The absolute yield of bacteriochlorophyll fluorescence in vivo

Abstract— –The method of Weber and Teale for determining absolute fluorescence quantum yield of dyes in solution was modified for determination of the yield of bacteriochlorophyll fluorescence from chromatophores and whole cells of photosynthetic bacteria. Measured yields ranged from about 1–6 per cent. The yield depended on intensity and wavelength of the exciting light. The higher yield at higher light intensity was interpreted as due to saturation of photosynthesis. The lower yield in some strains when excited at 810 nm was attributed to preferential excitation of the reaction center pigment P800. From this study and the lifetime measurements of others, the relation τ=Q.τ₀ was substantiated for the fluorescence of bacteriochlorophyll in vivo, τ being the actual lifetime, τ₀ the intrinsic lifetime as estimated from the absorption band area, and Q the quantum yield of fluorescence.     

Anisotropic fluorescence of Michler's ketone in liquid solutions

In solutions of Michler's ketone in ethanol at 295 K, the fluorescence is almost completely anisotropic. This phenomenon is caused by the unusually short lifetime of the excited singlet state due to a short intrinsic lifetime and processes leading to a very low quantum yield. The absorption, fluorescence and phosphorescence spectra, their degree of anisotropy and the values of the quantum yield at 295 K, 203 K and 100 K are reported.     

Fast and slow fluorescence emissions from pyrimidine vapor

The pressure and excitation-energy dependence of the fluorescence quantum yield and lifetime of pyrimidine vapor has been investigated in the pressure range 10^?3-10 torr. The results indicate that in conformity to the intermediate case the fluorescence of the isolated pyrimidine molecule consists of fast and slow components with lifetimes of the order of 1 ns and 10 μs, respectively. The total fluorescence quantum yield amounts to as high as 0.045. The yield of the slow fluorescence component decreases significantly with increasing excitation energy; this observation is interpreted as being due mainly to the lengthening of the radiative lifetime of that component.     

PHOTOCHEMICAL PROPERTIES OF PORPHYRIN c: AN AGENT FOR USE IN TUMOR PHOTOTHERAPY

Abstract— The absorption and fluorescence properties of porphyrin c (P c ), the porphyrin chromophore present in cytochrome c , have been determined in several solvents and micellar environments. In aqueous buffer solutions at pH 7.5 Pc may exist in both a fluorescent monomeric form with quantum yield of fluorescence, (Φ_f,) ∼ 0.03, and fluorescence lifetime, (τ_f) ∼ 8 ns, and as a non-fluorescent aggregate. The proportion of monomeric form is higher in organic solvents and micelles but is reduced with increasing porphyrin concentrations in aqueous solutions. Porphyrin c readily complexes with Zn²⁺ to produce a fluorescent chelate (Zn-P c ) with Φ_f, ∼ 0.02 and τ_f, ∼ 2 ns at pH 7.5. The yields of singlet excited oxygen formation from Pc and the Zn-P c complex are higher than observed for hematoporphyrin derivative (HpD). Both P c and Zn-P c are effective agents in tumor phototherapy and do not induce the prolonged cutaneous photosensitivity observed with the use of HpD.     

THE PHOTOPHYSICS OF MEROCYANINE 540. A COMPARATIVE STUDY IN ETHANOL AND IN LIPOSOMES

Abstract— Absorption and fluorescence emission spectra, fluorescence lifetimes, fluorescence quantum yields, photoisomerization quantum yields and triplet quantum yields were measured for Merocyanine 540 (MC540) in ethanol and in large unilamellar dimyristoyl phosphatidylcholine vesicles. The major differences in the photophysics between the two media are the increase of the fluorescence quantum yield from 0.15 in ethanol to 0.6 in vesicles at 25° C, and the appearance of a second fluorescence decay with a lifetime of 1.87 ns in the latter medium. Upper and lower limits for the photoisomerization quantum yields were determined by combining the data from laser flash photolysis and optoacoustic spectroscopy. The decrease in photoisomerization quantum yield upon incorporation of the dye into the lipid bilayer by a factor 2 suggests that this process competes directly with fluorescence. The temperature dependence of the fluorescence and photoisomerization quantum yields in solution supports this model. In both media MC540 has a very low triplet quantum yield with values 0.002 > (> ø_T > 0.02 in ethanol and 0.01 > ø_T- > 0.09 in liposomes Our data are consistent with the model whereby the dye is incorporated into the lipid bilayer as a monomer with two different orientations and this model is adopted on the basis of the biexponential behaviour of the fluorescence and photoisomer decay.     

