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

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 (O(2)(a(1)Delta(g))) 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.     

Direct measurement of the singlet oxygen lifetime in zeolites by near-IR phosphorescence.

Time-resolved near-IR phosphorescence spectroscopy was employed to determine the lifetime of singlet oxygen in Y-zeolites and porous silica and it was found to depend strongly on the alumina content of the zeolite.     

Quantitative determination of localized tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements.

This study describes the use of two-photon excitation phosphorescence lifetime measurements for quantitative oxygen determination in vivo. Doubling the excitation wavelength of Pd-porphyrin from visible light to the infrared allows for deeper tissue penetration and a more precise and confined selection of the excitation volume due to the nonlinear two-photon effect. By using a focused laser beam from a 1,064-nm Q-switched laser, providing 10-ns pulses of 10 mJ, albumin-bound Pd-porphyrin was effectively excited and oxygen-dependent decay of phosphorescence was observed. In vitro calibration of phosphorescence lifetime vs. oxygen tension was performed. The obtained calibration constants were kq = 356 Torr(-1) x s(-1) (quenching constant) and tau0 = 550 micros (lifetime at zero-oxygen conditions) at 37 degrees C. The phosphorescence intensity showed a squared dependency to the excitation intensity, typical for two-photon excitation. In vivo demonstration of two-photon excitation phosphorescence lifetime measurements is shown by step-wise PO2 measurements through the cortex of rat kidney. It is concluded that quantitative oxygen measurements can be made, both in vitro and in vivo, using two-photon excitation oxygen-dependent quenching of phosphorescence. The use of two-photon excitation has the potential to lead to new applications of the phosphorescence lifetime technique, e.g., noninvasive oxygen scanning in tissue at high spatial resolution. To our knowledge, this is the first report in which two-photon excitation is used in the setting of oxygen-dependent quenching of phosphorescence lifetime measurements.     

Photosensitization by hematoporphyrin: ESP evidence for free radical induction in unsaturated fatty acids and for singlet oxygen production.

Hematoporphyrin ability to photoreact by type I and type II mechanisms was investigated in some model systems. At room temperature, visible irradiation of hematoporphyrin-unsaturated fatty acids and hematoporphyrin-cholesterol systems resulted in the Electron Spin Resonance (ESR) spectrum of the hematoporphyrin free radical. Triplet state hematoporphyrin is shown to be involved in the electron transfer from the lipid moiety. Moreover an ESR method to monitor the singlet oxygen production by hematoporphyrin was used. β-carotene effect on both mechanisms (type I and type II) was tested.     

A study of solvent effects on the phosphorescence properties of flavins.

A combination of zero field triplet state techniques are used to study the excited electronic states of a series of flavin and flavin related molecules both in single crystals and glass matrices. Particular attention is given to the effects of solvent interaction on the triplet state properties of the flavin molecules. The total phosphorescence decay rate constants at 1.4 degrees K are reported for the flavin molecules in polar and nonpolar solvents. The rate constants are then correlated to the degree of solvent interaction. Results indicate possible complex formation between the isoalloxazine and adenine groups in FAD. Finally, the results and possible interpretation on the study of a flavoenzyme, L-amino acid oxidase are presented.     

Photosensitized formation of singlet oxygen by phycobiliproteins in neutral aqueous solutions

Phycobiliproteins (PBPs) are a type of promising sensitizers for photodynamic therapy (PDT). Upon irradiation (lambda>500nm) of an oxygen-saturated aqueous solution of phycobiliproteins, particularly, C-phycocyanin (C-PC), allophycocyanin (APC) or R-phycoerythrin (R-PE), the formation of singlet oxygen (1O2) was detected by using imidazole in the presence of p-nitrosodimethylaniline (RNO). The bleaching of RNO caused by the presence of imidazole in our system showed typical concentration dependence with a maximum at about 8mM imidazole, which is in agreement with the formation of 1O2. In addition, the generation of 1O2 was verified further in the presence of D2O and specific singlet oxygen quencher 1,4-diazabicyclo [2,2,2] octane (DABCO) and sodium azide (NaN3). Our experimental results indicated that APC possesses high ability to generate reactive oxygen species and the relative quantum yields of photogeneration of 1O2 by PBPs are as follows: APC > C-PC > R-PE.     

Singlet molecular oxygen in photobiochemical systems: IR phosphorescence studies.

