Two-photon excitation spectrum of fluorescence of the light-harvesting complex B800–850 from Allochromatium minutissimum within 1200–1500 (600–750) nm spectral range is not carotenoid mediated

Two types of peripheral light-harvesting complexes LH2 (B800–850) from photosynthetic purple bacterium Allochromatium minutissimum were studied. First type containing carotenoids was prepared from wild type cells. The other one was obtained from carotenoid depleted cells grown with diphenylamine. We have shown that under laser femtosecond excitation within absorption 1200–1500 nm wavelength range the two-photon excitation of LH2 complexes takes place. This can be observed as fluorescence of bacteriochlorophyll (BChl) spectral form B850 (BChl molecules of circular aggregate with strong exciton interaction in 850 nm spectral domain). LH2 fluorescence excitation spectra under two-photon excitation are the same for carotenoid-containing and carotenoidless preparations. In both cases the broad band with peak near 1350 (675) nm (FWHM ～ 240 (120) nm) was found. It is concluded that the broad band with peak near 1350 (675) nm in two-photon excitation spectra of LH2 complexes from Allochromatium minutissimum cannot be interpreted as two-photon excitation band of the optically forbidden S₀ → S₁ transition of carotenoids (rhodopin). Possible nature of this band is discussed.     

On indicator dilution and perfusion heterogeneity: A stochastic model

Unidirectional extraction of a substrate S in the capillaries following the arterial injection of a bolus containing S and a reference tracer R is assumed to follow first-order kinetics. If C_R and C_S denote normalized venous effluent concentrations of R and S, respectively, let L(t)=ln[C_R(t)⧸C_S(t)]. We derive a formula which expresses the experimental L(t) data in terms of the mean μ(t) and variance of the transit times of those capillaries which are contributing indicators at each sample time t. We examine the information thus contained in the L data about capillary and noncapillary transit times under several kinematic assumptions. We show that if the capillary and noncapillary transit times are stochastically independent with frequency functions h_c(t) and h_av(t), respectively, then the shapes of the graphs of L(t) and μ(t) depend on the variances and skewnesses of h_c(t) and h_av(t). Specifically, let r₂ be the ratio of the variance of h_c(t) to the variance of h_av(t), and let r₃ be the ratio of skewnesses in the same order. Then the graph of μ(t) is concave downward if r₂⧸r₃ > 1, concave upward if r₂⧸r₃< 1, and linear if r₂⧸r₃ = 1. If the fraction of S extracted is not too large, L(t) has nearly the same shape as μ(t), and therefore, L(t) contains information about h_c(t) and h_av(t).     

Time-dependent rotational rates of excited fluorophores. A linkage between fluorescence depolarization and solvent relaxation

It is generally assumed that the rotational diffusion coefficients of fluorophores are independent of time subsequent to excitation, and that the rotational diffusion coefficients of the ground and the excited states are the same. We now describe a linkage between the extent of solvent relaxation and the rate of fluorescence depolarization. Specifically, if a fluorophore displays time-dependent solvent relaxation it may also show a time-dependent decrease in its rotational rate. A decreased rate of rotation could result from the increased interaction with polar solvent molecules which occurs as a result of solvent relaxation. The decays of anisotropy predicted from our model closely mimic those often observed for fluorophores which are bound to macromolecules. For example, the decays are more complex than a single exponential, and the time-resolved anisotropy can display a limiting value which does not decay to zero. The effect of solvent relaxation upon the rates of rotational diffusion is expected to be most dramatic for solvent-sensitive fluorophores in a viscous environment. These conditions are frequently encountered for fluorophore-macromolecule complexes. Consideration of the linkage between solvent relaxation and rotational diffusion leads to two unusual predictions. First even spherical fluorophores in an isotropic environment could display multi- or nonexponential decays of fluorescence anisotropy. Secondly, for the special case in which the fluorophore dipole moment decreases upon excitation, the theory predicts that the anisotropy decay rate may increase with time subsequent to pulsed excitation. The predictions of this theory are consistent with published data on the effects of red-edge excitation upon the apparent rotational rates of fluorophores in polar solvents.     

