Stimulated fluorescence and fluorescence quenching in chlorophyll a and bacteriochlorophyll a

This communication deals with the photophysical processes that take place in chlorophyll solutions under intense nitrogen laser irradiation. The effect of the pump photon density on the fluorescence yield depends strongly on the geometry of the irradiation and the sampling set-up. If the fluorescence cell and sampling probe are placed close to the transverse arrangement used for obtaining laser output, line narrowing and gain, which are processes associated with high population inversions and stimulated fluorescence, are observed. A normal fluorescence spectrum and a decrease in fluorescence quantum yield with increasing pump power are observed in the fluorescence cells in oriented at an angle of 20–40° with respect to the transverse axis of the exciting beam. The decrease in quantum yield appears to result from absorption of the pump photons by the excited singlet of the chlorophylls, and it is suggested that an analogous mechanism may be responsible for the anomalous fluorescence quantum yield reported for in in vivo Chlorella vulgaris algae.     

Quenching of bacteriochlorophyll fluorescence in chlorosomes from Chloroflexus aurantiacus by exogenous quinones

The quenching of bacteriochlorophyll (BChl) c fluorescence in chlorosomes isolated from Chloroflexus aurantiacus was examined by the addition of various benzoquinones, naphthoquinones (NQ), and anthraquinones (AQ). Many quinones showed strong quenching in the micromolar or submicromolar range. The number of quinone molecules bound to the chlorosomes was estimated to be as small as one quinone molecule per 50 BChl c molecules. Quinones which exhibit a high quenching effect have sufficient hydrophobicity and one or more hydroxyl groups in the alpha positions of NQ and AQ. Chlorobiumquinone has been suggested to be essential for the endogenous quenching of chlorosome fluorescence in Chlorobium tepidum under oxic conditions. We suggest that the quenching effect of chlorobiumquinone in chlorosomes from Chl. tepidum is related to the 1'-oxo group neighboring the dicarbonyl group.     

Fundamental photoluminescence properties of pyrene carbonyl compounds through absolute fluorescence quantum yield measurement and density functional theory

We reviewed the photophysical properties of carbonyl-functionalized pyrene derivatives [i.e., pyrene with aldehyde (PA: 1-formylpyrene), ketone (PK: 1-acetylpyrene), carboxylic acid (PCA: 1-pyrenecarboxylic acid), and ester groups (PE: 1-methoxycarbonylpyrene)] using a measurement of absolute fluorescence quantum yield in various solvents and time-dependent density functional theory (TD-DFT) calculations. Here, we obtained new important data that fill in the gaps in existing datasets on these properties and help identify photoluminescence mechanisms. The results of the TD-DFT calculations were in agreement with the experimental results, and indicated that the low fluorescence of PA and PK is derived not only from intersystem crossing but also from internal conversion due to the proximity effect; this inference was also supported by the measurements of the photoluminescence spectra at low temperatures. In addition, factors leading efficiently to non-radiative processes were shown to be absent in PCA and PE. Thus, we successfully revised and systematized the photophysical properties of pyrene modified by carbonyl substitutes, including carboxamide groups, which were previously reported by us. Moreover, we showed that the photoluminescence properties of such compounds might be predictable by using TD-DFT calculations.     

Lifetimes of bacteriochlorophyll fluorescence in Rhodopseudomonas viridis and Heliobacterium chlorum at low temperatures

Fluorescence lifetimes of isolated membranes of Rhodopseudomonas viridis were measured in the temperature range of 77 K to 25 K. At room temperature, the main component of the fluorescence decay of bacteriochlorophyll (BChl) b had a time constant of 50 ps. In contrast to other purple bacteria, the emission at low temperature was spectrally homogeneous and showed essentially single lifetimes of 140 ps at 77 K and 180 ps at 25 K, with the primary electron donor in the oxidized state. Taking into account the relative fluorescence yields with open and closed reaction centers, we arrive at numbers of 125 ps and 215 ps, respectively, for open reaction centers. These numbers are significantly smaller than expected on the basis of measurements of the efficiency of charge separation, perhaps suggesting that the excitation decay in the absence of reaction centers is considerably faster at low temperature than at room temperature. At least four different spectral components with different lifetimes were observed at 25 K in the emission of Heliobacterium chlorum, a short-wavelength component of about 30 ps and three longer-wavelength components of about 100 ps, 300 ps, and 900 ps. This indicates a strong heterogeneity in the emitting pigment, BChl g-808. The component with the shortest lifetime does not appear to be affected by the redox state of the reaction center and might reflect energy transfer to BChl g species which are connected to the reaction center.     