Singlet molecular oxygen (1O2) is one of the most active intermediates involved in photosensitized oxygenation reactions in chemical and biological systems. Deactivation of singlet oxygen is accompanied by infrared phosphorescence (1270 nm) which is widely employed for 1O2 detection and study. This review considers techniques for phosphorescence detection, phosphorescence spectra, quantum yields and kinetics under laser excitation, the radiative and real 1O2 lifetimes in organic solvents and water, 1O2 quenching by biomolecules, and estimation of singlet oxygen lifetimes, diffusion lengths and phosphorescence quantum yields in blood plasma, cell cytoplasm, erythrocyte ghosts, retinal rod outer segments and chloroplast thylakoids. The experiments devoted to 1O2 phosphorescence detection in photosensitizer-containing living cells are discussed in detail. Information reviewed is important for understanding the mechanisms of photodestruction in biological systems and various applied problems of photobiology and photomedicine.     

Photosensitized formation of singlet oxygen by phycobiliproteins in neutral aqueous solutions

Phycobiliproteins (PBPs) are a type of promising sensitizers for photodynamic therapy (PDT). Upon irradiation (λ>500nm) of an oxygen-saturated aqueous solution of phycobiliproteins, particularly, C-phycocyanin (C-PC), allophycocyanin (APC) or R-phycoerythrin (R-PE), the formation of singlet oxygen (¹O₂) was detected by using imidazole in the presence of p-nitrosodimethylaniline (RNO). The bleaching of RNO caused by the presence of imidazole in our system showed typical concentration dependence with a maximum at about 8mM imidazole, which is in agreement with the formation of ¹O₂. In addition, the generation of ¹O₂ was verified further in the presence of D₂O and specific singlet oxygen quencher — 1,4-diazabicyclo [2,2,2] octane (DABCO) and sodium azide (NaN₃). Our experimental results indicated that APC possesses high ability to generate reactive oxygen species and the relative quantum yields of photogeneration of ¹O₂ by PBPs are as follows: APC > C-PC > R-PE.     

Direct observation of singlet oxygen phosphorescence at 1270 nm from L1210 leukemia cells exposed to polyporphyrin and light.

Near-infrared emission (1170-1475 nm) was studied from L1210 leukemia cells incubated with polyporphyrin (fractionated hematoporphyrin derivative), suspended in deuterium oxide buffer, and then exposed to light. Following pulsed laser excitation, the near-infrared emission decayed in two phases. The first phase of the emission (0-2 microseconds) was principally due to polyporphyrin fluorescence. The second phase of the emission (20-90 microseconds) was due mainly to singlet oxygen. Evidence supporting the assignment of the second phase emission to singlet oxygen included a spectral analysis showing a peak near 1270 nm and reductions in the second phase emission caused by the singlet oxygen quenchers, histidine, carnosine, and water. The second phase emission decayed in a biexponential manner with lifetimes of 4.5 +/- 0.5 and 49 +/- 4 microseconds. Most of the singlet oxygen in the second phase emission was likely due to singlet oxygen that was generated near the surface of the L1210 leukemia cells and then diffused into the deuterium oxide buffer. Direct measurements of singlet oxygen phosphorescence at 1270 nm may prove to be a useful analytical technique for studying photochemical generation of singlet oxygen in cultured cells.     

Detection of singlet (1O2) oxygen phosphorescence during chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide

Evidence for the production of singlet molecular oxygen (1O2) during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide has been obtained through the use of optical spectroscopy, oxygen electrode experiments, and electron spin resonance (ESR). ESR spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) demonstrate the production of the ethyl peroxyl free radical during the chloroperoxidase/ethyl hydroperoxide reaction. Oxygen and acetaldehyde concentrations suggest that the production of ethyl peroxyl radicals constitutes less than 2% of the decomposition of ethyl hydroperoxide at the concentrations of reactants used. The phosphorescence of 1O2 at 1268 nm was observed during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide in deuterium oxide buffer. Chloroperoxidase also catalyzes the decomposition of tert-butyl hydroperoxide to its corresponding peroxyl radical. Alkoxyl and alkyl-DMPO spin adducts were also detected. A much lower yield of 1O2 phosphorescence was observed during the chloroperoxidase-catalyzed decomposition of tert-butyl hydroperoxide. This phosphorescence probably arises through secondary production of alkyl peroxyl radicals. These results suggest that the initial enzyme-dependent production of ethyl peroxyl radicals is followed by enzyme-independent reaction of two peroxyl radicals through the tetroxide intermediate, as originally proposed by Russell (Russell, G. A. (1957) J. Am. Chem. Soc. 79, 3871-3877), to form acetaldehyde, ethyl alcohol, and molecular oxygen.     