Picosecond multiphoton scanning near-field optical microscopy.

We have implemented simultaneous picosecond pulsed two- and three-photon excitation of near-UV and visible absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 1064-nm emission from a pulsed Nd:YVO4 laser was used to excite the visible mitochondrial specific dye MitoTracker Orange CM-H2TMRos or a Cy3-labeled antibody by two-photon excitation, and the UV absorbing DNA dyes DAPI and the bisbenzimidazole BBI-342 by three-photon excitation, in a shared aperture SNOM using uncoated fiber tips. Both organelles in human breast adenocarcinoma cells (MCF 7) and specific protein bands on polytene chromosomes of Drosophila melanogaster doubly labeled with a UV and visible dye were readily imaged without photodamage to the specimens. The fluorescence intensities showed the expected nonlinear dependence on the excitation power over the range of 5-40 mW. An analysis of the dependence of fluorescence intensity on the tip-sample displacement normal to the sample surface revealed a higher-order function for the two-photon excitation compared to the one-photon mode. In addition, the sample photobleaching patterns corresponding to one- and two-photon modes revealed a greater lateral confinement of the excitation in the two-photon case. Thus, as in optical microscopy, two-photon excitation in SNOM is confined to a smaller volume.     

Three-photon excitation of N-acetyl-L-tyrosinamide.

We observed emission from the tyrosine derivative N-acetyl-L-tyrosinamide (NATyrA) when excited with the fundamental output of a femtosecond Ti:Sapphire laser from 780 to 855 nm. The dependence on incident laser power indicates a three-photon process. The emission spectra and intensity decay in glycerol-water (30:70) at 5 degrees C were found to be identical for one- and three-photon excitation. Also the excitation spectrum of three-photon-induced fluorescence of NATyrA corresponds to the one-photon excitation spectrum. The time-zero or fundamental anisotropy spectrum was reconstructed from the frequency-domain anisotropy decays. The three-photon anisotropies are similar or larger than the one-photon anisotropies. These three-photon anisotropies are surprising given the near zero values known for tyrosine with two-photon excitation. The observations indicate that one- and three-photon excitation directly populates the same singlet excited states(s). However, the origin of the anisotropies with multi-photon excitation of tyrosine remain unclear and unpredictable.     

Differential effects of senescence on the molecular organization of membranes in ripening tomato fruit

Changes in the molecular organization of membranes in pericarp cells of ripening tomato fruit were examined by fluorescence depolarization after labeling with fluorescent lipid-soluble probes. The fluorescent labels were partitioned into isolated protoplasts and purified plastids from fruit at various stages of senescence. Values for steady-state anisotropy (r_ss) of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled protoplasts rose progressively during the early stages of ripening over a time frame that overlapped the climacteric rise in ethylene production. This can be interpreted as reflecting a decrease in the lipid fluidity of primarily plasma membrane. By contrast, there was no significant change during ripening in r_ss for plastid membranes labeled with DPH, 1-[4-trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cis- or trans-parinaric acid. Nor was there any change during ripening in the limiting fluorescence anisotropy (r_oo) and order parameter (S) for plastids labeled with DPH or TMA-DPH, parameters that are corrected for any differences in lifetime. Some degree of lifetime heterogeneity, possibly reflecting structurally distinct domains, was discerned in both young and senescent plastids that had been labeled with DPH or TMA-DPH, but this also did not change as ripening progressed. Thus membranes of the pericarp cells sustain different fates as the tomato fruit ripens, implying that there are distinguishable mechanisms of membrane deterioration in senescing tissues.     