The Auger-Raman effect and theK-shell fluorescence yield of krypton

An attempt is made to measure the relative intensities of the resonant Raman scattering and the corresponding Auger process. A multiwire proportional counter is employed to distinguish the two processes. The experiment on krypton at theK-edge supports the conjectured constant fluorescence yield for photon energies below the edge, allowing a shift of less than ±10% of the above-edge value. The experiment leads to an improved method of determining fluorescence yields. TheK-shell fluorescence yield of krypton is remeasured to 0.660±0.007.     

Recent advances in instrumentation for absolute emission quantum yield measurements

The fluorescence and phosphorescence quantum yields (Φ) of standard solutions have been re-evaluated based on an absolute method using an integrating sphere equipped with a multichannel spectrometer. We have examined in detail the Φ value of ruthenium(II) tris(2,2′-bipyridine) complex which have been often used as the standards in the determination of quantum yields of transition-metal complexes. This revealed that the Φ values for [Ru(bpy)₃]²⁺ were 0.063 in deaerated H₂O, 0.040 in aerated H₂O, 0.095 in deaerated CH₃CN, and 0.018 in aerated CH₃CN, respectively, which are significantly higher than the previously accepted values.We have also examined the technical aspects in the determination of absolute emission quantum yields for lanthanide complexes and those of organic crystals of anthracene. For the accurate determination for lanthanide complexes, special care must be taken in the spectroscopic measurements because of their narrow absorption and emission bands. For organic crystals, the fluorescence quantum yields are reduced due to reabsorption, chemical impurities and structural defects. Our observations for highly purified anthracene crystals revealed that the lower limit value of Φ was 0.64.     

Photostability and the yield of triplet state generation of bacteriochlorophyll c and bacteriopheophytin c in solution

The paper deals with some fast and slow processes of excitation energy deactivation in bacteriochlorophyll c and bacteriopheophytin c. The experiments were carried out in the air, and in oxygen or nitrogen atmosphere at different temperatures. The parameters such as fluorescence yield, the yield of triplet state generation and photostability were estimated. On the basis of these parameters an attempt was made to preliminary select the pigments best suited for medical applications. It seems that the photosensitive and highly fluorescent bacteriochlorophyll c could be used as a fluorescence probe for diagnosis, whereas its derivative without the magnesium ion is better suited for the photochemical reactions occurring during therapeutic treatment.     

Energy transfer method for determination of absolute fluorescence quantum yields

A new method for the determination of absolute fluorescence quantum yields based on simple relative measurements is described. This energy transfer method is particularly suited for studies of dyes in systems of monomolecular layers. The application of the new method to an investigation of the deactivation of excited thiacyanine dye molecules in monolayers gave evidence for radiationless deactivation processes other than thermal equilibration occurring in higher vibronic levels of the excited singlet state.     

Laser induced fluorescence in NO2: absolute fluorescence cross sections and quantum yields

Spectrally resolved collision-free absolute fluorescence cross sections have been measured, for NO₂ excited, by the second harmonic of the Nd: YAG laser. The total cross section into the 523.0–650.0 nm range was found to be 2.3 × 10^?20 cm². The measured absorption cross section of 1.46 × 10^?19 cm² implies a quantum yield of 16% over this range.     

