Fluorescence Intensity Of Dye Research Articles

Analysis of Fluorescence Lifetime and Energy Transfer Efficiency in Single-Molecule Photon Trajectories of Fast-Folding Proteins.

In single-molecule Förster resonance energy transfer (FRET) spectroscopy, the dynamics of molecular processes are usually determined by analyzing the fluorescence intensity of donor and acceptor dyes. Since FRET efficiency is related to fluorescence lifetimes, additional information can be extracted by analyzing fluorescence intensity and lifetime together. For fast processes where individual states are not well separated in a trajectory, it is not easy to obtain the lifetime information. Here, we present analysis methods to utilize fluorescence lifetime information from single-molecule FRET experiments, and apply these methods to three fast-folding, two-state proteins. By constructing 2D FRET efficiency-lifetime histograms, the correlation can be visualized between the FRET efficiency and fluorescence lifetimes in the presence of the submicrosecond to millisecond dynamics. We extend the previously developed method for analyzing delay times of donor photons to include acceptor delay times. To determine the kinetics and lifetime parameters accurately, we used a maximum likelihood method. We found that acceptor blinking can lead to inaccurate parameters in the donor delay time analysis. This problem can be solved by incorporating acceptor blinking into a model. While the analysis of acceptor delay times is not affected by acceptor blinking, it is more sensitive to the shape of the delay time distribution resulting from a broad conformational distribution in the unfolded state.

Label-free and enzyme-free platform for the construction of advanced DNA logic devices based on the assembly of graphene oxide and DNA-templated AgNCs.

DNA-based molecular logic computation has drawn extensive attention in bioanalysis, intelligent diagnostics of diseases and other nanotechnology areas. Herein, taking 2-to-1 and 4-to-2 encoders and a 1-to-2 decoder as model molecular logic devices, we for the first time combined the quenching ability of GO (graphene oxide) to DNA-templated AgNCs with G-quadruplex-enhanced fluorescence intensity of porphyrin dyes for the construction of label-free and enzyme-free dual-output advanced DNA molecular logic devices. Also, through the application of negative logic conversion to an XOR logic gate and combined with an INHIBIT logic gate, we also operated a label-free and enzyme-free comparator.

89 EFFECT OF MEDIA, METABOLIC REGULATORS, AND STAGE OF DEVELOPMENT ON LIPID CONTENT AND MITOCHONDRIAL POLARITY OF IN VITRO-PRODUCED HOLSTEIN EMBRYOS

Current bovine embryo culture methods result in accumulation of lipids and reactive oxygen species, possibly due to sub-optimal metabolic regulation. These effects decrease the cryopreservation survival and implantation potential of in vitro-produced (IVP) embryos. Forskolin has been shown to decrease lipid accumulation, and vitamin K2 (Vit K2) is thought to decrease oxidative stress from in vitro conditions. The aims of this study were (1) to assess lipid content of embryos cultured with or without forskolin and Vit K2 in both continuous and sequential SOF-based medium, and (2) to examine individual and combined effects of forskolin and Vit K2 on mitochondrial polarity. For Experiment 1, a 2 × 2 × 2 factorial design was used to compare culture systems (continuous v. 3-step sequential), additives (no additive v. Vit K2 (0.5 mM at Day 3) plus forskolin (10 µM at Day 5), and blastocyst stage [6 (early) v. 7 (late)] on overall lipid content. For Experiment 2, mitochondrial polarity of stage 7 blastocysts was analysed from the following groups: no additive, Vit K2 (0.5 mM at Day 3), forskolin (10 µM at Day 5), and Vit K2 plus forskolin. IVP embryos (n = 199, Experiment 1; n = 45, Experiment 2) were produced by standard procedures and cultured at 38.5°C in 5% O2, 5% CO2, and 90% N2. For Experiment 1, embryos were stained with 1 μg mL–1 Nile Red, and two images per embryo were taken along the equatorial plane at 40× magnification. For Experiment 2, embryos were stained with 300 nM MitoTracker Red CMX-Rosamine, and 10 images per embryo were acquired by confocal microscopy with a 5-μm step size at 40× magnification. For both experiments, fluorescence intensity (FI) of each image was measured by Image PRO software with embryo controlled for and background fluorescence corrected. Data (Table 1) were analysed by ANOVA and means were compared by Tukey’s HSD. In Experiment 1, embryos cultured with forskolin and Vit K2 showed decreased lipid content in both the early and late stage (P < 0.05), with no effect from culture system (P > 0.05). In Experiment 2, forskolin and Vit K2 individually increased mitochondrial polarity (P < 0.05), but had no combined effect (P > 0.05). In conclusion, these data suggest that while a combination of forskolin and Vit K2 as media additives reduces lipid accumulation, the interaction between these metabolic regulators may negate their individual effects on mitochondrial polarity. Table 1.Fluorescence intensity of Nile Red and MitoTracker Red dyes between treatment groups

Directed Self-Assembly of Block Copolymer Micelles onto Topographically Patterned Surface.

