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1.
Molecules ; 24(8)2019 Apr 24.
Article in English | MEDLINE | ID: mdl-31022940

ABSTRACT

Light-driven phase change materials (PCMs) have received significant attention due to their capacity to convert visible light into thermal energy, storing it as latent heat. However, continuous photo-thermal conversion can cause the PCMs to reach high thermal equilibrium temperatures after phase transition. In our study, a novel light-driven phase change material system with temperature-control properties was constructed using a thermochromic compound. Thermochromic phase change materials (TC-PCMs) were prepared by introducing 2-anilino-6-dibutylamino-3-methylfluoran (ODB-2) and bisphenol A (BPA) into 1-hexadecanol (1-HD) in various proportions. Photo-thermal conversion performance was investigated with solar radiation (low power of 0.09 W/cm2) and a xenon lamp (at a high power of 0.14 W/cm2). The TC-PCMs showed a low equilibrium temperature due to variations in absorbance. Specifically, the temperature of TC-PCM180 (ODB-2, bisphenol A and 1-HD ratio 1:2:180) could stabilize at 54 °C approximately. TC-PCMs exhibited reversibility and repeatability after 20 irradiation and cooling cycles.


Subject(s)
Aniline Compounds/chemical synthesis , Benzhydryl Compounds/chemical synthesis , Fatty Alcohols/chemical synthesis , Fluoresceins/chemical synthesis , Phenols/chemical synthesis , Aniline Compounds/chemistry , Aniline Compounds/radiation effects , Benzhydryl Compounds/chemistry , Benzhydryl Compounds/radiation effects , Fatty Alcohols/chemistry , Fatty Alcohols/radiation effects , Fluoresceins/chemistry , Fluoresceins/radiation effects , Hot Temperature , Light , Phase Transition/radiation effects , Phenols/chemistry , Phenols/radiation effects , Temperature
2.
Analyst ; 144(5): 1704-1710, 2019 Feb 25.
Article in English | MEDLINE | ID: mdl-30657475

ABSTRACT

The superoxide anion (O2˙-) plays a crucial role in several physiological processes and many human diseases. Developing new methods for O2˙- detection in biological systems is very important. A FRET-based two-photon (TP) fluorescent probe with a ratiometric signal, TFR-O, was developed. A naphthalene derivative based TP fluorescent group was selected as the energy donor group, and a rhodol fluorescent group was chosen as the energy acceptor; the trifluoromethanesulfonate group was chosen as the recognition moiety. After reacting with O2˙-, the recognition moiety was removed and the fluorophore was released, leading to a fluorescence intensity decrease at the wavelength of 425 nm and a significant enhancement of the fluorescence intensity at 550 nm. The fluorescence intensity ratio between 550 and 425 nm (I550/I425) varied from 0.15 to 6.72, with the O2˙- concentration increasing from 0 to 50 µM. The detection limit of the TFR-O was 83 nM. Moreover, TFR-O was applied for detecting and imaging O2˙- in cells and liver tissues.


Subject(s)
Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Mesylates/chemistry , Naphthalenes/chemistry , Superoxides/analysis , Animals , Fluoresceins/chemical synthesis , Fluoresceins/radiation effects , Fluoresceins/toxicity , Fluorescence , Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/radiation effects , Fluorescent Dyes/toxicity , Limit of Detection , Liver/metabolism , Mesylates/chemical synthesis , Mesylates/radiation effects , Mesylates/toxicity , Mice , Naphthalenes/chemical synthesis , Naphthalenes/radiation effects , Naphthalenes/toxicity , Photons , RAW 264.7 Cells , Superoxides/metabolism
3.
Chem Commun (Camb) ; 54(66): 9238-9241, 2018 Aug 14.
Article in English | MEDLINE | ID: mdl-30066708

ABSTRACT

By combining the advantages of the photocaging technology and traditional analyte-responsive fluorescent probes, we designed and synthesized the first photocaged lysosomal-targeted fluorescent HOCl probe (PL-HA). The new caged PL-HA probe was capable of remote light-controlled recognition of HOCl in lysosomes.