Fluorescence imaging with two-photon evanescent wave excitation

We demonstrate broad-field, non-scanning, two-photon excitation fluorescence (2PEF) close to a glass/cell interface by total internal reflection of a femtosecond-pulsed infrared laser beam. We exploit the quadratic intensity dependence of 2PEF to provide non-linear evanescent wave (EW) excitation in a well-defined sample volume and to eliminate scattered background excitation. A simple model is shown to describe the resulting 2PEF intensity and to predict the effective excitation volume in terms of easily measurable beam, objective and interface properties. We demonstrate non-linear evanescent wave excitation at 860 nm of acridine orange-labelled secretory granules in live chromaffin cells, and excitation at 900 nm of TRITC-phalloidin-actin/GPI-GFP double-labelled fibroblasts. The confined excitation volume and the possibility of simultaneous multi-colour excitation of several fluorophores make EW 2PEF particularly advantageous for quantitative microscopy, imaging biochemistry inside live cells, or biosensing and screening applications in miniature high-density multi-well plates.Abbreviations 1PEF one-photon excited fluorescence - 2PEF two-photon excited fluorescence - APD avalanche photo diode - CHO Chinese hamster ovary - DMEM Dulbecco's modified Eagle's medium - EGFP enhanced green fluorescent protein - EW evanescent wave - FCS fetal calf serum - GPI glycosylphosphatidylinositol - TIR total internal reflectionThis paper is dedicated to the memory of Prof. Horst Harreis (1940–2002)     

Photolysis of the ion pair [Pt(NH3)5Cl]S2O8: reduction of platinum(IV) induced by outer-sphere charge transfer excitation

The ion pair [Pt^IV(NH₃)₅Cl]³⁺S₂O₈²⁻ shows a S₂O₈²⁻ → [Pt(NH₃)₅Cl]³⁺ outer-sphere charge transfer (OSCT) absorption at λ_max=267 nm. OSCT excitation leads to the reduction of Pt(IV) by S₂O₈²⁻ to Pt(II) with φ=3×10⁻³ at λ_irr=280 nm.     

Mercury(II) binding to s4U in E. coli tRNA(Val)

The accessibility of the s⁴U base in native tRNA^Val from E.coli was monitored by studying the binding of various mercurials. The relative binding order HgBr₂[unk]HgCl₂CH₃HgOAc[unk]CH₃HgCl[unk]PCMB parallels approximately the steric requirements of linear HgX₂ or RHgX compounds for S_N2 displacement by sulfur, although other factors are operative. Para-chloromercuri-benzoate (PCMB) does not bind the thiolated nucleotide unless the tertiary structure of the tRNA is opened up by removal of Mg²⁺ ions and heating to 40°. Under these conditions, equilibrium dialysis measurements using ¹⁴C-labeled PCMB showed one binding site (n = 0.93) with an association constant, K₁, of 9 × 10⁴M⁻¹.     

Mobility of fluorescent probe molecules in lipid bilayer vesicles as studied by steady-state and time-dependent nuclear Overhauser effect measurements in 1H-nuclear magnetic resonance spectroscopy

In order to elucidate the mobilities of the fluorophores of fluorescent 2- and 16-(9-anthroyloxy)palmitic acids (16-AP and 2-AP, respectively) in lipid bilayer vesicles, the steady-state and time-dependent nuclear Overhauser effects in ¹H-NMR spectroscopy, but not the fluorescence depolarization in fluorescence spectroscopy, have been measured. The steady-state nuclear Overhauser effect measurements showed an appreciable magnitude of negative nuclear Overhauser effects between the resonances due to the fluorophores of the two fluorescent probes and lipids. These results definitely mean that in lipid bilayers, the fluorophores (anthroyloxy ring) of the fluorescent probes experience other types of motions with much longer correlation times than those detected by the fluorescence depolarization measurements, since at the correlation time showed by the fluorescent method (1–2 · 10⁻⁹ s or less), no such transfer of the negative nuclear Overhauser effects is expected to occur. The correlation times of the fluorophores, as calculated from the cross-relaxation rates of the anthroyl ring protons of 16-AP and 2-AP, were 3.8 · 10⁻⁸ and 1.1 · 10⁻⁷ s, respectively. These values, respectively, compare favorably with those of the terminal methyl of acyl chains and the choline methyl carbons which were estimated by ¹³C T₂ relaxation times. Thus, it is concluded that the fluorophores of both 16-AP and 2-AP have a slow form of motion which moves with a similar time scale to those of lipids in addition to the faster one that causes fluorescence depolarization.     