Time-resolved spectroscopy of the excited singlet states of tirapazamine and desoxytirapazamine

Laser flash photolysis (LFP, 400 nm excitation) of the anti-cancer drug tirapazamine (TPZ) in acetonitrile produces the singlet excited-state S1 with lambda(max) = 544 nm. The lifetime of this state is 130 ps, in good agreement with the reported fluorescence lifetime. The excited state is reduced to the corresponding radical anion by KSCN or KI. The spectrum of the radical anion is in good agreement with previously reported pulse radiolysis studies and time-dependent density functional theory (TD-DFT) calculations. LFP of desoxytirapazamine (dTPZ) also produces the first excited singlet state, S1. The fluorescence quantum yield and lifetime (5.4 ns) of the dTPZ singlet excited state are both much greater than the corresponding values of TPZ. This is explained by DFT calculations that predict that cyclization of TPZ to form an oxaziridine is thermodynamically facile but that cyclization of dTPZ to form an oxadiaziridine is not. Thus, the S1 state of TPZ has a short lifetime and low fluorescence quantum yield due to ready cyclization whereas the cyclization of the S1 state of dTPZ is unimportant and does not limit either the fluorescence quantum yield or the fluorescence lifetime. This conclusion is confirmed by studies of dTPZ', an isomer of dTPZ containing the C=N-O moiety which has a low quantum yield and short fluorescence lifetime similar to that of TPZ.     

Fluorescence lifetime and quantum yield of diphenylketyl radical at low temperature

The fluorescence lifetimes of the diphenylketyl radical trapped in ethanol and EPA matrices are 16.8 ± 0.5 and 21.1 ± 0.6 ns, respectively in the temperature region of 77–130 K. The fluorescence quantum yield in ethanol matrix is 0.16 ± 0.6. These values are consistent with the radiative lifetime calculated from the absorption spectrum.     

Single-molecule fluorescence lifetime and anisotropy measurements of the red fluorescent protein,DsRed, in solution

Fluorescence lifetime and anisotropy measurements were made on the red fluorescent protein (DsRed) from tropical coral of the Discosoma genus, both at single-molecule and bulk concentrations. As expected from previous work, the fluorescence lifetime of DsRed in solution is dependent on laser power, decreasing from an average fluorescence lifetime in the beam of about 3.3 ns at low power (3.5 ns if one extrapolates to zero power) to about 2.1 ns at 28 kW/cm2. At the single-molecule level, exciting with 532 nm, 10 ps laser pulses at 80 MHz repetition rate, DsRed particles entering the laser beam initially have a lifetime of about 3.6 ns and convert to a form having a lifetime of about 3.0 ns with a quantum yield of photoconversion on the order of 10(-3) (calculated in terms of photons per DsRed tetramer). The particles then undergo additional photoconversion with a quantum yield of roughly 10(-5), generating a form with an average lifetime of 1.6 ns. These results may be explained by rapid photoconversion of one DsRed monomer in a tetramer, which acts as an energy transfer sink, resulting in a lower quantum yield for photoconversion of subsequent monomers. Multiparameter correlation and selective averaging can be used to identify DsRed in a mixture of fluorophores, in part exploiting the fact that fluorescent lifetime of DsRed changes as a function of excitation intensity.     

Analysis of Fluorescence Lifetime of Protochlorophyllide and Chlorophyllide in Isolated Etioplast Membranes Measured from Multifrequency Cross-correlation Phase Fluorometry

The fluorescence decays of protochlorophyllide (Pchlide) and of chlorophyllide (Chlide) in wheat etioplast membranes were analyzed using a multiexponential fluorescence decay model. Using different excitation wavelengths from 430 to 470 nm, we found that a triple-exponential model at 14°C and a double-exponential model at — 170°C were adequate to describe the Pchlide fluorescence decay. We discuss the origin of the three fluorescence lifetime components at 14°C on the basis of the dependence of their fractional intensities on the excitation wavelength and by correlating the fractional intensities with integrated fluorescence intensities of different Pchlide forms in steady-state fluorescence spectra. The fluorescence decay of the main Pchlide form, photoactive Pchlide-F657, is shown to have a complex character with a fast component of 0.25 ns and a slower component of about 2 ns. Two lifetime components of 2 ns and 5.5–6.0 ns are ascribed to the second photoactive form, Pchlide-F645, and to nonphotoactive Pchlide forms, respectively. In etioplast membranes preilluminated by a short saturating light pulse, we found a single 5.0 ns component for Chlide-F688 (the Chlide-NADPH: protochlorophyllide oxidoreductase [PORJ-NADP⁺complex) and an additional 1.6 ns component when the formation of Chlide-F696 (the Chlide-POR-NADPH complex) was promoted by exogenous NADPH. From the fluorescence lifetime results we evaluated the quantum yield of the primary photoreaction by Chlide-F696 as being 70%.     