Subject(s)
Fluoresceins/pharmacology , Fluorescent Dyes/pharmacology , Hypochlorous Acid/analysis , Lysosomes/metabolism , Animals , Fluoresceins/chemical synthesis , Fluoresceins/radiation effects , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/radiation effects , HeLa Cells , Humans , Hydrogen-Ion Concentration , Limit of Detection , Mice , Microscopy, Fluorescence , Morpholines/chemical synthesis , Morpholines/pharmacology , Morpholines/radiation effects , RAW 264.7 Cells , Ultraviolet Rays
4.
ACS Sens ; 3(9): 1622-1626, 2018 09 28.
Article in English | MEDLINE | ID: mdl-30145883

ABSTRACT

Reactive sulfur species play a very important role in modulating neural signal transmission. Hydrogen polysulfides (H2S n, n > 1) are recently suggested to be the actual signaling molecules. There are still few spatiotemporal controllable-based probes to detect H2S n. In this work, for the first time, we proposed the photocleavage product of the common photoremovable protecting group (2-nitrophenyl moiety) capable of trapping H2S n. Taking advantage of this, we constructed the probe H1 containing a photocontrollable group, a mitochondrial directing unit and a signal reporter fluorescein dye. H1 exhibited excellent fluorescence enhancement (50-fold) in response to H2S n under the aqueous buffer only after UV irradiation. H1 also showed high selectivity and sensitivity for H2S n over other reactive sulfur species, reactive oxygen species, and other analytes, especially biothoils including hydrogen sulfide, cysteine, homocysteine, and glutathione. We showed the utility of H1 to image H2S n in living cells with high spatiotemporal resolution.


Subject(s)
Fluorescent Dyes/chemistry , Mitochondria/metabolism , Sulfides/analysis , Fluoresceins/chemical synthesis , Fluoresceins/chemistry , Fluoresceins/radiation effects , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/radiation effects , Hep G2 Cells , Humans , Limit of Detection , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Nitrobenzoates/chemical synthesis , Nitrobenzoates/chemistry , Nitrobenzoates/radiation effects , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/radiation effects , Sulfides/metabolism , Ultraviolet Rays
5.
Chem Commun (Camb) ; 54(55): 7589-7592, 2018 Jul 14.
Article in English | MEDLINE | ID: mdl-29796477

ABSTRACT

A synthetic three-fluorophore system with two enzymatically cleavable linkers has been developed for the simultaneous detection of two proteases in a mixture. The probe was designed to afford single excitation/triple emission ratiometric detection through a fluorescence change during the cleavage of a peptide linker. The developed assays were verified for trypsin and chymotrypsin as the model enzymes.


Subject(s)
Aminocoumarins/chemistry , Chymotrypsin/analysis , Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Rhodamines/chemistry , Trypsin/analysis , Aminocoumarins/chemical synthesis , Aminocoumarins/radiation effects , Drug Stability , Enzyme Assays , Fluoresceins/chemical synthesis , Fluoresceins/radiation effects , Fluorescence , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/radiation effects , Hydrolysis , Lysine/analogs & derivatives , Lysine/chemistry , Phenylalanine/analogs & derivatives , Phenylalanine/chemistry , Rhodamines/chemical synthesis , Rhodamines/radiation effects
6.
Chem Commun (Camb) ; 53(92): 12434-12437, 2017 Nov 16.
Article in English | MEDLINE | ID: mdl-29099527

ABSTRACT

Intravital ablation of particular cell populations is necessary to decipher their roles under spatiotemporal conditions. Energy transfer-based photodynamic therapy presented a conditional range for specifically inducing the death of GFP expressing cells, with little effect on normal cells. This novel system enables easy access to the functional study of cells.


Subject(s)
Fluoresceins/radiation effects , Green Fluorescent Proteins/radiation effects , Photosensitizing Agents/radiation effects , Ablation Techniques/methods , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/radiation effects , Cell Line, Tumor , Energy Transfer , Female , Fluoresceins/chemistry , Green Fluorescent Proteins/chemistry , Humans , Mice, Inbred C57BL , Photochemotherapy/methods , Photosensitizing Agents/chemistry
7.
Bioorg Med Chem ; 24(18): 4199-4205, 2016 09 15.
Article in English | MEDLINE | ID: mdl-27430566

ABSTRACT

Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines.