Synthesis, structure and reactivity of complexes containing [M(S2)2] fragments (M=Ru, Os; (S2)=1,2-benzenedithiolate (2−))

Subsequent addition of 1,2-benzenedithiol (S₂-H₂) and nBuLi to a solution of [Ru(NO)Cl₃ · xMeOH] in THF afforded exclusively the monomeric species NBu₄[Ru^II(NO)(S₂)₂] (1). Formation of dimeric (NBu₄)₂[Ru^II(NO)(S₂)₂]₂ (2) has been confirmed when the deprotonated ligand S₂-Li₂ was added to [Ru(NO)Cl₃ · xMeOH] and allowed to stir for 30 h. The monomer 1 undergoes aerial oxidation to give (NBu₄)₂[Ru^IV(S₂)₃] (3). The reaction between RuCl₃ · xH₂O and S₂-H₂ in the presence of NaOMe, afforded the dinulear Ru^III species (NMe₄)₂[Ru^III(S₂)₂]₂ (4). A modified method for the preparation of 1 is being employed to synthesize the osmium analogue NBu₄[Os(NO)(S₂)₂] (5) effectively. The solid state structures of 1, 2 and 3 were determined by X-ray crystal structure analysis. A comparison of relevant bond distance data suggests that 1,2-benzenedithiolate acts as an “innocent” ligand.     

A model of photosystem II for the analysis of fast fluorescence rise in plant leaves

The polyphasic patterns of fluorescence induction rise in pea leaves in vivo and after the treatment with ionophores have been studied using a Plant Efficiency Analyzer. To analyze in detail photosystem II (PS II) electron transfer processes, an extended PS II model was applied, which included the sums of exponential functions to specify explicitly the light-driven formation of the transmembrane electric potential (ΔΨ(t)) as well as pH in the lumen (pH_L(t)) and stroma (pH_S(t)). PS II model parameters and numerical coefficients in ΔΨ(t), pH_L(t), and pH_S(t) were evaluated to fit fluorescence induction data for different experimental conditions: leaf in vivo or after ionophore treatment at low or high light intensity. The model imitated changes in the pattern of fluorescence induction rise due to the elimination of transmembrane potential in the presence of ionophores, when ΔΨ = 0 and pH_L(t), pH_S(t) changed to small extent relative to control values in vivo, with maximum ΔΨ(t) ∼ 90 mV and ΔΨ(t) ∼ 40 mV for the stationary state at ΔpH ≅ 1.8. As the light intensity was increased from 300 to 1200 μmol m⁻² s⁻¹, the heat dissipation rate constants increased threefold for nonradiative recombination of P680⁺Phe⁻ and by ∼30% for P680⁺Q_A⁻. The parameters ΔΨ, pH_S and pH_L were analyzed as factors of PS II redox state populations and fluorescence yield. The kinetic mechanism of fluorescence quenching is discussed, which is related with light-induced lumen acidification, when ⁺Q_A⁻ and P680⁺ recombination probability increases to regulate the Q_A reduction.     

A Birefringence Relaxation Determination of Rotational Diffusion of Magnetotactic Bacteria

The orientational relaxation of the magnetotactic bacterium Aquaspirillum magnetotacticum is observed by the decay of the optical birefringence upon switching off an aligning magnetic field. The data yield a rotational diffusion constant D_r [unk] 0.13 s^-1 and information about cell sizes that is consistent with optical microscopy data.     

Effects of bee venom melittin on the order and dynamics of dimyristoylphosphatidylcholine unilamellar and multilamellar vesicles