In vivo fluorescence imaging of bone-resorbing osteoclasts

Osteoclasts are giant polykaryons responsible for bone resorption. Because an enhancement or loss of osteoclast function leads to bone diseases such as osteoporosis and osteopetrosis, real-time imaging of osteoclast activity in vivo can be of great help for the evaluation of drugs. Herein, pH-activatable chemical probes BAp-M and BAp-E have been developed for the detection of bone-resorbing osteoclasts in vivo. Their acid dissociation constants (pK(a)) were determined as 4.5 and 6.2 by fluorometry in various pH solutions. These pK(a) values should be appropriate to perform selective imaging of bone-resorbing osteoclasts, because synthesized probes cannot fluoresce intrinsically at physiological pH and the pH in the resorption pit is lowered to about 4.5. Furthermore, BAp-M and BAp-E have a bisphosphonate moiety that enabled the probes to localize on bone tissues. The hydroxyapatite (HA) binding assay in vitro was, therefore, performed to confirm the tight binding of the probes to the bone tissues. Our probes showed intense fluorescence at low pH values but no fluorescence signal under physiological pH conditions on HA. Finally, we applied the probes to in vivo imaging of osteoclasts by using intravital two-photon microscopy. As expected, the fluorescence signals of the probes were locally observed between the osteoclasts and bone tissues, that is, in resorption pits. These results indicate that our pH-activatable probes will prove to be a powerful tool for the selective detection of bone-resorbing osteoclasts in vivo, because this is the first instance where in vivo imaging has been conducted in a low-pH region created by bone-resorbing osteoclasts.     

Photo-electrochemical control of photosystem II chlorophyll fluorescence in vivo

The effect of electric field on chlorophyll fluorescence is considered on the basis of the reversible radical pair model. The hypothesis is presented that the electric fields generated by photosynthetic charge separation in reaction centers and propagated laterally through the thylakoid lumen are associated with changes in chlorophyll fluorescence yield.     

Laser-induced fluorescence in a furnace: A viable approach to absolute analysis?

The possibility of performing absolute analysis by laser-induced atomic fluorescence in a graphite tube atomizer is discussed. At the sensitivities which are typical of these measurements, i.e., at absolute levels of the order of femtograms, it is argued that the lack of certified reference materials and the practical limitations in preparing standard solutions constitute the major incentives to the development of absolute analysis. The various theoretical and experimental parameters needed in the procedure of converting a measured fluorescence signal, e.g., a voltage, into the corresponding number of emitting atoms are discussed and evaluated with respect to a typical determination of a toxic element like thallium.     

In vitro and in vivo fluorescence monitoring of photosensitizers

The use of steady state fluorescence spectroscopy in the detection and monitoring of potential photochemotherapeutic agents is examined. Problems associated with both in vitro and in vivo fluorescence measurements are investigated, and typical data are presented. Recent results on the use of fluorescence in pharmacokinetic studies are discussed, and the relative merits of in vitro vs. in vivo methods are outlined.     

Self-absorption effects on alpha-induced atmospheric nitrogen fluorescence yield

Nitrogen fluorescence induced by radiation can be used to detect the presence of radioactive contamination in the environment. Contamination quantification from the fluorescence signal requires: the source’s effective alpha spectrum; the specific radiation quantum fluorescence efficiency; optical attenuation length in air of the fluorescence signal; the absolute throughput and quantum efficiency of the optical instrumentation; calibration of the instrumentation; and radiation transport modeling of the “effective” array exposure rate given the alpha particle spectrum. Field testing conducted on optical instrumentation measured the nitrogen fluorescence yield generated by ²⁴¹Am alpha emissions. Laboratory studies of ²⁴¹Am via alpha spectrometry determined whether the presence of solids on source surfaces produced sufficient self-absorption to decrease fluorescence. Results from the laboratory studies provided correction to the effective alpha-source activity values in a model of the earlier optical-sensor field measurements, and determined the air fluorescence efficiency of alpha particles generated by the ²⁴¹Am sources used in the field experiments.     