Subject(s)
Fluorescent Dyes/chemistry , Peptide Nucleic Acids/chemistry , Rhodamines/chemistry , Cell Line, Tumor , Flow Cytometry , Fluoresceins/chemistry , Fluoresceins/radiation effects , Fluorescent Dyes/radiation effects , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Microscopy, Fluorescence , Peptide Nucleic Acids/chemical synthesis , Peptide Nucleic Acids/radiation effects , Photobleaching , RNA, Double-Stranded/chemistry , Rhodamines/radiation effects
8.
J Physiol ; 593(3): 521-39, 2015 Feb 01.
Article in English | MEDLINE | ID: mdl-25433071

ABSTRACT

KEY POINTS: The renin-angiotensin system plays a key role in cardiovascular physiology and its overactivation has been implicated in the pathogenesis of several major cardiovascular diseases. There is growing evidence that angiotensin II (Ang-II) may function as an intracellular peptide to activate intracellular/nuclear receptors and their downstream signalling effectors independently of cell surface receptors. Current methods used to study intracrine Ang-II signalling are limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we present novel photoreleasable Ang-II analogues used to probe intracellular actions with spatial and temporal precision. The photorelease of intracellular Ang-II causes nuclear and cytosolic calcium mobilization and initiates the de novo synthesis of RNA in cardiac cells, demonstrating the application of the method. ABSTRACT: Several lines of evidence suggest that intracellular angiotensin II (Ang-II) contributes to the regulation of cardiac contractility, renal salt reabsorption, vascular tone and metabolism; however, work on intracrine Ang-II signalling has been limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we aimed to synthesize and characterize cell-permeant Ang-II analogues that are inactive without uncaging, but release active Ang-II upon exposure to a flash of UV-light, and act as novel tools for use in the study of intracrine Ang-II physiology. We prepared three novel caged Ang-II analogues, [Tyr(DMNB)(4)]Ang-II, Ang-II-ODMNB and [Tyr(DMNB)(4)]Ang-II-ODMNB, based upon the incorporation of the photolabile moiety 4,5-dimethoxy-2-nitrobenzyl (DMNB). Compared to Ang-II, the caged Ang-II analogues showed 2-3 orders of magnitude reduced affinity toward both angiotensin type-1 (AT1R) and type-2 (AT2R) receptors in competition binding assays, and greatly-reduced potency in contraction assays of rat thoracic aorta. After receiving UV-irradiation, all three caged Ang-II analogues released Ang-II and potently induced the contraction of rat thoracic aorta. [Tyr(DMNB)(4)]Ang-II showed the most rapid photolysis upon UV-irradiation and was the focus of subsequent characterization. Whereas Ang-II and photolysed [Tyr(DMNB)(4)]Ang-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R), caged [Tyr(DMNB)(4)]Ang-II did not. Cellular uptake of [Tyr(DMNB)(4)]Ang-II was 4-fold greater than that of Ang-II and significantly greater than uptake driven by the positive-control HIV TAT(48-60) peptide. Intracellular photolysis of [Tyr(DMNB)(4)]Ang-II induced an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n), and initiated 18S rRNA and nuclear factor kappa B mRNA synthesis in adult cardiac cells. We conclude that caged Ang-II analogues represent powerful new tools for use in the selective study of intracrine signalling via Ang-II.


Subject(s)
Angiotensin II/analogs & derivatives , Calcium Signaling , Receptors, Angiotensin/metabolism , Ultraviolet Rays , Angiotensin Receptor Antagonists/pharmacology , Animals , Fluoresceins/radiation effects , Fluorescent Dyes/radiation effects , HEK293 Cells , Humans , Male , Microscopy, Fluorescence/methods , Rats , Rats, Sprague-Dawley , Receptors, Angiotensin/agonists
9.
Ultrasound Med Biol ; 38(3): 476-86, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22264409

ABSTRACT

The ultrasound exposure parameters that maximize drug release from dierucoyl-phosphatidylcholine (DEPC)-based liposomes were studied using two transducers operating at 300 kHz and 1 MHz. Fluorescent calcein was used as a model drug, and the release from liposomes in solution was measured using a spectrophotometer. The release of calcein was more efficient at 300 kHz than at 1 MHz, with thresholds of peak negative pressures of 0.9 MPa and 1.9 MPa, respectively. Above this threshold, the release increased with increasing peak negative pressure, mechanical index (MI), and duty cycle. The amount of drug released followed first-order kinetics and increased with exposure time to a maximal release. To increase the release further, the MI had to be increased. The results demonstrate that the MI and the overall exposure time are the major parameters that determine the drug's release. The drug's release is probably due to mechanical (cavitation) rather than thermal effects, and that was also confirmed by the detection of hydroxide radicals.


