Fluorescence Lifetime Methods Research Articles

Detection of Multiple-Heated Edible Oil Based on Fluorescence Spectroscopy and Lifetime Method

Detection of Multiple-Heated Edible Oil Based on Fluorescence Spectroscopy and Lifetime Method

Effect of BaO on the structure and luminescence properties of Eu3+doped GSRG glass

Eu3+ doped GeO2-SiO2-RO (R=Mg, Ca, Ba)-Ga2O3 (GSRG) glass was prepared by high temperature melting method. Through XRD, absorption spectrum, fluorescence spectrum, fluorescence lifetime, infrared spectrum and other test methods, the influence of BaO on the structure and luminescence properties of Eu3+ doped GSRG glass was studied. The results show that BaO in RO (R=Mg, Ca, Ba) can make Eu3+ doped GSRG glass have higher luminous intensity. As BaO gradually replaces MgO, the luminous intensity of glass first increases and then decreases with the increase of BaO concentration. The luminous intensity reaches its peak when the BaO concentration reaches 25 mol%. Meanwhile, the transition from germanium-oxygen tetrahedron [GeO4] to germanium-oxygen octahedron [GeO6] appeared in the glass structure, again by [GeO6] shift back to [GeO4]. The CIE chromaticity coordinates show that the color temperature of prepared glass is 1700–1800 K, and the color purity is above 98%.

Study of Interaction Between Bovine Serum Albumin and Dolutegravir Intermediate: Fluorescence and Molecular Docking Analysis

Novel (4R,12aS)-7-methoxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido-[1',2':-4,5]-pyrazino[2,1-b][1,3]oxazine-9-carboxylic acid (L) was synthesized and characterised. The interaction between bovine serum albumin (BSA) with L was scrutinized by steady-state fluorescence spectroscopy, fluorescence anisotropy, fluorescence lifetime, and molecular docking methods. The fluorescence titration experiments of BSA resulted in fluorescence quenching with the incremental addition of L. The conformational binding of L to BSA has been investigated by molecular docking analysis. The molecular probe's best conformation showed the affinity as free binding energy release of -7.93 Kcal/mol. The docking analysis confirms that ligand binds in the near vicinity of TRP-213 in the binding pocket of subdomain IIA.

Na2YMg2(VO4)3:Er3+,Yb3+ phosphors: Up-conversion and optical thermometry

Herein, Na2YMg2(VO4)3:Er3+,Yb3+ phosphors are reported for up-conversion (UC) luminescent material as optical thermometry relied on fluorescence intensity ratio (FIR) and fluorescence lifetime. Their structures, UC luminescent properties and temperature sensing properties have been systematically investigated. Heavy doping of Er3+ and Yb3+ ions with maximal content of 59% is realized, which is beneficial to obtain highly efficient UC luminescence. Excited by 980 nm laser, Er3+ and Er3+,Yb3+ doped phosphors give 525, 550 and 660 nm emissions, which are originated from 2H11/2, 4S3/2 and 4F9/2 → 4I15/2 transitions of Er3+ ions, respectively. The reasonable UC mechanism is discussed in detail. What's more, the UC emissions of all samples dominate in green area from thermally coupled 2H11/2/4S3/2 levels of Er3+ ions, which is helpful to obtain better temperature sensing performances. And the maximal relative sensitivities of FIR and fluorescence lifetime methods reach 1.104 %K−1 (303 K) and 0.087 %K−1 (412 K), respectively. All results manifest that Na2YMg2(VO4)3:Er3+,Yb3+ phosphors are good potential alternative for optical temperature sensors.

Study on the interaction between pyridoxal and CopC by multi-spectroscopy and docking methods.

The interaction between pyridoxal hydrochloride (HQ) and apoCopC was investigated using Fourier transform infrared spectroscopy (FTIR), isothermal titration calorimetry (ITC), circular dichroism (CD), fluorescence spectroscopy, three-dimensional (3D) fluorescence spectroscopy, fluorescence lifetime, TNS fluorescence and docking methods. FTIR, CD, TNS fluorescence and fluorescence lifetime experiments suggested that the apoCopC conformation was altered by HQ with an increase in the random coil content and a reduction in the β-sheet content. In addition, the data from fluorescence spectroscopy, 3D fluorescence spectroscopy and molecular docking revealed that the binding site of HQ was located in the hydrophobic area of apoCopC, and a redshift of the HQ fluorescence spectra was observed. Furthermore, ITC and fluorescence quenching data manifested that the binding ratio of HQ and apoCopC was 1:1, and the forming constant was calculated to be (7.06 ± 0.21) × 105 M-1. The thermodynamic parameters ΔH and ΔS suggested that the formation of a CopC-HQ complex depended on the hydrophobic force. Furthermore, the average binding distance between tryptophan in apoCopC and HQ was determined by means of Förster non-radioactive resonance energy transfer and molecular docking. The results agreed well with each other. As a redox switch in the modulation of copper, the interaction of apoCopC with small molecules will affect the action of the redox switch. These findings could provide useful information to illustrate the copper regulation mechanism.

