Liquid chromatography with fluorescence detection has been used in analyses demanding high sensitivity and selectivity. As majority systems rely on reversed-phase columns with water being the main component of the mobile phase, fluorescent compounds with emission maxima higher than 500 nm might be dynamically quenched. A simple replacement of H2O with D2O enhanced the sensitivity for selected compounds by 10-200%. Affected compounds included an anti-cancer drug doxorubicin, a luminescent probe fluorescein, and naturally occurring forms of vitamin B2. Similar levels of enhancement were obtained by fluorescence spectrometry. Such simple yet effective approach may greatly improve HPLC analyses coupled to fluorescence detection.
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
