Time-resolved methods in biophysics. 7. Photon counting vs. analog time-resolved singlet oxygen phosphorescence detection

Grup d'Enginyeria Molecular, Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017, Barcelona, Spain.
Photochemical and Photobiological Sciences (Impact Factor: 2.94). 10/2008; 7(9):1003-10. DOI: 10.1039/b804333g
Source: PubMed

ABSTRACT Two recent advances in optoelectronics, namely novel near-IR sensitive photomultipliers and inexpensive yet powerful diode-pumped solid-state lasers working at kHz repetition rate, enable the time-resolved detection of singlet oxygen (O2(a1Deltag)) phosphorescence in photon counting mode, thereby boosting the time-resolution, sensitivity, and dynamic range of this well-established detection technique. Principles underlying this novel approach and selected examples of applications are provided in this perspective, which illustrate the advantages over the conventional analog detection mode.

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Available from: Peter Kapusta, Nov 19, 2014
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    • "To filter first and third harmonics of laser radiation, 50-cm water filter and dichroic mirrors (BK7series, Eksma Optics, Vilnius, Lithuania) were used. Near-infrared luminescence was measured perpendicularly to the excitation beam in a photon counting mode [20] "
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    ABSTRACT: Antimicrobial photodynamic therapy (PDT) is used for the eradication of pathogenic microbial cells and involves the light-excitation of dyes in the presence of O2 yielding reactive oxygen species including the hydroxyl radical (OH) and singlet oxygen ((1)O2). In order to chemically enhance PDT by the formation of longer-lived radical species, we asked whether thiocyanate (SCN(-)) could potentiate the methylene blue (MB) and light mediated killing of the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli. SCN(-) enhanced PDT (10µM MB, 5J/cm(2) 660nmhv) killing in a concentration dependent manner of S. aureus by 2.5 log10 to a maximum of 4.2 log10 at 10mM (p<0.001) and increased killing of E. coli by 3.6 log10 to a maximum of 5.0 log10 at 10mM (p<0.01). We determined that SCN(-) rapidly depleted O2 from an irradiated MB system, reacting exclusively with (1)O2, without quenching the MB excited triplet state. SCN(-) reacted with (1)O2, producing the sulfur trioxide radical anion (a sulfur-centered radical demonstrated by EPR spin-trapping). We found that MB-PDT of SCN(-) in solution produced both sulfite and cyanide anions, and that addition of each of these salts separately enhanced MB-PDT killing of bacteria. We were unable to detect EPR signals of OH, which, together with kinetic data, strongly suggests that MB, known to produce OH and (1)O2, may, under the conditions used, preferentially form (1)O2.
    Free Radical Biology and Medicine 08/2013; 65. DOI:10.1016/j.freeradbiomed.2013.08.162 · 5.71 Impact Factor
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    • "The fluorescence decays were analyzed using the PicoQuant FluoFit 4.0 data analysis software. Singlet oxygen phosphorescence was detected using a customized PicoQuant Fluotime 200 described elsewhere [24]. Singlet oxygen quantum yield (Φ Δ ) was determined from the comparison of the phosphorescence signal amplitudes of optically-matched solutions of the sample to that of a reference, in this case, 5,10,15,20-tetrakis (N-methyl-4-pyridylium)-21H,23H-porphine (TMPyP) (Φ Δ, H2O = 0.74, [25]). "
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    ABSTRACT: BACKGROUND: The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments. METHODS: Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence. RESULTS: m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments. CONCLUSIONS: Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism. GENERAL SIGNIFICANCE: These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.
    Biochimica et Biophysica Acta 05/2013; 1830(10). DOI:10.1016/j.bbagen.2013.05.021 · 4.66 Impact Factor
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    • "To filter first and third harmonics of laser radiation, 50-cm water filter and dichroic mirrors (BK7series, Eksma Optics, Vilnius, Lithuania) were used. Near-infrared luminescence was measured perpendicularly to the excitation beam in a photon counting mode [20] "
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