The effects of bee venom melittin on the order and dynamics of dimyristoylphosphatidylcholine unilamellar and multilamellar vesicles at a protein-to-lipid molar ratio of 1:60 have been investigated by employing the techniques of nanosecond emission anisotropy with 1,6-diphenyl-1,3,5-hexatriene as the fluorescent probe, enhancement by polar groups of the weakly allowed 0-0 vibronic transition in the fluorescence spectrum of pyrene, and Raman spectroscopy. The emission anisotropy results, which are found to be consistent with the wobble-in-cone model, show that the protein induces an increase in the order parameter, S, of the acyl chains of unilamellar vesicles below, at, and above their phase transition temperature, T_t, and it decreases strongly the diffusion rate, D_w, only below T_t. On the other hand, for multilamellar vesicles, the protein induces a decrease in S only at T_t and does not affect D_w. These effects are consistent with the observed changes in the degree of enhancement of the 0-0 vibronic transition of pyrene. Moreover, the protein broadens the thermal transition profile of multilamellar vesicles but sharpens dramatically that of unilamellar vesicles and fuses them without changing significantly the T_t in either case. On the other hand, the Raman data detect a decrease in the inter- and intramolecular order of the acyl chains of multilamellar vesicles below T_t and a decrease of only the former above T_t. This disparity between the Raman and the nanosecond emission anisotropy data is discussed in terms of differences in the time scales of the two techniques and in the state of aggregation of the lipid-bound melittin. The data for the enhancement of the 0-0 vibronic transition of pyrene suggest that, for a melittin-to-lipid ratio of 1:60, the size or structure of channels formed in the bilayer by melittin does not allow the penetration of a neutral molecule the size of pyrene deeply into the bilayer.     

Anisotropy-based sensing with reference fluorophores

We describe a new approach to fluorescence sensing based on measurements of steady-state anisotropies in the presence of reference fluorophores with known anisotropies. The basic concept is that the anisotropy of a mixture reflects a weighted average of the anisotropies of the emitting species. By use of reference fluorophores the starting anisotropy can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the analyte result in changes in anisotropy. A wide dynamic range of anisotropies is available because of the freedom to select high or low starting values. Anisotropy-based sensing was demonstrated for pH using 6-carboxyfluorescein and for protein affinity or immunoassay using an oriented film with high anisotropy and a protein labeled with a metal-ligand complex. The latter measurements were performed with a simple light-emitting diode excitation source without an excitation polarizer. The sensitive range of the assay can be adjusted by changing the intensity of the reference fluorophore. Anisotropy-based sensing can have numerous applications in clinical and analytical chemistry.     

Photobleaching in two-photon excitation microscopy

The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study thin samples may be limited by increased photobleaching.     

A ferromagnetically coupled copper(II) trinuclear secondary building unit as precursor for the preparation of molecule-based magnets

The compounds 3D-{(Ph₄P)₂[ZnCu₃(Hmesox)₃Cl]·2.5H₂O} (1) and 3D-{(Ph₄P)₂[NiCu₃(Hmesox)₃Cl]·2.5H₂O} (2) have been prepared and their structure and magnetic properties investigated (H₄mesox = mesoxalic acid, 2-dihydroxymalonic acid). The compounds are obtained by means of the same procedure followed to prepare 3D-{(Ph₄P)₂[MnCu₃(Hmesox)₃Cl]·3.5H₂O}and 3D-{(Ph₄P)₂[CoCu₃(Hmesox)₃Cl]}, to which 1 and 2 are isostructural as deducted from the X-ray powder diffraction patterns and IR spectra. During the synthesis the {Cu₃(Hmesox)₃Cl}⁴⁻ unit is generated which acts as ferromagnetically coupled secondary building unit (SBU) to give 3D chiral networks with (10,3)-a topology, the [Zn{Cu₃(C₃HO₆)₃Cl}]²ⁿ⁻ and [Ni{Cu₃(C₃HO₆)₃Cl}]²ⁿ⁻, in 1 and 2, respectively. The tetraphenylphosphonium cations are located in the voids of the 3D anion framework giving a supramolecular 3D cation net with the same (10,3)-a topology as the anion framework and both can be thought of forming interpenetrating supramolecular and covalent (10,3)-a nets. Because of the different nature of the interpenetrated 3D (10,3)-a frameworks a single crystal of 1 and 2 is chiral and enantiopure. The analysis of the magnetic properties in 1 by means of the isotropic spin Hamiltonian, H = −J(S₁S₂ + S₂S₃ + S₁S₃), reveal a ferromagnetic coupling with J = +11.2 cm⁻¹ in the copper(II) trinuclear unit of 1. Compound 2 exhibits long-range magnetic ordering with a T_c of 7.0 K due to a ferrimagnetic coupling between the ferromagnetically coupled copper(II) ions of the trinuclear unit, antiferromagnetically coupled to the Ni(II) ions.     