Photophysical Properties of Pyrazinopsoralen,A New Monofunctional Psoralen*

Abstract— Pyrazinopsoralen (PzPs), a new monofunctional psoralen, has a UV absorption spectrum similar to other psoralens except that it absorbs more strongly in the long-UVA than 8-methoxypsoralen. The solvent effects on the UV absorption and fluorescence emission spectra indicate that the lowest excited singlet state is the π,π* state like other psoralen derivatives. It shows a much lower fluorescence quantum yield (0.0008 in ethanol at room temperature) than the other psoralens as expected by the increased proximity effect (vibronic perturbation) due to close ¹(n,π*) to ¹(π,π*) states. The fluorescence lifetime was 1.05 ns in methylcyclohexane with a single exponential decay, while more than two components were observed in other solvents with the short-lived component being the major (>95%). The triplet state of PzPs could not be detected by phosphorescence, laser flash excitation (T-T absorption) and singlet oxygen formation probably due to very low φ_isc, or short lifetime of the triplet state (τ_T) caused by the fast T₁→ S₀ intersystem crossing.     

FLUORESCENCE AND THE LOCATION OF TRYPTOPHAN RESIDUES IN PROTEIN MOLECULES

Abstract— Fluorescence spectra of a number of native and denaturated proteins have been analysed, using spectral band width (ΔΛ), spectral maximum position (Λ_m), fluorescence quenching by external ionic quenchers, lifetime (b), and quantum yield ( q ) and its changes upon denaturation. The results enabled a model of fluorescence properties of tryptophan residues in the proteins to be substantiated by considering the existence of three discrete spectral classes, one buried in nonpolar regions of the protein (Λ_m 330–332 nm, ΔΛ= 48–49nm, q 0.11, τ= 2.1 ns) and two on the surface. One of the latter is completely exposed to water (Λ_m# 350–353 nm, ΔΛ= 59–61 nm, q # 0.2, τ= 5.4 ns); the other is in limited contact with water which is probably immobilized by bonding at the macromolecular surface (Λ_m# 340–342 nm, ΔΛ= 53–55 nm, q # 0.3, = 4.4 ns). Some quantitative predictions from the model, for (a) the fraction of fluorescence that is quenched by ionic quenchers, (b) the mean values of quantum yield, and (c) the mean values of fluorescence lifetime for various proteins, show good concordance with independent experimentally determined values.     

Excited state geometry of and radiationless processes in the lowest B3u(nπ*) singlet state of s-tetrazine

High resolution absorption and laser induced emission spectra of the lowest B_3u(nπ^*) singlet state of s-tetrazine-h₂ and -d₂ in a benzene crystal at 1.8 K are presented and discussed. The absorption spectrum with origin at 17231 cm^?1 (h₂) is dominated by a progression in ν_6a and a Herzberg-Teller origin which has been assigned as ν₁. The absence of ν₁ in the emission spectrum is explained as being due to a destructive vibronic interference effect. The Franck-Condon envelope of the unique ν_6a progression in emission is used for a determination of the excited state structure and the limitations of this procedure are examined. Direct lifetime measurements using a dye laser and single photon counting techniques show the fluorescence lifetime of s-tetrazine-h₂ and -d₂ to be shorter than 1.5 ns. From a deconvolution of the emission pulse of dimethyl s-tetrazine its fluorescence lifetime in the gas phase is found to be 6.0 ± 0.3 ns. Through a comparison of the fluorescence quantum yield of s-tetrazine-h₂ and dimethyl s-tetrazine we calculate for s-tetrazine-h₂ a fluorescence lifetime of 1.5 ± 0.2 ns and a fluoresence quantum yield of 1.8 × 10^?3. The ratio of the emissive lifetimes of s-tetrazine-d₂ and -h₂ was measured from relative fluorescence yields and found to be 1.18 ± 0.05. Photodissociation quantum yield studies on s-tetrazine-h₂, -d₂ and dimethyl for excitation into the origin of the ¹B_3u(nπ^*) state show this yield to be in the range of 1.3 ± 0.3, and this could explain the low fluorescence yields of the s-tetrazines. The fluorescence quantum yields in the gas phase are found to vary among the vibronic levels of the ¹B_3u state. This finding is in agreement with earlier measurements by Vemulapalli and Cassen, but the report by these authors that such quantum yield variations also occurred in the rovibronic structure is not confirmed.     

