Fluorescence Decay Time Research Articles

Conformational and Functional Disturbances of Serum Albumin Are Characteristic for First Episode Drug-naive Schizophrenic Patients.

Objective Conformational changes of human serum albumin (HSA) can disturb its main functions. Aim To reveal serum albumin disturbances using the high-tech approaches in first episode schizophrenia. Methods First-episode drug-naive schizophrenic (FES) patients were investigated. There were employed laser time resolved subnanosecond fluorescence spectroscopy (LTRSFS) using specific albumin fluorescent probe and high resolution nuclear magnetic resonance (NMR) 1 H spectroscopy. Results There were revealed 3 binding sites in albumin molecule with fluorescent decay time of 1, 3 and 9 nanoseconds (A1, A3 and A9 sites, respectively) in healthy volunteers using LTRSFS approach. There was found significant decrease of fluorescent decay time of probe bound to albumin A3 site of FES patients as compared with controls. It points out on the conformational changes in HSA molecule in FES patients. NMR 1 H spectra of blood serum and its albumin fraction of healthy donors and FES patients were studied. There were shown clear differences in NMR 1 H spectra between these two groups that points out to the conformational changes of albumin molecule in FES patients and disturbances in albumin transport functions. The reaction ability of thiol groups of albumin molecules in FES patients was significantly lower in comparison with healthy persons. It also points out on disturbances of antioxidant albumin properties in FES patients. Conclusion High-tech approaches can be useful for the development of new biomarkers for diagnostic and predicting methods and methods for the evaluation of the efficacy of different types of therapy of mental disorders.

Bright or dark immune complexes of anti-TAMRA antibodies for adapted fluorescence-based bioanalysis.

Fluorescence labels, for example fluorescein or rhodamin derivatives, are widely used in bioanalysis applications including lateral-flow assays, PCR, and fluorescence microscopy. Depending on the layout of the particular application, fluorescence quenching or enhancement may be desired as the detection principle. Especially for multiplexed applications or high-brightness requirements, a tunable fluorescence probe can be beneficial. The alterations in the photophysics of rhodamine derivatives upon binding to two different anti-TAMRA antibodies were investigated by absorption and fluorescence-spectroscopy techniques, especially determining the fluorescence decay time and steady-state and time-resolved fluorescence anisotropy. Two monoclonal anti-TAMRA antibodies were generated by the hybridoma technique. Although surface-plasmon-resonance measurements clearly proved the high affinity of both antibodies towards 5-TAMRA, the observed effects on the fluorescence of rhodamine derivatives were very different. Depending on the anti-TAMRA antibody either a strong fluorescence quenching (G71-DC7) or a distinct fluorescence enhancement (G71-BE11) upon formation of the immune complex was observed. Additional rhodamine derivatives were used to gain further information on the binding interaction. The data reveal that such haptens as 5-TAMRA could generate different paratopes with equal binding affinities but different binding interactions, which provide the opportunity to adapt bioanalysis methods including immunoassays for optimized detection principles for the same hapten depending on the specific requirements.

Fluorescence properties of Schiff base – N,N′-bis(salicylidene) – 1,2-Phenylenediamine in presence of bile acid host

Fluorescence properties of Schiff base – N,N′-bis(salicylidene) – 1,2-phenylenediamine (LH2) is used to study the micelles formed by aggregation of different important bile acids like cholic acid, deoxycholic acid, chenodeoxycholic acid and glycocholic acid by steady state and picosecond time-resolved fluorescence spectroscopy. The fluorescence band intensity was found out to increase with concomitant red shift with gradual addition of different bile acids. Binding constant of the probe with different bile acids as well as critical micelle concentration was obtained from the variation of fluorescence intensity on increasing concentration of bile acids in the medium. The increase in fluorescence quantum yields, fluorescence decay times and substantial decrease in nonradiative decay rate constants in bile acids micellar environment points to the restricted motion of the fluorophore inside the micellar subdomains.