Using Fractional Intensities of Time-resolved Fluorescence to Sensitively Quantify NADH/NAD+ with Genetically Encoded Fluorescent Biosensors

In this paper, we propose a novel and sensitive ratiometric analysis method that uses the fractional intensities of time-resolved fluorescence of genetically encoded fluorescent NADH/NAD+ biosensors, Peredox, SoNar, and Frex. When the conformations of the biosensors change upon NADH/NAD+ binding, the fractional intensities (αiτi) have opposite changing trends. Their ratios could be exploited to quantify NADH/NAD+ levels with a larger dynamic range and higher resolution versus commonly used fluorescence intensity and lifetime methods. Moreover, only one excitation and one emission wavelength are required for this ratiometric measurement. This eliminates problems of traditional excitation-ratiometric and emission-ratiometric methods. This method could be used to simplify the design and achieve highly sensitive analyte quantification of genetically encoded fluorescent biosensors. Wide potential applications could be developed for imaging live cell metabolism based on this new method.

Dual mode monitoring probe for mitochondrial viscosity in single cell

Viscosity strongly influences intracellular transportation of mass and signal, and although bulk viscosity can be well measured with macroscopic samples quantities, microscale measurements in live cells still remain a challenge. By introducing a CHO group into the meso-position of the pentamethine chain, a thiazole-Cy5 dye based two-photon fluorescence probe, Mito-V, was developed. It exhibited a selective response to solution viscosity by fluorescence ratio and lifetime methods. More importantly, Mito-V can localize in mitochondria and quantitatively image its viscosity changes by the dual fluorescence mode during the apoptosis.

Intracellular Calcium Ion Concentration Measurement Using a Phase-modulation Fluorescence Lifetime Method with Compensation for Phase Shift due to the Presence of Proteins

The calcium ion concentration in cells was measured by a phase-modulation fluorescence lifetime method with compensation for proteins. A high-accuracy measurement of the calcium ion concentration is best realized by fluorescence lifetime measurements, because the fluorescence lifetime is independent of the fluorescence intensity. The fluorescence intensity is easily varied by the scattering of excitation and emission light in cells, photobleaching, the concentration of fluorochromes, and wavelength dispersion of optical elements. A phase-modulation fluorescence lifetime measurement, however, provides high accuracy and precision, and can measure not only the calcium ion concentration, but also other ion concentrations, such as that of magnesium, sodium, and potassium. We have examined the phase-modulation fluorescence lifetime shift using protein compensation in cells, and have measured the calcium ion concentration in cells stimulated with bradykinin.

Photophysics of Untethered ZnTPP–Fullerene Complexes in Solution

The spectroscopy and dynamic behavior of the self-assembled, Soret-excited zinc tetraphenylporphyrin (ZnTPP) plus fullerene (C(60)) model system in solution has been examined using steady state fluorescence quenching, nanosecond time-correlated single photon counting, picosecond fluorescence upconversion, and picosecond transient absorption methods. Evidence of ground state complexation is presented. Steady-state quenching of the S(2) and S(1) fluorescence of ZnTPP by C(60) reveals that the quenching processes only occur in the excited complexes, are ultrafast, and proceed at different rates in the two states. Only uncomplexed ZnTPP is observed by fluorescence lifetime methods; the locally excited complexes are either dark or, more likely, rapidly relax to products that do not radiate strongly. Both short-range (Dexter) energy transfer and electron transfer relaxation mechanisms are evaluated. Picosecond transient absorption data obtained from the subtle differences between the spectra of Soret-excited ZnTPP with and without a large excess of added C(60) reveal the formation, on a subpicosecond time scale, of relatively long-lived charge-separated species. Soret excitation of ZnTPP···C(60) does not produce a quantitative yield of species in the lower S(1) excited state.