Time-resolved Luminescence and Singlet Oxygen Formation After Illumination of the Hypericin–Low-density Lipoprotein Complex

Time-resolved fluorescence and phosphorescence study of hypericin (Hyp) in complex with low-density lipoproteins (LDL) as well as the evolution of singlet oxygen formation and annihilation after illumination of Hyp/LDL complexes at room temperature are presented in this work. The observed shortening of the fluorescence lifetime of Hyp at high Hyp/LDL molar ratios (>25:1) proves the self-quenching of the excited singlet state of monomeric Hyp at these concentration ratios. The very short lifetime (∼0.5 ns) of Hyp fluorescence at very high Hyp/LDL ratios (>150:1) suggests that at high local Hyp concentration inside LDL molecules fast and ultrafast nonradiative decay processes from excited singlet state of Hyp become more important. Contrary to the lifetime of the singlet excited state, the lifetime (its shorter component) of Hyp phosphorescence is not dependent on Hyp/LDL ratio in the studied concentration range. The amount of singlet oxygen produced as well as the integral intensity of Hyp phosphorescence after illumination of Hyp/LDL complexes resemble the dependence of the concentration of molecules of Hyp in monomeric state on Hyp/LDL until a concentration ratio of 60:1. This fact confirms that only monomeric Hyp is able to produce the excited triplet state of Hyp, which in aerobic conditions leads to singlet oxygen production. The value of singlet oxygen lifetime (∼8 μs) after its formation from the excited triplet state of Hyp in LDL proves that molecules of singlet oxygen remain for a certain period of time inside LDL particles and are not immediately released to the aqueous surrounding. That Hyp exists in the complex with LDL in the monodeprotonated state is also demonstrated.     

Fluorescence properties of fluoranthene as a function of temperature and environment

The fluoranthene fluorescence properties were investigated in a variety of environments, i.e. in different solvents and temperatures, in the solid state, and in the vapor phase. The emission maximum was found to be independent of environment. The absorption spectrum in different solvents exhibits only minor changes. In solution, the fluorescence lifetime shows a slight inverse relationship to the solvent dielectric constant. With water/methanol mixtures of varying composition as the solvent, the lifetime decreases linearly with increasing mole fraction of water. At 77K, the fluoranthene fluorescence lifetime in frozen polar and nonpolar solvents are the same within experimental error. In hexane the fluorescence lifetime is independent of temperature (77±3 and 82±7 ns, at room temperature and 77 K, respectively). In methanol the lifetime is 64±3 ns at room temperature and increases linearly to 80±4 ns at 77 K. In the vapor phase the lifetime is 32±1 ns. No fluorescence quantum yield change was observed for either S₁ or S₂ manifold excitation.     

THE FLUORESCENCE PROPERTIES OF ORTHO AMINOBENZOATE ANESTHETICS IN DEFINED SOLVENTS AND PHOSPHOLIPID VESICLES

Abstract— The fluorescence properties of three ortho aminobenzoate local anesthetics have been determined in a variety of solvents. Results from these studies have been used to deduce how these drugs interact with phosphatidylcholine bilayers. The emission energy, fluorescence quantum yield and lifetime exhibited a biphasic dependence on solvent polarity. In aprotic solvents, alcohols and in ethanol-water mixtures containing less than 40% water, quantum yields and lifetimes were high (approximately 0.55 and 8.5 ns respectively). In ethanol-water mixtures containing >40% water, the strong fluorescence quenching was primarily due to an increase in the rate of non-radiative deactivation of the excited state. Both the radiative ( k_r ) and non-radiative ( k_nr ) rate constants show a biphasic dependence on solvent polarity. These studies suggest the presence of two singlet excited states for these molecules, a polar singlet excited state, S_1-p and a charge transfer excited state, S_1-ct with the latter predominating in ethanol-water mixture containing >40% water. In egg phosphatidylcholine bilayers, the fluorescence, lifetime and quantum yield are consistent with the view that these drugs are localized within the lipid head group region where the charge-transfer excited state can be stabilized by intermolecular hydrogen bonding.     