Luminescent Properties and Energy Transfer Mechanism of Gd3+ and Eu2+ Co-doped Phosphate Glasses

The phosphate glasses doped with Eu2+, Gd3+, respectively, and co-doped with Gd3+ and Eu2+ were prepared by high-temperature melting method. The transmission spectra, the excitation spectra, the emission spectra and the fluorescent decay time were investigated. The energy transfer process between Gd3+ and Eu2+ was studied. From the excitation spectra and the emission spectra of the phosphate glasses doped with Eu2+, we observed that the emission intensity of Eu2+ shows higher for 0.02 mol% Eu2+-doped phosphate glass. According to the excitation spectra and the emission spectra and the fluorescence decay curves, when the concentration of Eu2+ was 0.02 mol%, the optimal concentration of Gd3+ was 0.3 mol%. Based on Dexter theory, it is shown that the energy transfer between Gd3+ and Eu2+ was nonradiation energy transfer by analyzing the energy-level diagram. The fluorescence decay curves of Gd3+ were expressed by the Inokuti–Hirayama’s model and were used to analyze energy transfer mechanism between Gd3+ and Eu2+. And the energy transfer efficiency was also calculated.

In situ induced metal-enhanced fluorescence: A new strategy for biosensing the total acetylcholinesterase activity in sub-microliter human whole blood

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities (i.e., total AChE) in human blood are biomarkers for theranostic monitoring of organophosphate neurotoxin-poisoned patients. We developed an ultra-sensitive method to detect the total AChE activity in sub-microliter human whole blood based on in situ induced metal-enhanced fluorescence (MEF). Both AChE and BChE can catalyze the hydrolysis of the acetylthiocholine (ATCh) substrate and produce positively-charged thiocholine (TCh). TCh can reverse the negatively-charged surface of core–shell Ag@SiO2 nanoparticles (NPs). The negatively-charged fluorescent dye (8-hydroxypyrene-1,3,6-trisulfonic acid, HPTS) is then confined to the surface of Ag@SiO2 NPs and generates an enhanced fluorescence signal in situ. Changes in the surface charge of Ag@SiO2 NPs are monitored by Zeta potential, and the MEF effect is confirmed by the measurements of fluorescence time decay. AChE activity has a dynamic range of 0U/mL to 0.005U/mL and a detection limit of 0.05mU/mL. The total AChE activity in the sub-microliter human whole blood could be determined; the results were further validated. Therefore, combining the AChE catalytic reaction with MEF provides a simple, ultra-sensitive, and cost-effective “in situ MEF” approach to determine the total AChE activity in human whole blood sample down to sub-microliters without matrix interferences. The strategy also allows potential usage in other tissues and other fields.

Structural and luminescence properties of Gd2Si2O7:Ce prepared by solution combustion followed by heat treatment

A method comprising solution combustion followed by a heat treatment has been employed to synthesize cerium doped gadolinium pyrosilicate (Gd2Si2O7:Ce, or GPS:Ce) compounds. The powder obtained after the combustion was annealed at 1200°C for 4h and 1600°C for 3h to synthesize triclinic and orthorhombic phases of the GPS, respectively. Structural and morphological characterizations of the synthesized compounds were carried out using X-ray diffraction and electron microscopy (SEM, TEM) techniques. A change in the enthalpy was observed in the differential thermal analysis data as a consequence of triclinic to orthorhombic phase transition in the GPS. Luminescence spectra and fluorescence decay time were measured at room temperature to characterize emission centers created in GPS compounds doped with trivalent rare earth ion (Ce3+). The triclinic GPS:Ce phase exhibited photoluminescence peaks at 379nm and 410nm while for the orthorhombic phase emissions at 353nm and 380nm were observed. A multi-component exponential decay pattern of the luminescence is observed for both the GPS:Ce phases. In addition, samples of the orthorhombic GPS:Ce were found to exhibit X-ray excited luminescence (XEL).

Two-photon imaging of intact living plants during freezing with a flexible multiphoton tomograph

We describe the combination of a flexible multiphoton tomograph (MPTflex) with a heating and cooling stage. The stage allows temperature control in the range of (−196 °C) (77 K) to +600 °C (873 K) with selectable heating/freezing rates between 0.01 K min−1 and 150 K min−1. To illustrate the imaging capabilities of the combined system, fluorescence intensity and lifetime of intrinsic molecules from a plant leaf were imaged with submicron resolution during freezing in vivo without detaching the leaf from the plant. An increase of fluorescence intensity and decay times with decreasing temperature was observed. The measurements illustrate the usefulness of multiphoton imaging as a non-invasive online tool to investigate temperature-induced effects. The flexible multiphoton tomograph with its adjustable mechano-optical arm and scan head allows imaging at otherwise hardly accessible sample regions.