We report a facile method to control directed self-assembly (DSA) of spherical micelles of block copolymers (BCPs) by topographically patterned surface. A cylinder-forming polystyrene-block-poly(2-vinylpyridine) copolymer [Mn,PS = 175 kg/mol, Mn,P2VP = 70 kg/mol, and polydipersity index (PDI) = 1.08] was phase-separated on a thin film of poly(vinyl alcohol) (PVA) by solvent annealing. By additional treatment with ethanol as a preferential solvent for P2VP block, the surface of BCP thin film was reconstructed to produce nanopores. Nanoporous structures in BCP thin films were transferred to the underlying hydrophilic PVA film by reactive ion etching (RIE). Then spherical BCP micelles were quickly self-assembled within the nanopores in the PVA layer due to topographical contrast and surface energy difference during spin-coating. Consequently, the site-selective array of BCP micelles was utilized as templates to achieve heterogeneous organization of nanoparticles and organic fluorescent dyes over a large area. In addition, it was observed that those heterogeneous assemblies showed a remarkable decrease in fluorescence intensity of organic dyes.

Effect of carbamazepine and gabapentin on excitability in the trigeminal subnucleus caudalis of neonatal rats using a voltage-sensitive dye imaging technique.

BackgroundThe antiepileptic drugs carbamazepine and gabapentin are effective in treating neuropathic pain and trigeminal neuralgia. In the present study, to analyze the effects of carbamazepine and gabapentin on neuronal excitation in the spinal trigeminal subnucleus caudalis (Sp5c) in the medulla oblongata, we recorded temporal changes in nociceptive afferent activity in the Sp5c of trigeminal nerve-attached brainstem slices of neonatal rats using a voltage-sensitive dye imaging technique.ResultsElectrical stimulation of the trigeminal nerve rootlet evoked changes in the fluorescence intensity of dye in the Sp5c. The optical signals were composed of two phases, a fast component with a sharp peak followed by a long-lasting component with a period of more than 500 ms. This evoked excitation was not influenced by administration of carbamazepine (10, 100 and 1,000 μM) or gabapentin (1 and 10 μM), but was increased by administration of 100 μM gabapentin. This evoked excitation was increased further in low Mg2+ (0.8 mM) conditions, and this effect of low Mg2+ concentration was antagonized by 30 μM DL-2-amino-5-phosphonopentanoic acid (AP5), a N-methyl-d-aspartate (NMDA) receptor blocker. The increased excitation in low Mg2+ conditions was also antagonized by carbamazepine (1,000 μM) and gabapentin (100 μM).ConclusionCarbamazepine and gabapentin did not decrease electrically evoked excitation in the Sp5c in control conditions. Further excitation in low Mg2+ conditions was antagonized by the NMDA receptor blocker AP5. Carbamazepine and gabapentin had similar effects to AP5 on evoked excitation in the Sp5c in low Mg2+ conditions. Thus, we concluded that carbamazepine and gabapentin may act by blocking NMDA receptors in the Sp5c, which contributes to its anti-hypersensitivity in neuropathic pain.

Potential interactions of calcium-sensitive reagents with zinc ion in different cultured cells.

BackgroundSeveral chemicals have been widely used to evaluate the involvement of free Ca2+ in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca2+-sensitive reagents in diverse cultured cells.Methodology/Principal FindingsIn rat astrocytic C6 glioma cells loaded with the fluorescent Ca2+ dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca2+ chelator EGTA irrespective of added CaCl2. The addition of the Ca2+ ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn2+ ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters.Conclusions/SignificanceTaken together, comprehensive analysis is absolutely required for the demonstration of a variety of physiological and pathological responses mediated by Ca2+ in diverse cells enriched of Zn2+.