Supermolecular System of Perylene Bismide Dyes Incorporated into Si-HMS for Solar Energy Conversion

Si-HMS were synthesized and characterized by XRD and N2 adsorption isotherm. Perylene derivative has been incorporated into its channels by impregnation method. And the resulting composite was characterized by absorption, fluorescence emission and fluorescence lifetime methods. The results indicated that perylene molecules in HMS presented spectra with broad bands, loss of vibrational structure, red shift of maxima and elongated lifetime.

Fluorescence lifetime study of ECFP/EYFP labeled MBP in Förster resonance energy transfer

This paper studies the conformational change of the binding protein by a fluorescence lifetime method. As a model protein, maltose binding protein (MBP) where enhanced cyan protein (ECFP) and enhanced yellow fluorescent protein (EYFP) were genetically fused to act as a donor and an acceptor in Forster resonance energy transfer (FRET) was used. The ECFP and the EYFP were linked to the C-terminal and N-terminal regions of the MBP, respectively. In order to investigate the conformational change of the MBP, the lifetime of the ECFP, which acts as a donor in the ECFP:MBP:EYFP fusion protein, was analyzed during the FRET process. We observed that two lifetime components exist when the ECFP is linked to the MBP and that the lifetime of the ECFP is shortened when ECFP:MBP:EYFP protein undergoes a conformational change as a result of the maltose binding. In addition, we observed that the lifetime of the donor is gradually shorter in the ECFP:MBP:EYFP fusion protein as the maltose concentration increases. By a lifetime analysis and simulation study, we found that the participant rate of the ECFP:MBP:EYFP protein in FRET is the main cause of the donor lifetime shortening in relation to the increase of the maltose concentration.

Complimentary Fluorescence Lifetime and Raman Microscopy Methods for Cellular Imaging

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Partition of tocopheryl glucopyranoside into liposome membranes studied by fluorescence methods

Vitamin E is poorly soluble in aqueous solutions. Enhanced physiological activity is expected from synthesized glycosidic tocopherol derivatives. We investigated binding, location and interactions of newly synthesized DL-α-tocopheryl β D glucopyranoside (II) in phosphatidylcholine liposomes using fluorescence emission, anisotropy and lifetime methods. In liposomes emission maximum and fluorescence lifetime of glucoside were similar to those observed in methanol. High fluorescence anisotropy value indicates that tocopheryl glucoside is located in restricted mobility region of the membrane. Thermodynamic calculation indicated efficient partition of (II) into membrane. The energy minimization calculations of electrostatic potential distribution of (II) and solvation energies performed with Gaussian program confirmed strong affinity of glucosidic moiety for ionic interactions and supported proposed model of interactions. The all obtained data indicate that DL-α-tocopheryl β-glucoside is embedded into the membrane interior whereas sugar moiety protrudes above the water/lipid interface of the membrane surface.

Resolution of (+)-cinchonine and (−)-cinchonidine by phase-modulation fluorescence spectroscopy

The different fluorescence lifetime profiles of the optical isomers (+)-cinchonine (CN) and (−)-cinchonidine (CD) have been used to resolve their mixtures, on the basis of phase and modulation fluorescence lifetime measurements. To resolve mixtures of these compounds two distinct approaches have been used: simultaneous equations from phase-resolved and modulation-based fluorescence lifetime measurements. Results indicate that frequency domain fluorimetry is useful to resolve stereomeric compounds for determination without physical separation. The precision of the measurements gives RSD values of 5.2% and 4.0% for CD and CN, respectively. Detection limits with the modulation fluorescence lifetime method were 11.3 and 17.2 μM for CD and CN, respectively.

Quantitative measurement of protease activity with correction of probe delivery and tissue absorption effects

Proteases play important roles in a variety of pathologies from heart disease to cancer. Quantitative measurement of protease activity is possible using a novel spectrally matched dual fluorophore probe and a small animal lifetime imager. The recorded fluorescence from an activatable fluorophore, one that changes its fluorescent amplitude after biological target interaction, is also influenced by other factors including imaging probe delivery and optical tissue absorption of excitation and emission light. Fluorescence from a second spectrally matched constant (non-activatable) fluorophore on each nanoparticle platform can be used to correct for both probe delivery and tissue absorption. The fluorescence from each fluorophore is separated using fluorescence lifetime methods.