PsbS is required for systemic acquired acclimation and post-excess-light-stress optimization of chlorophyll fluorescence decay times in Arabidopsis

Systemic acquired acclimation (SAA) is an important light acclimatory mechanism that depends on the global adjustments of non-photochemical quenching and chloroplast retrograde signaling. As the exact regulation of these processes is not known, we measured time-resolved fluorescence of chlorophyll a in Arabidopsis thaliana leaves exposed to excess light, in leaves undergoing SAA, and in leaves after excess light episode. We compare the behavior induced in wild-type plants with null mutant of non-photochemical quenching (npq4–1). The wild type rosettes exhibit a small reduction of fluorescence decay times in leaves directly exposed to excess light and in leaves undergoing SAA in ambient low light. However in npq4–1 exposition to excess light results in much faster fluorescence decay, which is insensitive to excitation power. At the same time npq4–1 leaves undergoing SAA displayed intermediate fluorescence decay. The npq4–1 plants also lost the ability to optimize florescence decay, and thus chlorophyll a dynamics up to 2 h after excess light episode. The fluorescence decay dynamics in both WT and npq4–1 can be described by a set of 3 maximum decay times. Based on the results, we concluded that functional PsbS is required for optimization of absorbed photon fate and optimal light acclimatory responses such as SAA or after excess light stress.

Bright, water-soluble CeF3 photo-, cathodo-, and X-ray luminescent nanoparticles

Bright, water-soluble CeF3 nanoparticles with small size and narrow size distribution have been synthesized using a simple co-precipitation method without any ligands. Size control of nanoparticles from 13 ± 2 to 9 ± 2 nm was achieved by varying the reaction time. Colloidal properties have been found to vary with pH and, independently, with dilution. The photoluminescence of the as-synthesized nanoparticles shows a highly photostable UV/Visible fluorescence band due to allowed 5d–4f transitions, also observed in the X-ray luminescence spectrum. This band is suitable for X-ray excitation of a range of photosensitizers. The photoluminescence quantum yield of nanoparticles was also determined to be 31 %. Using the measured fluorescence decay time of 25 ns, the radiative lifetime of Ce in CeF3 was found to be 80.6 ns. Both photoluminescence and cathodoluminescence emission are affected by the reaction time and measurement temperature. Electron-beam-induced defect annealing is also observed.

Nanodiamond Emitters of Single Photons

Luminescence properties of single color centers were studied in nanodiamonds of different origin. It was found that single photon emitters could be realized even in molecularsized diamond (less than 2 nm) capable of housing stable luminescent center “silicon-vacancy.” First results on incorporation of single-photon emitters based on luminescent nanodiamonds in plasmonic nanoantennas to enhance the photon count rate and directionality, diminish the fluorescence decay time, and provide polarization selectivity are presented.

Spectroscopic and structural properties of Na3RE(PO4)2:Yb orthophosphates synthesised by hydrothermal method (RE = Y, Gd)

Na3RE(PO4)2 orthophosphates, where RE=Y and Gd, doped with Yb3+ ions were synthesised by hydrothermal method and characterised by X-ray diffraction, microscopic, IR and Raman techniques. The emission, excitation spectra were recorded at room temperature and fluorescence decay time was determined. As-synthesised samples crystallise in a trigonal modification and during calcination at 500°C they transform to a monoclinic or orthorhombic symmetry (for yttrium and gadolinium compounds, respectively). Finally, the samples calcined at 700°C do not reveal the trigonal symmetry. The morphology of particles for all samples is similar and resembles rice grains. All the obtained for the studied orthophosphates data were compared to those previously reported for the same compounds synthesised by Pechini method.

Confocal FLIM of Genetically Encoded FRET Sensors for Quantitative Ca2+ Imaging

Fluorescence lifetime imaging (FLIM) is a powerful imaging mode that can be combined with confocal imaging. Changes in the fluorescence decay time of a donor in an intramolecular Förster resonance energy transfer (FRET)-based biosensor provide intrinsic quantitative data. Here, we describe a protocol using both the Ca(2+) sensor TN-XL, which uses troponin C, as the Ca(2+)-sensing unit, and the FLIM technology based on time-correlated single-photon counting.