Subject(s)
Delayed-Action Preparations/chemistry , Delayed-Action Preparations/radiation effects , Fluoresceins/chemistry , Liposomes/chemistry , Liposomes/radiation effects , Sonication/methods , Diffusion/radiation effects , Dose-Response Relationship, Radiation , Fluoresceins/radiation effects , Radiation Dosage
10.
Free Radic Res ; 45(2): 139-46, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20942573

ABSTRACT

During investigation of UVA-induced oxidative stress in HaCaT keratinocytes with dihydrorhodamine 123 (DHR123) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), exaggerated baseline values were observed within control samples, suggesting a mechanism of probe oxidation and subsequent change in fluorescence intensity (FI) independent of cellular ROS generation. The effects of diluent, UVA pre-treatment and loading protocols upon the FI of the probes have therefore been investigated. The study confirmed the capacity of Dulbecco's Modified Eagle's Medium (DMEM) to confer fluorescence intensity changes in both probes, most notably DCF-DA. In addition, UVA pre-treatment compromises the effectiveness of DHR123 and DCF-DA to detect ROS generated in a cell-free system. In vitro data shows a greater UVA-induced FI increase in HaCaT cells loaded with probe before rather than after UVA treatment. This study has important implications for future research, the understanding of previous studies and associated confounding effects using DHR123 and DCF-DA as ROS sensitive probes.


Subject(s)
Fluoresceins/metabolism , Reactive Oxygen Species/metabolism , Rhodamines/metabolism , Artifacts , Cell-Free System/metabolism , Cell-Free System/radiation effects , Cells, Cultured , Culture Media/chemistry , Culture Media/metabolism , Culture Media/radiation effects , Fluoresceins/chemistry , Fluoresceins/radiation effects , Fluorometry , Humans , Keratinocytes/metabolism , Keratinocytes/radiation effects , Oxidation-Reduction/radiation effects , Oxidative Stress/radiation effects , Rhodamines/chemistry , Rhodamines/radiation effects , Ultraviolet Rays/adverse effects , Xanthine Oxidase/metabolism
11.
Opt Express ; 18(2): 938-46, 2010 Jan 18.
Article in English | MEDLINE | ID: mdl-20173916

ABSTRACT

An image-patterned molecular delivery system for mammalian cells is demonstrated by pulsed laser irradiation of gold particles immobilized on a substrate below a cell monolayer. Patterned cavitation bubble nucleation was captured using a time-resolved imaging system and molecular delivery verified by observing the uptake of a membrane-impermeable fluorescent dye, calcein. Delivery efficiency as high as 90% was observed and multiplexed, patterned dye delivery was demonstrated.


Subject(s)
Cell Membrane/chemistry , Drug Delivery Systems/methods , Fluoresceins/chemistry , Gold/chemistry , Gold/radiation effects , Kidney/chemistry , Optical Tweezers , Cell Membrane/radiation effects , Diffusion/radiation effects , Fluoresceins/radiation effects , HeLa Cells , Humans , Kidney/radiation effects , Light
12.
Photochem Photobiol Sci ; 7(3): 299-302, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18389146

ABSTRACT

The UV/Vis absorption and fluorescence properties of dibenzofluorescein (DBFL) in organic solvents were measured and used to shed light on the possible presence of its tautomers or various prototropic forms. DBFL in aprotic solvents mainly exists in two tautomeric forms, viz. quinoid and lactone, but neither are efficiently fluorescent. In protic solvents, such as methanol and ethanol, both the monoanion and neutral quinoid are present and showed the highest fluorescence quantum yield. In contrast, DBFL is fully dissociated to the monoanion and dianion in deionized water.


Subject(s)
Acetone/chemistry , Acetonitriles/chemistry , Alcohols/chemistry , Dimethyl Sulfoxide/chemistry , Ether/chemistry , Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Fluoresceins/isolation & purification , Fluoresceins/radiation effects , Fluorescence , Fluorescent Dyes/isolation & purification , Fluorescent Dyes/radiation effects , Hydrogen Bonding , Molecular Conformation , Photochemistry , Sensitivity and Specificity , Solvents/chemistry , Spectrometry, Fluorescence/methods , Spectrophotometry, Ultraviolet/methods , Stereoisomerism , Ultraviolet Rays
13.
Photochem Photobiol ; 84(5): 1238-43, 2008.
Article in English | MEDLINE | ID: mdl-18422880