In vivo fluorescence lifetime tomography

Local molecular and physiological processes can be imaged in vivo through perturbations in the fluorescence lifetime (FLT) of optical imaging agents. In addition to providing functional information, FLT methods can quantify specific molecular events and multiplex diagnostic and prognostic information. We have developed a fluorescence lifetime diffuse optical tomography (DOT) system for in vivo preclinical imaging. Data is captured using a time-resolved intensified charge coupled device (ICCD) system to measure fluorescence excitation and emission in the time domain. Data is then converted to the frequency domain, and we simultaneously reconstruct images of yield and lifetime using an extension to the normalized Born approach. By using differential phase measurements, we demonstrate DOT imaging of short lifetimes (from 350 ps) with high precision (+/-5 ps). Furthermore, this system retains the efficiency, speed, and flexibility of transmission geometry DOT. We demonstrate feasibility of FLT-DOT through a progressive series of experiments. Lifetime range and repeatability are first measured in phantoms. Imaging of subcutaneous implants then verifies the FLT-DOT approach in vivo in the presence of inhomogeneous optical properties. Use in a common research scenario is ultimately demonstrated by imaging accumulation of a targeted near-infrared (NIR) fluorescent-labeled peptide probe (cypate-RGD) in a mouse with a subcutaneous tumor.

Frequency-Domain Fluorescence Lifetime Measurements via Frequency Segmentation and Recombination as Applied to Pyrene with Dissolved Humic Materials

In this study, the association behavior of pyrene with different dissolved humic materials (DHM) was investigated utilizing the recently developed segmented frequency-domain fluorescence lifetime method. The humic materials involved in this study consisted of three commercially available International Humic Substances Society standards (Suwannee River fulvic acid reference, SRFAR, Leonardite humic acid standard, LHAS, and Florida peat humic acid standard, FPHAS), the peat derived Amherst humic acid (AHA), and a chemically bleached Amherst humic acid (BAHA). It was found that the three commercial humic materials displayed three lifetime components, while both Amherst samples displayed only two lifetime components. In addition, it was found that the chemical bleaching procedure preferentially removed red wavelength emitting fluorophores from AHA. In regards to pyrene association with the DHM, different behavior was found for all commercially available humics, while AHA and BAHA, which displayed strikingly similar behavior in terms of fluorescence lifetimes. It was also found that there was an enhancement of pyrene's measured lifetime (combined with a decrease in pyrene emission) in the presence of FPHAS. The implications of this long lifetime are discussed in terms of (1) quenching mechanism and (2) use of the fluorescence quenching method used to determine the binding of compounds to DHM.

On-chip, time-correlated, fluorescence lifetime extraction algorithms and error analysis

A new, simple, and hardware-only fluorescence-lifetime-imaging microscopy (FLIM) is proposed to implement on-chip lifetime extractions, and their signal-to-noise-ratio based on statistics theory is also deduced. The results are compared with Monte Carlo simulations, giving good agreement. Compared with the commonly used iterative least-squares method or the maximum-likelihood-estimation- (MLE-) based, general purpose FLIM analysis software, our algorithm offers direct calculation of fluorescence lifetime based on the collected photon counts stored in on-chip counters and therefore delivers faster analysis for real-time applications, such as clinical diagnosis. Error analysis considering timing jitter based on statistics theory is carried out for the proposed algorithms and is also compared with MLE to obtain optimized channel width or measurement window and bit resolution of the time-to-digital converters for a given accuracy. A multi-exponential, pipelined fluorescence lifetime method based on the proposed algorithms is also introduced. The performance of the proposed methods has been tested on mono-exponential and four-exponential decay experimental data.

Probing DNA Hybridization in Homogeneous Solution and at Interfaces via Measurement of the Intrinsic Fluorescence Decay Time of a Single Label

The hybridization of DNA oligomers including molecular beacons can be detected by measurement of either the decay time or the intensity of a single fluorescent label attached to the end of the respective oligonucleotide. The method works both in solution and solid phase and can distinguish between fully complementary and mismatch sequences as demonstrated for a 15-mer oligonucleotide and a 25-mer molecular beacon. The fluorescence lifetime method is advantageous in (a) requiring a single label (and therefore a single labeling step) only; and (b), being based on measurement of a self-referenced magnitude that is hardly affected by parameters such as fluctuations in light intensity that make measurement of intensity more prone to interferences.

Limit of accuracy for fluorescence lifetime temperature sensing

Limit of accuracy for the fluorescence lifetime (FL) temperature-sensing method performed and marked that at lower temperature (<400 K) it works well but for higher temperatures FL method does not found suitable. The non-radiative relaxation rate is noted to vary with increase in the temperature.