Effect of nanosize micelles of ionic and neutral surfactants on the photophysics of protonated 6-methoxyquinoline: Time-resolved fluorescence study

The excited state dynamic studies have been carried out to investigate the effects of micellar surface charge on the photophysics of protonated 6-methoxyquinoline (6MQ+) in anionic, sodium dodecylsulphate (SDS), cationic, cetyltrimethylammonium bromide (CTAB) and neutral, triton X-100 (TX100) surfactant at premicellar, micellar and postmicellar concentrations in aqueous phase at room temperature. At premicellar concentrations of SDS, there is a slight decrease in emission intensity and at micellar and postmicellar concentrations, increase in emission intensity and blue shift of spectrum has been observed. The blue shift in fluorescence spectrum and slight increase in quantum yield are attributed to incorporation of solute molecule to the micelles. Edge excitation red shift (EERS) in fluorescence maximum of 6MQ+ has been observed in all the surfactant solutions studied. The EERS has been ascribed in terms of solvent relaxation process. In SDS surfactant system, due to heterogeneous restricted motion of solvent molecules, the solvent viscosity increases which results in an increase in net magnitude of EERS. The fluorescence decay components of 6MQ+ fit with multi exponential functions in all the micellar systems studied. The location of the probe molecule in micellar systems is justified by a variety of spectral parameters such as refractive index, dielectric constant, ET (30), EERS, average fluorescence decay time, radiative and non radiative rate constants, and rotational relaxation time.

Preferential molecular encapsulation of an ICT fluorescence probe in the supramolecular cage of cucurbit[7]uril and β-cyclodextrin: an experimental and theoretical approach.

Supramolecular interaction between an intramolecular charge transfer (ICT) probe, N,N-dimethylaminonaphthyl-(acrylo)-nitrile (DMANAN), and two well-recognized macrocyclic hosts, cucurbit[7]uril (CB7) and β-cyclodextrin (β-CD), has been studied in aqueous medium by absorption, emission, time-resolved measurements, and (1)H NMR spectroscopic methods. The changes in the profiles of the fluorescence spectra illustrate significant modifications in fluorescence intensity, decay time, and quantum yield upon confinement of probe within the hydrophobic cavity of the hosts. Using the Benesi-Hildebrand relationship, the stoichiometric ratio as well as the binding constant of the host-guest complexation has been estimated. The stable inclusion complexes of the probe with different hosts have been supported by DFT and ONIOM based quantum chemical calculations. These methods of measurement establish that the acceptor group of the probe resides inside the hydrophobic cavity of the macrocycle. The competitive binding of metal ions and cationic surfactants to CB7 has been excellently mapped with this guest fluorosensor.

Combustion synthesis and photoluminescence properties of a novel blue-green-emitting Er3+ and Li+ co-doped LaNbTiO6

A single-phase blue-green light emitting phosphor, LaNbTiO6: Er3+, Li+, was synthesized by using a simple and facile sol–gel combustion approach. Its structure and photoluminescence (PL) properties were investigated as a function of Er3+ and Li+ ion concentration. The luminous mechanisms of the Er3+ doping and Er3+/Li+ co-doping in the LaNbTiO6 host were also discussed, including the intra-4f-transitions of Er3+ and the charge compensation of Li+. The incorporation of Li+ ions into LaNbTiO6 lattice further altered the local structure and symmetry of the crystals field around Er3+. Concentration quenching occurred when Er3+ and Li+ reached certain levels. Furthermore, the critical transfer distance of Er3+ → Er3+ in the phosphor was calculated. The absolute quantum efficiencies and the fluorescence decay time of the phosphors were studied. The phosphor produced blue-green light, presenting CIE chromaticity coordinates of (0.211, 0.277) and (0.214, 0.320).

Optical properties of phosphate glasses co-doped with Yb3+ and silver nanoparticles

We report the fabrication and spectroscopic characterization of Yb3+-doped phosphate glass, also containing silver nitrate. Scanning electron microscopy (SEM) provides the evidence of the formation of silver nano-particles (SNPs), which are formed as a consequence of melting and thermal decomposition of AgNO3. Absorption spectra of the samples in the visible-to-near-infrared spectral range reveal the presence of bands centered at 410 nm associated with the SNP-plasmon resonance, and at 976 nm due to the Yb3+. Under 916-nm laser-diode pumping, the effect of the SNP reflects that: i) the fluorescence in the 950-nm–1150-nm spectral range is strongly enhanced(∼ 30 times), while the fluorescence decay time associated with the 2F5/2 → 2F7/2 transition of Yb3+ increases 25%, and ii) the basic lasing properties (saturation pumping intensity, the emission and absorption cross sections) are substantially improved.

Flavin adenine dinucleotide structural motifs: from solution to gas phase.