Torsion-dependent fluorescence switching of amyloid-binding dye NIAD-4

Fluorescence intensity and quantum yield of organic dyes often depend on their internal rotation in the excited electronic state. NIAD-4 is a novel amyloid-binding dye and a possible substitute for Thioflavin T in amyloid tagging. Its structure suggests that it should act like a molecular rotor whose photophysical properties are strongly governed by internal rotation, although little is known about the photophysics of this molecule to date. We investigated the molecular rotor property of NIAD-4 by measuring the viscosity dependence of fluorescence intensity against the molecular rotor model. The origin of torsion-dependent fluorescence switching of NIAD-4 is discussed.

Response of unilamellar DPPC and DPPC:SM vesicles to hypo and hyper osmotic shocks: A comparison

DPPC and DPPC:SM large unilamellar vesicles (LUVs), prepared by extrusion, readily respond to osmotic shocks (hypo- and hyper-osmotic) by water influx/efflux (evaluated by changes in turbidity) and by entrapped calcein liberation (measured by an increase in dye fluorescence intensity). On the other hand, small unilamellar vesicles (SUVs) prepared by sonication are almost osmotically insensitive. LUVs water transport, both in hypo- and hyper-osmotic conditions, takes place faster than calcein ejection towards the external solvent. Similarly, response to a hypotonic imbalance is faster than that associated to a hypertonic stress. This difference is particularly noticeable for the increase in calcein fluorescence intensity and can be related to the large reorganization of the bilayer needed to form pores and/or to adsorb the dye to the inner leaflet of the vesicle after water efflux. Conversely, addition of SM to the vesicles barely modify the rate of calcein permeation across the bilayer.

Fluorescence properties of indolenium carbocyanine dyes in solid state

The fluorescence intensity of indolenium carbocyanine dyes in the solid state depended on both the alkyl group on the nitrogen atoms in the indolenium rings and the kind of counter anions. The N-propyl derivative having a bis(trifluoromethylsulfonyl)imide anion, whose fluorescence maximum was observed at 657 nm with shoulder peaks, exhibited the highest fluorescence quantum yield of 0.31. The wave deconvolution of fluorescence spectrum indicated three components assigned to the S1,0→S0,0, S1,0→S0,1, and S1,0→S0,2 transitions. The X-ray structure of this compound indicates an isolated monomer-type packing, whereas the other derivatives shows the isolated dimer-type packing or consequent π/π stacking. The cyanine dye having the least π/π interactions among adjacent cationic fluorophores could exhibit the most intense fluorescence in the solid state.

Quantifying Molecular Transport through Lipid Electropores Induced by Nanosecond Pulsed Electric Fields

High amplitude, nanosecond-duration, pulsed electric fields (nsPEFs) induce the formation of nanometer-diameter pores in cell membranes. These transient lipid electropores facilitate the transport of normally impermeant ions and small molecules across the membrane. Quantitative measurements of molecular transport through lipid electropores are essential for the validation of electroporation models and for optimizing the nsPEF dose for application-specific responses. We calibrated the fluorescence intensity of the membrane integrity marker dye YO-PRO-1 in dense cell lysates using a fast confocal microscope in order to quantify the time-dependent molecular influx of YO-PRO-1 into living cells after nsPEF exposure. Using this method we were able to measure the number of YO-PRO-1 molecules that enter U-937 human histiocytic lymphoma cells following electropermeabilization by a single 6 ns, 20 MV/m pulse.

Effect ofBacillus cereusHemolysin II on Hepatocyte Cells

We investigated the efficiency of increasing the permeability (permeabilization) of cell membranes in primary liver cells by Bacillus cereus hemolysin II. An assessment of the degree of permeabilization was car ried out by measuring the fluorescence intensity of various low molecular weight dyes, which enter through pores into hepatocyte cells cultivated with hemolysin. We uncovered a high efficacy of hemolysin HlyII action on hepatocyte cell walls, which exceeded the effect of nonionic detergent, digitonin, which is commonly employed for pore formation in various cell membranes. Our results also point to the reversibility of membrane permeabilization in primary hepatocytes. The data obtained in this study can be utilized for assessments of pore-forming activity, in studies of hepatic mechanisms of action, and also the determination of the liver toxicity for different low molecular weight drugs.

Spectrophotometric, spectrofluorimetric, and potentiometric assays of cetyltrimethylammonium bromide in industrial wastewater samples.