ABSTRACT

There are controversial reports in the literature concerning the reactivity of singlet oxygen ((1)O(2)) with the redox probe 2',7'-dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which (1)O(2) is quantitatively generated in the presence of DCFH, we were able to show that the formation rate of the fluorescent molecule derived from DCFH oxidation, which is 2',7'-dichlorofluorescein (DCF), increases in D(2)O and decreases in sodium azide, proving the direct role of (1)O(2) in this process. We have also prepared solutions in which either (1)O(2) or dication (MB(2+)) and semi-reduced (MB) radicals of the sensitizer and subsequently super-oxide radical (O(2)(-)) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O(2)(-), indicating that both (1)O(2) and MB(2+) react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB(2+) than in the presence of (1)O(2), considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of (1)O(2) with DCFH is actually larger than that of MB(2+). We conclude that DCFH can continue to be used as a probe to monitor general redox misbalance induced in biologic systems by oxidizing radicals and (1)O(2).


Subject(s)
Fluoresceins/chemistry , Fluoresceins/chemical synthesis , Singlet Oxygen/chemistry , Fluoresceins/radiation effects , Free Radicals/chemistry , Free Radicals/radiation effects , Lasers , Molecular Structure , Oxidation-Reduction , Singlet Oxygen/radiation effects
14.
Free Radic Res ; 40(9): 959-65, 2006 Sep.
Article in English | MEDLINE | ID: mdl-17015276

ABSTRACT

Dichlorofluorescin (DCFH), a widely used fluorescent probe for reactive oxygen species (ROS) was decomposed completely and generated two distinct fluorescent products by photo-irradiation at 254 nm for 30 min. In the previous study, we had shown that one was dichlorofluorescein (DCF), a well known oxidized product of DCFH. In this study we investigated the other product and identified it as monochlorofluorescein (MCF) by 1H-NMR and fast atom bombardment/mass spectrum (FAB/MS) analyses. MCF was generated by photo-irradiation, but not by ROS. On the other hand, DCF was produced by both photo-irradiation and ROS. MCF showed similar fluorescent emission spectrum to DCF, however, its fluorescence intensity was more than that of DCF. The kinetic study suggested that MCF was not generated from DCF but from monochlorofluorescin, which might be generated from DCFH by photo-irradiation.


Subject(s)
Fluoresceins/chemistry , Fluoresceins/radiation effects , Ultraviolet Rays , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy/methods , Molecular Structure , Sensitivity and Specificity , Spectrometry, Mass, Fast Atom Bombardment/methods
15.
Photochem Photobiol ; 81(2): 358-66, 2005.
Article in English | MEDLINE | ID: mdl-15623352

ABSTRACT

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique used to selectively inactivate proteins of interest to elucidate their biological function. CALI has potential applications to a wide array of biological questions, and its efficiency allows for high-throughput application. A solid understanding of its underlying photochemical mechanism is still missing. In this study, we address the CALI mechanism using a simplified model system consisting of the enzyme beta-galactosidase as target protein and the common dye fluorescein. We demonstrate that protein photoinactivation is independent from dye photobleaching and provide evidence that the first singlet state of the chromophore is the relevant transient state for the initiation of CALI. Furthermore, the inactivation process was shown to be dependent on oxygen and likely to be based on photooxidation of the target protein via singlet oxygen. The simple model system used in this study may be further applied to identify and optimize other CALI chromophores.


Subject(s)
Lasers , beta-Galactosidase/antagonists & inhibitors , Absorption , Coloring Agents/chemistry , Fluoresceins/chemistry , Fluoresceins/radiation effects , Light , Models, Chemical , Oxygen/chemistry , Photochemistry , Singlet Oxygen/radiation effects , Time Factors , beta-Galactosidase/chemistry , beta-Galactosidase/radiation effects
16.
Free Radic Biol Med ; 30(12): 1418-25, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11390187

ABSTRACT

Cell culture media (RPMI 1640, Dulbecco's Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5'-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS). The production of ROS was light dependent. The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin. These observations point to exposure of cells to ROS under in vitro culture conditions.