Flavin adenine dinucleotide (FAD) is involved in important metabolic reactions where the biological function is intrinsically related to changes in conformation. In the present work, FAD conformational changes were studied in solution and in gas phase by measuring the fluorescence decay time and ion-neutral collision cross sections (CCS, in a trapped ion mobility spectrometer, TIMS) as a function of the solvent conditions (i.e., organic content) and gas-phase collisional partner (i.e., N2 doped with organic molecules). Changes in the fluorescence decay suggest that FAD can exist in four conformations in solution, where the abundance of the extended conformations increases with the organic content. TIMS-MS experiments showed that FAD can exist in the gas phase as deprotonated (M = C27H31N9O15P2) and protonated forms (M = C27H33N9O15P2) and that multiple conformations (up to 12) can be observed as a function of the starting solution for the [M + H]+ and [M + Na]+molecular ions. In addition, changes in the relative abundances of the gas-phase structures were observed from a “stack” to a “close” conformation when organic molecules were introduced in the TIMS cell as collision partners. Candidate structures optimized at the DFT/B3LYP/6-31G(d,p) were proposed for each IMS band, and results showed that the most abundant IMS band corresponds to the most stable candidate structure. Solution and gas-phase experiments suggest that the driving force that stabilizes the different conformations is based on the interaction of the adenine and isoalloxazine rings that can be tailored by the “solvation” effect created with the organic molecules.

Optical and thermal characterization of microcrystalline Na3RE(PO4)2:Yb orthophosphates synthesized by Pechini method (RE = Y, La, Gd)

Na3RE(PO4)2 orthophosphates (RE=Y, La, Gd) doped with Yb3+ ions were synthesized by Pechini method and characterized by differential scanning calorimetry/thermogravimetric analysis, X-ray diffraction, IR and Raman spectroscopies as well as optical techniques (emission spectra and fluorescence decay time). All measurements, apart from DSC, were performed at room temperature. The spectroscopic properties were discussed in terms of the structure of the obtained materials. They show that lanthanum and gadolinium phosphates synthesized by Pechini method crystallize in the orthorhombic structure, similar to that of Na3Ce(PO4)2. For the first time double sodium yttrium orthophosphate has been obtained in monoclinic modification, that could be compared to one of the Na3Gd(PO4)2 polymorph. Annealing of the obtained orthophosphates at 700°C leads to grain of ∼200nm. The microstructure of the powders annealed at 700°C strongly depends on RE3+ cations.

Photobleaching of fluorescein as a probe for oxidative stress in single cells

BackgroundROS are involved in the regulation of many physiological and pathological processes. Apoptosis and necrosis are processes that are induced by changes in concentrations of Reactive Oxygen Species (ROS). This study aims to detect and quantify the cellular response to changing ROS concentrations in the scope of apoptosis and necrosis. MethodsPhotobleaching of the fluorescent substrate fluorescein is used as a probe to detect the response of individual Jurkat-T-lymphocytes and Prostate-Cancer-3(PC-3) cells to oxidative stress, induced by hydrogen peroxide (H2O2). A kinetic model is proposed to describe changes in intracellular dye quantities due to photobleaching, dye hydrolysis, influx and leakage, yielding a single time-dependent decaying exponent+constant. ResultsFluorescein photobleaching is controlled and used to detect intracellular ROS. An increase in the decay time of fluorescence of intracellular fluorescein (slow photobleaching) was measured from cells incubated with H2O2 at 50μM. At higher H2O2 concentrations a decrease in the decay time was measured (fast photobleaching), in contrast to in vitro results with fluorescein and H2O2 in phosphate buffer saline (PBS), where the addition of H2O2 decreases the decay time, regardless of the irradiation dose used. ConclusionsThe anomalous, ROS-concentration dependent reduction of the photobleaching rate in cells, as opposed to solutions, might indicate on the regulation of the activity of intracellular oxidative-stress protective mechanisms, as seen earlier with other methods. SignificanceAssessing photobleaching via the time decay of the fluorescence intensity of an ROS-sensitive fluorophore may be adapted to monitor oxidative stress or ROS-related processes in cells.

Asante Calcium Green and Asante Calcium Red--novel calcium indicators for two-photon fluorescence lifetime imaging.

For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720–900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca2+-free and Ca2+-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca2+-dependent way, unraveling in vitro dissociation constants K D of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca2+-free and Ca2+-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K D of 180 nM was determined. Thus, quantitative [Ca2+]i recordings were realized, unraveling a reversible dopamine-induced [Ca2+]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca2+ indicator dye for 2P-FLIM recordings applicable in diverse biological systems.