Time-resolved fluorescence
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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 confocal microspectrofluorometry and fluorescence microscopy imaging were applied to monitor the cellular uptake of fluorescent-labeled oligonucleotides (ONs) delivered by a porphyrin molecule. The fate of porphyrin-ON complexes inside living cells has also been monitored. Due to intrinsic fluorescence of the porphyrin and sensitivity of its characteristics to microenvironment, multicomponent analysis of time-resolved fluorescence provides unique information about stability of the porphyrin-ON complexes, ON interactions with their target sequences, and ON and porphyrin distributions after delivery inside the cells. Time-resolved confocal microspectrofluorometry indeed delivers additional information compared with fluorescence confocal microscopy imaging widely employed to study ON uptake.
- MeSH
- antisense oligonukleotidy chemie MeSH
- buněčné jádro metabolismus MeSH
- buňky 3T3 MeSH
- časové faktory MeSH
- financování organizované MeSH
- fluorescence MeSH
- fluorescenční mikroskopie metody přístrojové vybavení MeSH
- kationty MeSH
- konfokální mikroskopie metody přístrojové vybavení MeSH
- lékové transportní systémy MeSH
- melanom experimentální MeSH
- myši MeSH
- oligonukleotidy chemie MeSH
- porfyriny chemie MeSH
- senzitivita a specificita MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
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
- apoproteiny chemie MeSH
- časové faktory MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční spektrometrie metody MeSH
- myoglobin chemie MeSH
- naftalensulfonany chemie MeSH
- rezonanční přenos fluorescenční energie MeSH
- rozpouštědla chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural 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
- buňky 3T3 MeSH
- časové faktory MeSH
- fibroblasty chemie cytologie MeSH
- fluorescence * MeSH
- fluorescenční mikroskopie MeSH
- fotochemoterapie MeSH
- fotosenzibilizující látky chemie MeSH
- indoly chemie MeSH
- kultivované buňky MeSH
- myši MeSH
- optické zobrazování * MeSH
- organokovové sloučeniny chemie MeSH
- singletový kyslík analýza chemie MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Time-resolved microspectrofluorimetry and fluorescence microscopy imaging-two complementary fluorescence techniques-provide important information about the intracellular distribution, level of uptake and binding/interactions inside living cell of the labeled molecule of interest. They were employed to monitor the "fate" of AS1411 aptamer labeled by ATTO 425 in human living cells. Confocal microspectrofluorimeter adapted for time-resolved intracellular fluorescence measurements by using a phase-modulation principle with homodyne data acquisition was employed to obtain emission spectra and to determine fluorescence lifetimes in U-87 MG tumor brain cells and Hs68 non-tumor foreskin cells. Acquired spectra from both the intracellular space and the reference solutions were treated to observe the aptamer localization and its interaction with biological structures inside the living cell. The emission spectra and the maximum emission wavelengths coming from the cells are practically identical, however significant lifetime lengthening was observed for tumor cell line in comparison to non-tumor one.
- MeSH
- aptamery nukleotidové metabolismus MeSH
- časové faktory MeSH
- fluorescenční mikroskopie metody MeSH
- fluorescenční spektrometrie metody MeSH
- intracelulární prostor genetika metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- oligodeoxyribonukleotidy genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The present work provides a proof-of-concept that the singlet oxygen-sensitized delayed fluorescence (SOSDF) can be detected from individual living mammalian cells in a time-resolved microscopy experiment. To this end, 3T3 mouse fibroblasts incubated with 100 μM TPPS4 or TMPyP were used and the microsecond kinetics of the delayed fluorescence (DF) were recorded. The analysis revealed that SOSDF is the major component of the overall DF signal. The microscopy approach enables precise control of experimental conditions - the DF kinetics are clearly influenced by the presence of the (1)O2 quencher (sodium azide), H2O/D2O exchange, and the oxygen concentration. Analysis of SOSDF kinetics, which was reconstructed as a difference DF kinetics between the unquenched and the NaN3-quenched samples, provides a cellular (1)O2 lifetime of τΔ = 1-2 μs and a TPPS4 triplet lifetime of τT = 22 ± 5 μs in agreement with previously published values. The short SOSDF acquisition times, typically in the range of tens of seconds, enable us to study the dynamic cellular processes. It is shown that SOSDF lifetimes increase during PDT-like treatment, which may provide valuable information about changes of the intracellular microenvironment. SOSDF is proposed and evaluated as an alternative tool for (1)O2 detection in biological systems.
- MeSH
- analýza jednotlivých buněk přístrojové vybavení metody MeSH
- azid sodný chemie MeSH
- buňky 3T3 MeSH
- design vybavení MeSH
- fibroblasty chemie MeSH
- fluorescence * MeSH
- kinetika MeSH
- kyslík chemie MeSH
- mikroskopie přístrojové vybavení metody MeSH
- myši MeSH
- oxid deuteria chemie MeSH
- singletový kyslík chemie MeSH
- voda chemie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