A molecular hybrid producing simultaneously singlet oxygen and nitric oxide by single photon excitation with green light

Combination of photosensitizers (PS) for photodynamic therapy with NO photodonors (NOPD) is opening intriguing horizons towards new and still underexplored multimodal anticancer and antibacterial treatments not based on “conventional” drugs and entirely controlled by light stimuli. In this contribution, we report an intriguing molecular hybrid based on a BODIPY light-harvesting antenna that acts simultaneously as PS and NOPD upon single photon excitation with the highly biocompatible green light. The presented hybrid offers a combination of superior advantages with respect to the other rare cases reported to date, meeting most of the key criteria for both PSs and NOPDs in the same molecular entity such as: (i) capability to generate ¹O₂ and NO with single photon excitation of biocompatible visible light, (ii) excellent ¹O₂ quantum yield and NO quantum efficiency, (iii) photogeneration of NO independent from the presence of oxygen, (iv) large light harvesting properties in the green region. Furthermore, this compound together with its stable photoproduct, is well tolerated by both normal and cancer cells in the dark and exhibits bimodal photomortality of cancer cells under green light excitation due to the combined action of the cytotoxic ¹O₂ and NO.     

Enzymatic recognition of DNA modifications induced by singlet oxygen and photosensitizers.

DNA modifications induced either by photosensitization (illumination in the presence of methylene blue) or by chemically generated singlet oxygen (thermal decomposition of an 1,4-etheno-2,3-benzodioxin) are recognized and incised by repair endonucleases present in crude bacterial cell extracts. Only a small fraction of the incised modifications are sites of base loss (AP-sites) sensitive to exonuclease III, endonuclease IV from E. coli or to the UV-endonuclease from M. luteus. Cell extracts from E. coli strains overproducing or defective in endonuclease III recognize the modifications induced by illumination in the presence of methylene blue just as well as do those from wild-type E. coli strains. This indicates that dihydropyrimidine derivatives, which are characteristic of hydroxyl radical-induced DNA modifications, are absent. In contrast, most of the modifications induced are not recognized by a cell extract from a fpg strain defective in formamidopyrimidine-DNA glycosylase FPG protein). Furthermore, incision by a cell extract from an E. coli strain overproducing FPG protein takes place at much lower protein concentration than with the wild-type strain. Experiments with purified FPG protein confirm that this enzyme is responsible for the recognition of singlet oxygen-induced DNA base modifications.     

Regulation of formation of covalently bound flavins in liver and cerebrum by thyroid hormones.

The effects of thyroxine (T₄) and triiodothyronine (T₃) treatment upon the formation of [2-¹⁴C]flavins bound covalently to tissue proteins in liver and cerebrum were measured 1 h after a subcutaneous injection of [2-¹⁴C]riboflavin in male rats of different ages. In livers of rats of ages 2, 3, and 12 months, T₄ (100 μg/100 g body wt) and T₃ (25 μg/100 g body wt) in daily intraperitoneal doses for 7 days each increased incorporation into covalently bound flavins 50% above that in saline-treated controls. In newborn rats, T₄ in doses of 10 μg/rat for 7 days increased incorporation similarly to that in adults. In adult rats doses of T₃ from 2.5 to 25 μg/100 g body wt were nearly as effective as larger doses of T₃ and T₄ in increasing the formation of covalently bound flavins in liver. In cerebra of newborn rats, T₄ was ineffective in increasing the formation of covalently bound flavins. However, in cerebra of rats of ages 2, 3, and 12 months, both T₃ and T₄ consistently increased the formation of covalently bound flavins. Doses of T₃ from 2.5 to 25 μg/100 g body wt produced significant increases. These findings are of interest in view of our previous demonstration that the formation of flavin adenine dinucleotide, the major tissue flavin, is not increased in rat brain even by massive doses of thyroid hormones. The present results indicate that the formation of the fraction of flavins bound covalently to tissue proteins differs from the usual pattern of brain metabolism of adult rats in being subject to control by thyroid hormones.