Photophysical behavior of a dimeric cyanine dye (BOBO-1) within cationic liposomes

This study is aimed at establishing optimal conditions for the use of 2,2'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl-1(4H)-pyridinyl-4-ylidenemethy-lidyne]]bis[3-methyl]-tetraiodide (BOBO-1) as a fluorescent probe in the characterization of lipid/DNA complexes (lipoplexes). The fluorescence spectra, anisotropy, fluorescence lifetimes and fluorescence quantum yields of this dimeric cyanine dye in plasmid DNA (2694 base pairs) with and without cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]), are reported. The photophysical behavior of the dye in the absence of lipid was studied for several dye/DNA ratios using both supercoiled and relaxed plasmid. At dye/DNA ratios (d/b) below 0.01 the fluorescence intensity increases linearly, whereas lifetime and anisotropy values of the dye are constant (tau approximately 2.5 ns and = 0.20). By agarose gel electrophoresis it was verified that up to d/b = 0.01 DNA conformation is not considerably modified, whereas for d/b = 0.05-0.06 a single heavy band appears on the gel. For these and higher dye/DNA ratios the fluorescence intensity, anisotropy and average lifetime values decrease with an increase in BOBO-1 concentration. When cationic liposomes are added to the BOBO-1/DNA complex, an additional effect is noticed: The difference in the environment probed by BOBO-1 bound to DNA leads to a decrease in quantum yield and average lifetime values, and a redshift is apparent in the emission spectrum. For fluorescence measurements including energy transfer (FRET), a d/b ratio of 0.01 seems to be adequate because no considerable change on DNA conformation is detected, a considerable fluorescent signal is still measured after lipoplex formation, and energy migration is not efficient.     

EFFECTS OF PIGMENT HETEROGENEITY ON FLUORESCENCE IN PHOTOSYNTHETIC UNITS

A computer solution of the Pauli Master Equation is used to study the fluorescence quantum yield of a two-dimensional random host-trap-anti-trap system. Calculations predict that both the quantum yield and prompt fluorescence lifetime from photosynthetic units increase as pigment heterogeneity increases. From the temperature dependent action spectrum of fluorescence the pairwise Forster transfer rate is predicted to decrease with decreasing temperature.     

Spectral,lifetime and photochemical characteristics of 4-acetoxy-chalcone in viscous media

Both fluorescence and excitation spectra of 4-acetoxy-chalcone (4-AC) are bathochromically shifted as the medium polarity increases. The fluorescence quantum yields are sensitive to medium viscosity and increase sharply as the medium viscosity increases due to prohibition of radiationless deactivation pathways. The fluorescence lifetime of 4-AC in glycerol has been measured as τ = 1.9 ns. The photochemical quantum yields (thought to be a trans-cis photo-isomerization) are also sensitive to medium viscosity with minimum φ_c values obtained in highly viscous solvents. The effect of medium viscosity on the quenching of 4-AC fluorescence using picric acid as a quencher has been studied. A static-type quenching mechanism is proposed to account for the quenching efficiency as the medium viscosity increases.     

Observation of ultrafast Q-band fluorescence in horse heart cytochrome c in reduced and oxidized forms

The dynamics of fluorescence from horse heart cytochrome c is investigated in reduced (ferrous) and oxidized (ferric) forms by a streak camera and an up-conversion technique under B-band excitation at 415 nm. In the reduced form, we found the Q-band emission at 550 and 600 nm originated from the S(1) state in a short time range. A very broad continuum observed from 440 to 660 nm had only shown a slow component and was assigned to impurity. In the reduced form, the lifetime of S(1) was determined to be 120 fs by using the up-conversion technique. In the oxidized form, the S(1) lifetime was estimated to be 21 fs. These values are consistent with the values estimated from the quantum yield in order of their magnitude.     

Anomalous fluorescence of aceanthrylene: An example of second excited state emission (S2 → S0)

An anomalous fluorescence with a quantum yield of 0.017 near 24000 cm^?1 is detected from hexane solutions of aceanthrylene. Excitation and synchronous scan spectra verify that the second excited state with a lifetime of 4.3 ns is the source of the fluorescence. The absorption spectrum is simulated by PPP SCF CI computations.