Influence of glucose-templated proline mimetics on the β-turn conformation of the peptide fragment Ac-Leu-d-Phe-Pro-Val-NMe2 found in Gramicidin S

The synthesis of tetrapeptide-based β-turn mimetics containing spirocyclic glucose-templated 3-hydroxyproline hybrids Glc3′(S)-5′(R)(CH₂OH)HypH and Glc3′(S)-5′(S)(CH₂OH)HypH as proline mimetics is presented. NMR-based conformational analysis of Ac-Leu-d-Phe-[Glc3′(S)-5′(R)(CH₂OH)HypH]-Val-NMe₂ and Ac-Leu-d-Phe-[Glc3′(S)-5′(S)(CH₂OH)HypH]-Val-NMe₂ demonstrates the presence of β-turn conformations. Different turn structures were observed by changing the stereochemistry at 5′-position of Glc3′(S)-5′(R)(CH₂OH)HypH. The major prolyl amide cis isomer of glucose-protected tetrapeptide Ac-Leu-d-Phe-[Glc(MOM)₄3′(S)-5′(R)(CH₂OMOM)HypH]-Val-NMe₂11 and glucose unprotected Ac-Leu-d-Phe-[Glc3′(S)-5′(R)(CH₂OH)HypH]-Val-NMe₂13 forms a type VI β-turn conformation. In contrast, the major prolyl amide trans rotamer of tetrapeptide Ac-Leu-d-Phe-[Glc(MOM)₄3′(S)-5′(S)(CH₂OMOM)HypH]-Val-NMe₂12 conserves a similar β-turn conformation as the Gramicidin S-based peptide fragment Ac-Leu-d-Phe-Pro-Val-NMe₂16.     

Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM)

We describe a novel variant of fluorescence lifetime imaging microscopy (FLIM), denoted anisotropy-FLIM or rFLIM, which enables the wide-field measurement of the anisotropy decay of fluorophores on a pixel-by-pixel basis. We adapted existing frequency-domain FLIM technology for rFLIM by introducing linear polarizers in the excitation and emission paths. The phase delay and intensity ratios (AC and DC) between the polarized components of the fluorescence signal are recorded, leading to estimations of rotational correlation times and limiting anisotropies. Theory is developed that allows all the parameters of the hindered rotator model to be extracted from measurements carried out at a single modulation frequency. Two-dimensional image detection with a sensitive CCD camera provides wide-field imaging of dynamic depolarization with parallel interrogation of different compartments of a complex biological structure such as a cell. The concepts and technique of rFLIM are illustrated with a fluorophore-solvent (fluorescein-glycerol) system as a model for isotropic rotational dynamics and with bacteria expressing enhanced green fluorescent protein (EGFP) exhibiting depolarization due to homotransfer of electronic excitation energy (emFRET). The frequency-domain formalism was extended to cover the phenomenon of emFRET and yielded data consistent with a concentration depolarization mechanism resulting from the high intracellular concentration of EGFP. These investigations establish rFLIM as a powerful tool for cellular imaging based on rotational dynamics and molecular proximity.     

Influence of excitation light intensity and leaf age on the slow chlorophyll fluorescence transient in radish

The temporal characteristics of the slow phase of chlorophyll fluorescence induction-T _0.5 (half-decay time) and t _min (an integral-based index of the variable emission rate)-as well as the popular amplitude index F _P/F _S were determined at different excitation light intensities (I _ex [400?C500 nm] of 20?C80 W/m²) in dark-adapted leaves of different age (3?C24 days) taken from radish plants grown under continuous light of 100 W/m² PAR. All the profiles thus obtained were mutually consistent, and the age-related variations were minimized at I _ex > 40 W/m²; at that the age-averaged temporal indices proved to be more light-responsive than the standard amplitude ratio.