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
Singlet oxygen is a highly reactive species which is involved in a number of processes, including photodynamic therapy of cancer. Its very weak near-infrared emission makes imaging of singlet oxygen in biological systems a long-term challenge. We address this challenge by introducing Singlet Oxygen Feedback Delayed Fluorescence (SOFDF) as a novel modality for semi-direct microscopic time-resolved wide-field imaging of singlet oxygen in biological systems. SOFDF has been investigated in individual fibroblast cells incubated with a well-known photosensitizer aluminium phthalocyanine tetrasulfonate. The SOFDF emission from the cells is several orders of magnitude stronger and much more readily detectable than the very weak near-infrared phosphorescence of singlet oxygen. Moreover, the analysis of SOFDF kinetics enables us to estimate the lifetimes of the involved excited states. Real-time SOFDF images with micrometer spatial resolution and submicrosecond temporal-resolution have been recorded. Interestingly, a steep decrease in the SOFDF intensity after the photodynamically induced release of a photosensitizer from lysosomes has been demonstrated. This effect could be potentially employed as a valuable diagnostic tool for monitoring and dosimetry in photodynamic therapy.
- MeSH
- 3T3 Cells MeSH
- Time Factors MeSH
- Fibroblasts chemistry cytology MeSH
- Fluorescence * MeSH
- Microscopy, Fluorescence MeSH
- Photochemotherapy MeSH
- Photosensitizing Agents chemistry MeSH
- Indoles chemistry MeSH
- Cells, Cultured MeSH
- Mice MeSH
- Optical Imaging * MeSH
- Organometallic Compounds chemistry MeSH
- Singlet Oxygen analysis chemistry MeSH
- Cell Survival MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
