The phasor method of treating fluorescence lifetime data provides a facile and convenient approach to characterize lifetime heterogeneity and to detect the presence of excited state reactions such as solvent relaxation and Förster resonance energy transfer. The method uses a plot of M sin(Φ) versus M cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. A principal advantage of the phasor method is that it provides a model-less approach to time-resolved data amenable to visual inspection. Although the phasor approach has been recently applied to fluorescence lifetime imaging microscopy, it has not been used extensively for cuvette studies. In the current study, we explore the applications of the method to in vitro samples. The phasors of binary and ternary mixtures of fluorescent dyes demonstrate the utility of the method for investigating complex mixtures. Data from excited state reactions, such as dipolar relaxation in membrane and protein systems and also energy transfer from the tryptophan residue to the chromophore in enhanced green fluorescent protein, are also presented.
- MeSH
- Apoproteins chemistry MeSH
- Time Factors MeSH
- Fluorescent Dyes chemistry MeSH
- Spectrometry, Fluorescence methods MeSH
- Myoglobin chemistry MeSH
- Naphthalenesulfonates chemistry MeSH
- Fluorescence Resonance Energy Transfer MeSH
- Solvents chemistry MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
We present a method for the determination of the lignan enterolactone in plasma (serum). This compound, produced by intestinal bacteria from matairesinol and secoisolariciresinol in fiber-rich food, is a biomarker related to the intake of a healthy diet. The method is based on time-resolved fluoroimmunoassay using a europium chelate as a label. After synthesis of 5'-O-carboxymethoxyenterolactone the compound is coupled to bovine serum albumin and then used as antigen in immunization of rabbits. The tracer with the europium chelate is synthesized using the same 5'-derivative of enterolactone. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). No antiserum cross-reactivity with available lignans, isoflavonoids, or flavonoids could be detected. The intraassay and interassay coefficients of variation at different concentrations vary 4.6-6.0 and 5.5-9.9, respectively. The working range of the assay is 1.5-540 nmol/liter. We measured enterolactone in serum/plasma of 224 Finnish subjects: 98.8% of the subjects had values <100 nmol/liter, 38.0% had 20-39.9 nmol/liter, and 34.4% had <20 nmol/liter. Copyright 1998 Academic Press.
- MeSH
- Fluoroimmunoassay * methods MeSH
- 4-Butyrolactone * analogs & derivatives blood MeSH
- Humans MeSH
- Lignans * blood MeSH
- Gas Chromatography-Mass Spectrometry MeSH
- Sensitivity and Specificity MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
INTRODUCTION: Competitive binding assays can be used to decipher not only the binding kinetics of studied ligands but also the binding site preference. Such assays are an essential step in the characterization of radioligands. However, the currently used competition assays require high concentrations of usually expensive ligands and still provide only binding site preference. By employing the time-resolved competition assay presented in this paper, binding characteristics including binding site preference can be obtained using less ligand. METHODS: To demonstrate the appropriateness of the time-resolved competition assay, we developed an assay in which the ligand binding was interrupted with a competitor. Experiments were performed on human carcinoma cell lines expressing epidermal growth factor receptor (EGFR). The targeting of the receptor was performed with radio-iodinated epidermal growth factor (EGF). The employed competitors involved either natural ligand transforming growth factor alpha (TGF-α) or anti-EGFR antibodies cetuximab and panitumumab targeting the same EGFR domain. RESULTS: Radio-iodinated EGF bound to EGFR was displaced with either low concentrations of cetuximab or high concentrations of panitumumab. In the case of TGF-α, we observed no competitive displacement of bound EGF at either high or low concentrations. When comparing the time-resolved competition assay with a manual competition assay, the resulting data of measured inhibition constants were in agreement. DISCUSSION: The results summarised in this study confirm the appropriateness of the time-resolved competition assay for assessing ligand binding properties. The assay has the potential to complement or replace conventional competition assays for determining binding site preference in the future.
- MeSH
- Time Factors MeSH
- Epidermal Growth Factor chemistry metabolism MeSH
- ErbB Receptors antagonists & inhibitors chemistry metabolism MeSH
- Antibodies, Monoclonal, Humanized chemistry pharmacology MeSH
- Binding, Competitive drug effects MeSH
- Humans MeSH
- Ligands MeSH
- Antibodies, Monoclonal chemistry pharmacology MeSH
- Tumor Cells, Cultured MeSH
- Substrate Specificity MeSH
- Transforming Growth Factor alpha chemistry metabolism MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Glycan biochips and biosensors are potentially important tools for detection of glycan – protein interactions. Among several applications they can be used for rapid and precise diagnosis of various diseases and infections. Two major detection techniques available for construction of glycan biochips and biosensors – fluorescent labelled and label-free methods are discussed.
