In addition, the influence of blue and yellow light, exciting chlorophyll a and phycobilisomes, respectively, ivas investigated. All results were compared to a white light control. Complementary chromatic adaptation in terms of an enhanced ratio of phycoerythrin to phycocyanin under green light conditions was observed. Red light (mainly absorbed by chlorophyll a) and green light (mainly absorbed by phycobilisomes) caused an increase of the antenna system, which was not preferentially excited. Yellow and blue light led to intermediate states comparable to each other and white light. Growth was reduced under all light qualities in comparison to white light, especially under conditions preferably exciting phycobilisomes (green light‐adapted algae had a 58% lower growth rate compared to white light‐adapted algae). Red and blue light‐adapted algae showed maximal photosynthetic capacity with white light excitation and significantly lower values with green light excitation. In contrast, green and yellow light‐adapted algae exhibited comparable photosynthetic capacities at all excitation wavelengths. Low‐temperature fluorescence emission analysis showed an increase of photosystem II emission in red light‐adapted algae and a decrease in green light‐adapted algae. A small increase of photosystem I emission teas also found in green light‐adapted algae, but this was much less than the photosystem II emission increase observed in red light‐adapted algae (both compared to phycobilisome emission). Efficiency of energy transfer from phycobilisomes to photosystem II was higher in red than in green light‐adapted algae. The opposite was found for the energy transfer efficiency from phycobilisomes to photosystem I. Zeaxanthin content increased in green and blue light‐adapted algae compared to red, white, and yellow light‐adapted algae. Results are discussed in comparison to published data on unicellular red algae and cyanobacteria.

Analysis of the 77 K fluorescence emission and excitation spectra of isolated etioplast inner membranes

Low-temperature fluorescence emission and excitation analyses revealed four discrete spectral forms of protochlorophyllide and/or protochlorophyll in the etioplast inner membranes isolated from leaves of wheat ( Triticum aestivum cv. Kosack). The emission spectra were recorded with various excitation wavelengths between 400 and 500 nm. The excitation spectra were recorded for fluorescence emitted at various wavelengths between 620 and 740 nm. The spectra were analysed by a combination of various computer-aided methods including differential analysis, gaussian resolution and topological plots. The four pigment forms can be characterized by their different excitation (Ex) and emission (Em) bands. Adding the satellite bands of the Qy (0-0) transitions, the emission vibrational (Emv) bands, with correlated amplitudes gave the following delineation: Ex436-Em633-Emv690, Ex443-Em645-Emv711, Ex451-Em657-Emv727, and Ex463-Em671-Emv740. The differences in the spectral properties of the four pigment forms are discussed in relation to possible differences in their structural arrangement, association to NADPH-protochlorophyllide oxidoreductase and localization in prolamellar bodies and/or prothylakoids.