This work deals with spectrophotometric, spectrofluorimetric, and potentiometric analyses of cetyltrimethylammonium bromide (CTAB) cationic detergent. The spectrophotometric procedure depends on measuring the absorbance of its binary complex with eosin yellow in Britton-Robinson buffer (pH 4) at Lambda max 547 nm in the range of 2.0-14.0 microg/mL with an accuracy of 100.15 +/- 0.54%. The spectrofluorimetric procedure depends on determining the quenching of the fluorescence intensity of fluorescein dye by CTAB in the presence of borate buffer at Lambda em = 500 nm, Lambda ex = 304 nm, in the range of 2.90-14.50 microg/mL with an accuracy of 99.81 + 0.33%. The electrochemical procedure describes an ionophore-based technique using a graphite sensor to measure 0.036 microg/mL and showed an accuracy of 100.11 +/- 0.61%. The experimental conditions affecting each of the three suggested procedures were studied and optimized. All the developed procedures were validated and satisfactorily applied for the determination of CTAB in industrial wastewater samples.

Fluorescence based fibre optic pH sensor for the pH 10–13 range suitable for corrosion monitoring in concrete structures

The design, development and evaluation of an optical fibre pH sensor for monitoring pH in the alkaline region are discussed in detail in this paper. The design of this specific pH sensor is based on the pH induced change in fluorescence intensity of a coumarin imidazole dye which is covalently attached to a polymer network and then fixed to the distal end of an optical fibre. The sensor provides a response over a pH range of 10.0–13.2 with an acceptable response rate of around 50min, having shown a very good stability over a period of longer than 20 months thus far. The sensor has also demonstrated little cross-sensitivity to ionic strength (IS) and also excellent photostability through a series of laboratory tests. These features make this type of sensor potentially well suited for in situ long term monitoring of pH in concrete structures, to enhance structural monitoring in the civil engineering sector.

Brilliant UltraViolet™ Dyes: A new set of high sensitivity fluorescence reagents for multicolor flow cytometry with a UV laser (P3301)

Abstract While UV lasers have been available for many years, their utility has been mainly restricted to DNA/RNA binding fluorochromes (e.g. DAPI, Hoescht 33342) for studying cell cycle or stem cell side populations. The low fluorescence intensity of standard fluorescent dyes, such as AlexaFluor™ 350 or AMCA, have made them impractical for routine immunological flow analyses. Here we report the development of a novel class of highly fluorescent polymeric reporters which are excitable by a UV (355nm) laser. The introduction of these dyes has the potential to dramatically extend the functionality of the UV laser adding up to 6 totally new colors for multi-color cytometric flow studies. The first Brilliant UltraViolet (BUV) polymer has an excitation maximum near 355nm with emission below 405nm. Cross laser excitation is minimal requiring very little compensation into the Brilliant Violet 421 channel. Furthermore these polymers can serve as the basis for developing a wide range of bright, ultraviolet excitable tandem reagents, which span the entire spectrum from UV to the near IR. Data will be presented to demonstrate how the superior staining performance and unique excitation / emission profiles of these new dyes offer a broader pallet of colors for those designing multi-parameter flow panels. Comparative flow performance will be presented as will multicolor staining panels which highlight their compatibility with conventional flow reagents.

Hydrogen peroxide modulates the contractile response induced by phenylephrine in renal hypertensive rat aorta

Reactive oxygen species (ROS) can increase contractile responses in hypertensive rat vessels. This study aimed to evaluate the role of the endothelial cells (EC) and ROS to the contraction induced by phenylephrine (PE) in aorta from renal hypertensive (2K‐1C) and normotensive rat (2K). Concentration‐effect curves for PE were constructed in intact endothelium (E+) and denuded aorta (E−), with or without Catalase. Expression of phosphorylated Ser1177eNOS was evaluated by Western‐Blot. In isolated EC incubated with PE, it was measured the fluorescence intensity (FI) of the ROS sensitive dye (DHE) by flow citometry, with or without Catalase. The H2O2 production was measured through assay kit. The maximum contraction to PE was decreased only in 2K‐1C E+, which was partially reversed by Catalase. PE induces higher Ser1177phosphorylation of eNOS in 2K‐1C aorta than in 2K. PE increased the FI only in EC from 2K‐1C, and it was abolished by Catalase. PE stimulated higher H2O2 production in 2K‐1C (E+) than in 2K (E+). In conclusion, the reduced contraction to PE in E+ rat aorta from 2K‐1C is due to high eNOS‐Ser1177 phosphorylation and H2O2 production in the EC. Supported by FAPESP, CAPES and CNPq.