- MeSH
- Bacterial Infections * diagnosis MeSH
- Biosensing Techniques * methods instrumentation utilization MeSH
- Chemistry Techniques, Analytical MeSH
- Diagnostic Imaging methods utilization MeSH
- DNA Probes * MeSH
- Fluoroimmunoassay methods utilization MeSH
- Humans MeSH
- Optical Imaging methods utilization MeSH
- Polysaccharides analysis chemistry MeSH
- Surface Plasmon Resonance methods instrumentation utilization MeSH
- Proteins chemistry MeSH
- Spectrum Analysis methods utilization MeSH
- Virus Diseases * diagnosis MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Fluorescence methods are versatile tools for obtaining dynamic and topological information about biomembranes because the molecular interactions taking place in lipid membranes frequently occur on the same timescale as fluorescence emission. The fluorescence intensity decay, in particular, is a powerful reporter of the molecular environment of a fluorophore. The fluorescence lifetime can be sensitive to the local polarity, hydration, viscosity, and/or presence of fluorescence quenchers/energy acceptors within several nanometers of the vicinity of a fluorophore. Illustrative examples of how time-resolved fluorescence measurements can provide more valuable and detailed information about a system than the time-integrated (steady-state) approach will be presented in this review: 1), determination of membrane polarity and mobility using time-dependent spectral shifts; 2), identification of submicroscopic domains by fluorescence lifetime imaging microscopy; 3), elucidation of membrane leakage mechanisms from dye self-quenching assays; and 4), evaluation of nanodomain sizes by time-resolved Förster resonance energy transfer measurements.
Time-resolved X-ray crystallography experiments were first performed in the 1980s, yet they remained a niche technique for decades. With the recent advent of X-ray free electron laser (XFEL) sources and serial crystallographic techniques, time-resolved crystallography has received renewed interest and has become more accessible to a wider user base. Despite this, time-resolved structures represent < 1 % of models deposited in the world-wide Protein Data Bank, indicating that the tools and techniques currently available require further development before such experiments can become truly routine. In this chapter, we demonstrate how applying data multiplexing to time-resolved crystallography can enhance the achievable time resolution at moderately intense monochromatic X-ray sources, ranging from synchrotrons to bench-top sources. We discuss the principles of multiplexing, where this technique may be advantageous, potential pitfalls, and experimental design considerations.
Experimental and theoretical foundations for femtosecond time-resolved circular dichroism (TRCD) spectroscopy of excitonic systems are presented. In this method, the system is pumped with linearly polarized light and the signal is defined as the difference between the transient absorption spectrum probed with left and with right circularly polarized light. We present a new experimental setup with a polarization grating as key element to generate circularly polarized pulses. Herein the positive (negative) first order of the diffracted light is left-(right-)circularly polarized and serves as a probe pulse in a TRCD experiment. The grating is capable of transferring ultrashort broadband pulses ranging from 470 nm to 720 nm into two separate beams with opposite ellipticity. By applying a specific chopping scheme we can switch between left and right circular polarizations and detect transient absorption (TA) and TRCD spectra on a shot-to-shot basis simultaneously. We perform experiments on a squaraine polymer, investigating excitonic dynamics, and we develop a general theory for TRCD experiments of excitonically coupled systems that we then apply to describe the experimental data in this particular example. At a magic angle of 54.7° between the pump-pulse polarization and the propagation direction of the probe pulse, the TRCD and TA signals become particularly simple to analyze, since the orientational average over random orientations of complexes factorizes into that of the interaction with the pump and the probe pulse, and the intrinsic electric quadrupole contributions to the TRCD signal average to zero for isotropic samples. Application of exciton theory to linear absorption and to linear circular dichroism spectra of squaraine polymers reveals the presence of two fractions of polymer conformations, a dominant helical conformation with close interpigment distances that are suggested to lead to short-range contributions to site energy shifts and excitonic couplings of the squaraine molecules, and a fraction of unfolded random coils. Theory demonstrates that TRCD spectra of selectively excited helices can resolve state populations that are practically invisible in TA spectroscopy due to the small dipole strength of these states. A qualitative interpretation of TRCD and TA spectra in the spectral window investigated experimentally is offered. The 1 ps time component found in these spectra is related to the slow part of exciton relaxation obtained between states of the helix in the low-energy half of the exciton manifold. The dominant 140 ps time constant reflects the decay of excited states to the electronic ground state.
- Publication type
- Journal Article MeSH
We describe a low-cost, easy to use binocular instrument to acquire retinal video sequences of both eyes simultaneously. After image registration, cardiac cycle-induced pulsatile light attenuation changes can be measured quantitatively with high spatial and temporal resolution. Parameters such as amplitude, pulse form, and time shift between light attenuation changes can be calculated and compared between eye sides. Deviation from inter-eye symmetry can be not only an early sign of beginning eye diseases such as glaucoma but also a sign of pathological changes in the carotid arteries; hence, this method can improve the early detection of pathological changes. Important features compared to existing monocular instruments are a narrow band light source with the wavelength close to the peak of the blood extinction, and a proportional relationship of image intensity and light intensity, which are the main requirements for quantitative evaluation.
- Publication type
- Journal Article MeSH