Subject(s)
Culture Media/radiation effects , Light , Reactive Oxygen Species , Azides/pharmacology , Culture Media/chemistry , Cyclic N-Oxides , Electron Spin Resonance Spectroscopy , Fluoresceins/radiation effects , Fluorescent Dyes/radiation effects , Folic Acid/radiation effects , Hydrolysis , Hydroxyl Radical , Oxidation-Reduction , Oxygen , Phenolsulfonphthalein/radiation effects , Photochemistry , Piperazines/pharmacology , Pyridoxine/radiation effects , Rhodamines/radiation effects , Riboflavin/radiation effects , Singlet Oxygen , Spin Labels , Superoxides/analysis , Tryptophan/radiation effects , Tyrosine/radiation effects
17.
Eur J Biochem ; 267(17): 5585-92, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10951218

ABSTRACT

Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial function by microscopy fluorescence imaging. It was found to have a strong photosensitizing action that prevalently involves the generation of reactive oxygen species (ROS). The photooxidation properties of calcein in solution were studied in the presence of histidine and tryptophan as oxidizable substrates. The photodegradation of histidine was mainly mediated by singlet oxygen (1O2), as shown by the inhibitory effect of sodium azide, a specific 1O2 scavenger. On the other hand, mixed photosensitization mechanisms were present when tryptophan was used as the target of the calcein-stimulated photoprocess. In addition to 1O2, hydroxyl radicals and hydrogen peroxide were involved as reactive species, as shown by using mannitol and catalase as scavengers. The calcein-photosensitized alterations of mitochondria as a potential source of artifacts in confocal microscopy studies of cells were considered. Irradiation of isolated mitochondria with visible light (500-600 nm) in the presence of calcein induced opening of the permeability transition (PT) pore. The extent of the mitochondrial membrane photodamage, however, was modulated by the nature of the calcein environment. Thus, pore opening was triggered at short irradiation times and low dye concentrations when calcein was dissolved in the bulk medium. On the contrary, calcein concentrated in the matrix space was rather inefficient as photosensitizer even at concentrations 10 times higher than those present in the external medium.


Subject(s)
Fluoresceins/metabolism , Mitochondria, Liver/metabolism , Reactive Oxygen Species/metabolism , Animals , Fluoresceins/radiation effects , Kinetics , Light , Oxidation-Reduction , Rats , Scattering, Radiation
18.
Free Radic Biol Med ; 26(1-2): 148-61, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9890650

ABSTRACT

The photoreduction of 2'-7'-dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (lambda > 300 nm) 2'-7'-dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/*OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.


Subject(s)
Fluoresceins/chemistry , Fluoresceins/radiation effects , Fluorescent Dyes/chemistry , Fluorescent Dyes/radiation effects , Aerobiosis , Anaerobiosis , Catalase/metabolism , Catalase/pharmacology , Electron Spin Resonance Spectroscopy , Fluoresceins/metabolism , Fluorescent Dyes/metabolism , Free Radicals/chemistry , Free Radicals/metabolism , Free Radicals/radiation effects , Glutathione/metabolism , In Vitro Techniques , NAD/metabolism , Oxidation-Reduction , Oxidative Stress , Oxygen Consumption/drug effects , Photochemistry , Solutions , Superoxide Dismutase/metabolism , Superoxide Dismutase/pharmacology
19.
Biophys J ; 70(6): 2959-68, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8744334

ABSTRACT

The investigation in this report aimed at providing photophysical evidence that the long-lived triplet excited state plays an important role in the non-single-exponential photobleaching kinetics of fluorescein in microscopy. Experiments demonstrated that a thiol-containing reducing agent, mercaptoethylamine (MEA or cysteamine), was the most effective, among other commonly known radical quenchers or singlet oxygen scavengers, in suppressing photobleaching of fluorescein while not reducing the fluorescence quantum yield. The protective effect against photobleaching of fluorescein in the bound state was also found in microscopy. The antibleaching effect of MEA let to a series of experiments using time-delayed fluorescence spectroscopy and nanosecond laser flash photolysis. The combined results showed that MEA directly quenched the triplet excited state and the semioxidized radical form of fluorescein without affecting the singlet excited state. The triplet lifetime of fluorescein was reduced upon adding MEA. It demonstrated that photobleaching of fluorescein in microscopy is related to the accumulation of the long-lived triplet excited state of fluorescein and that by quenching the triplet excited state and the semioxidized form of fluorescein to restore the dye molecules to the singlet ground state, photobleaching can be reduced.


Subject(s)
Fluoresceins/chemistry , Fluoresceins/radiation effects , Microscopy, Fluorescence , Biophysical Phenomena , Biophysics , Cysteamine , Fluorescein , Free Radicals , Kinetics , Photochemistry , Photolysis , Radiation-Protective Agents , Solutions
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