Supramolecular Interaction of Coumarin 1 Dye with Cucurbit[7]uril as Host: Combined Experimental and Theoretical Study

Molecules of the coumarin family have fluorescence characteristics that are highly sensitive to their environment, and thus, they have been used as fluorescent sensors in chemical and biological systems. However, the very poor fluorescence yield of most coumarin dyes in aqueous media limits their applications. We have adopted a supramolecular strategy to improve the fluorescence intensity of coumarin dye through its interaction with the relatively new host cucurbit[7]uril (CB[7]). The virtually nonfluorescent coumarin 1 (Φ(f) = 0.04) was converted into a highly fluorescent (Φ(f) = 0.52) entity in water upon addition of the nonfluorescent host CB[7]. Various spectroscopy techniques, namely, UV-vis absorption and steady-state and time-resolved fluorescence spectroscopies, established the formation of a strong 1:1 dye-CB[7] inclusion complex with a high binding constant of (1.2 ± 0.1) × 10(5) M(-1) for the dye. The stable inclusion complex of the neutral molecule was supported by density-functional-theory- (DFT-) based quantum chemical calculations. Energy decomposition analysis of various interaction factors in the host-guest complex revealed that key components providing stability to the complex were electrostatic, polarization, and charge-transfer energies. These new results on the formation of a strong inclusion complex of the versatile fluorophore coumarin 1 with the nontoxic host CB[7] could lead to the design of efficient molecular-scale biological probes, sensors, and photostable aqueous UV dye lasers.

Simple strategies towards bright polymer particles via one-step staining procedures

In order to develop simple and versatile procedures for the preparation of red emissive particles, various one-step swelling procedures for the loading of fluorophores into nanometer- and micrometer-sized polystyrene particles were systematically assessed. Parameters studied for model dyes from common dye classes include the composition of the swelling medium, dye charge and polarity, dye concentration, and particle surface chemistry. The dye loading procedures were compared based upon the efficiency of dye incorporation, fluorescence intensity, and colloidal stability of the resulting particles as well as the absence of dye leaking as determined by absorption and fluorescence spectroscopy, flow cytometry, and measurements of zeta potentials. In addition, for the first time, the influence of the amount of incorporated dye on the absolute fluorescence quantum yield and brightness of the fluorescent particles was investigated for selected chromophores in differently sized particles using a custom-made calibrated integrating sphere setup. Our results demonstrate the general suitability of these one-step loading procedures for efficient particle staining with neutral, zwitterionic, and charged fluorophores like oxazines, coumarines, squaraines, xanthenes, and cyanines emitting in the visible and near infrared. Dye polarity was identified as a suitable tool to estimate the loading efficiency of fluorophores into these polymer particles.

Aggregation induced emission in the rotatable molecules: the essential role of molecular interaction

Aggregation-induced emission (AIE) compounds, dye2 and dye3, with a rotatable N–N single bond and aza-crown-ether, have been synthesized. The fluorescence intensities of both dyes are very weak in THF, while become extraordinarily strong in a mixture of H2O–THF (v/v 95%). They are increased by 88.9 and 70.7 times, respectively, indicating enhanced blue-green fluorescence emissions. The λem of these two compounds in different states has been well studied. Besides this, dye1 and dye2 were characterized by single crystal X-ray structural determination. The results show that molecular interactions are formed in the particles, which considerably restrict the intramolecular vibration and rotation.

Restricting charge transfer in dye-graphene system

The absorbance and the fluorescence intensity of dye have been found to decrease on increasing the concentration of either (i) functionalized hydrogen-induced exfoliated multilayer graphene (f-HEG) or (ii) silver nanoparticles (AgNP). The observed static-quenching is due to the weak interaction between the dye and the AgNP or f-HEG in the ground state. In contrast, the spectra of the dye are unaffected by the presence of AgNP-decorated graphene. Pre-existing charge transfer interaction between the metal nanoparticles and graphene has been proposed to be mechanism responsible for this behavior.

Visualization of synchronous propagation of plant electrical signals using an optical recording method

Optical recording is widely used in electrophysiology research of animal cells, providing a good reference for the measurement of plant electrical signals at the multi-cell level. The aim of this research is to develop a method for monitoring the dynamic synchronous electrical activity in plant cells. Using a leaf strip of Vicia faba L as the specimen, a voltage-sensitive dye (VSD) and a stereo fluorescent microscope were applied to acquire an optical image time series, considering the different biological nature of the plant cell from the animal cell. Subsequently, the synchronous variation trend of the dye fluorescence intensity was extracted and observed through image analysis using self-designed signal processing procedures. This proved the feasibility of a visualization method of synchronous propagation of plant electrical signals.