铝试剂的荧光光谱与荧光量子产率

首次研究了铝试剂的荧光光谱和荧光量子产率,发现pH3至pH12条件下,用紫外光照射铝试剂溶液可以产生荧光,最大激发波长和最大发射波长分别为297nm和409nm,荧光强度与铝试剂浓度之间存在良好的线性关系,线性范围为0.01～3μg/mL,检测下限为0.01μg/mL,以硫酸奎宁为参比,测得铝试剂的荧光量子产率为0.16。     

Light-intensity dependence of the in vivo fluorescence lifetime of chlorophyll

Abstract— Phase-fluorometer measurements of the fluorescence lifetime, τ, from chlorophyll in Chlorella, Bishop's 8 and 11 Scenedesmus mutants, sugarbeet leaf and chloroplast fragments demonstrate that: τ is independent of modulation frequency at 27 and 14 mc. in the experimental-wavelength range from 650 to 735 nm (with blue or blue-green excitation); with Chlorelfa and chloroplast fragments τ rises hyperbolically with intensity to τ_max about 2 nsec and 0·7 nsec respectively; DCMU poisoned Chlorella and sugarbeet leaf as well as the mutants have τ values near 2 nsec; the lifetime-incident intensity relationship for Chlorella and chloroplast fragments is quantitatively similar to the incident-intensity dependence of fluorescence yield and oxygen evolution and thus supports the hypothesis that these three measuring variables are controlled by the concentration of ‘open’ trapping systems; τ is independent of emission wave-length to suggest that fluorescence is dominated by a single chlorophyll species. The reaction velocity-lifetime correlation indicates that fluorescence behavior is directly controlled by system II.     

Steric control of bacteriochlorophyll ligation

The axial coordination of central Mg(2+) ion in chlorophylls is of great structural and functional importance for virtually all photosynthetic chlorophyll proteins; however, little thermodynamic data are available on the ligand binding to these pigments. In the present study, spectral deconvolution of the bacteriochlorophyll Q(X) band serves to determine the ligand binding equilibria and relationships between thermodynamic parameters of ligand binding, ligand properties, and steric interactions occurring within the pigment. On the basis of the temperature effects on coordination, DeltaH degrees, DeltaS degrees, and DeltaG degrees of binding various types of ligands (acetone, dimethylformamide, imidazole, and pyridine) to diastereoisomeric bacteriochlorophylls were derived from respective van't Hoff's plots. At ambient temperatures, only ligation by imidazole and pyridine occurs spontaneously while DeltaG degrees becomes positive for ligation by acetone and dimethylformamide, due to a relatively large entropic effect, which is dominating when the energetic effects of ligation are small. It reflects, in quantitative terms, the control of the equatorial coordination of the Mg(2+) ion via the axial coordination: a "hard" free Mg(2+) ion is made into a softer center through the coordination of tetrapyrrole. Pigment structural features have comparable effects on the energetic and entropic contributions to the difference of ligation free energy between the diastereoisomers of bacteriochlorophyll. DeltaS degrees and DeltaH degrees values are consistently lower for the S epimer, most likely due to the steric crowding between bulky substituents. The two epimers show a 5 J.mol(-1).K(-1) difference in DeltaS degrees values, regardless of the ligand type, while the difference in DeltaH degrees amounts to 1.7 kJ.mol(-1), depending on the ligand. Such steric control of ligation would relate to the partial diastereoselectivity of chlorophyll self-assembly and, in particular, the very high diastereoselectivity of the ligation of chlorophylls in photosynthetic proteins.     

In vivo fluorescence spectroscopy of nonmelanoma skin cancer

In vivo and ex vivo tissue autofluorescence (endogenous fluorescence) have been employed to investigate the presence of markers that could be used to detect tissue abnormalities and/or malignancies. We present a study of the autofluorescence of normal skin and tumor in vivo, conducted on 18 patients diagnosed with nonmelanoma skin cancers (NMSC). We observed that both in basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) the endogenous fluorescence due to tryptophan residues was more intense in tumor than in normal tissue, probably due to epidermal thickening and/or hyperproliferation. Conversely, the fluorescence intensity associated with dermal collagen crosslinks was generally lower in tumors than in the surrounding normal tissue, probably because of degradation or erosion of the connective tissue due to enzymes released by the tumor. The decrease of collagen fluorescence in the connective tissue adjacent to the tumor loci was validated by fluorescence imaging on fresh-frozen tissue sections obtained from 33 NMSC excised specimens. Our results suggest that endogenous fluorescence of NMSC, excited in the UV region of the spectrum, has characteristic features that are different from normal tissue and may be exploited for noninvasive diagnostics and for the detection of tumor margins.