Fluorescence studies on R-phycoerythrin and C-phycoerythrin

Biliproteins are photosynthetic light-harvesting proteins, which transfer excitons with high efficiencies over relatively long distances until they arrive at a photosynthetic reaction center. Purified R-phycoerythrin (isolated from a red alga) and C-phycoerythrin (isolated from a cyanobacterium), each of which contains several chromophores, were studied by a combination of fluorescence emission, fluorescence excitation polarization, and absorption methods. The polarization spectra of both these biliproteins showed that there was a minimum of two spectrally distinct sensitizing chromophores, which, after absorbing photons, transfer excitons to the lowest-energy (fluorescing) chromophores. Some of these spectroscopic data were used to deconvolute the absorption spectra into the spectra of the two sensitizing and one fluorescing chromophores. It was shown that the higher-energy sensitizing chromophore could readily transfer its excitation energy to the fluorescing chromophore using the lower-energy sensitizing chromophore as an intermediary. However, there was sufficient spectral overlap between the higher-energy sensitizing chromophore and the fluorescing chromophore so that direct transfer between them could not be ruled out.     

Spin polarization transfer during intramolecular triplet-triplet energy transfer as studied by time resolved EPR spectroscopy

Spin polarization conservation during intramolecular triplet-triplet energy transfer was studied for the phthalimide derivatives. It is shown that spin polarization transfer is useful for determining the conformation and the nature of the lowest triple (T₁) states of the donor and acceptor moieties. The polarization pattern of the acceptor triplet state was well reproduced taking into account the polarization of the donor and the mutual orientation of the donor and acceptor moieties. This technique clarified the order of the triplet sublevels in energy of the phthalimide chromophore. The T₁ states of stilbene derivatives, which have a low quantum yield of intersystem crossing under direct excitation, were also detected.     

pH-Dependent fluorescence and singlet energy transfer in water-soluble polymers containing eosin and phenol red chromophores

The pH-dependent fluorescence and energy transfer properties of water-soluble polyacrylamide labeled with a donor (eosin) and acceptor (phenol red) were studied. Eosin emission intensity decreases with pH increases. This observation corresponds to the expectation that the fluorescence intensity of eosin should diminish with pH increases resulting from increased spectral overlap between dyes. The lower energy transfer efficiency compared to a pH sensor made with the same polymer, but with a cross-linker, can be explained by the larger distances between the two chromophores resulting from conformational flexibility and electrostatic repulsion of the chromophore ions.     

Two-Photon Excited Fluorescence Energy Transfer: A Study Based on Oligonucleotide Rulers

The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor–acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation [1]. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules.     

Fluorescence anisotropy of cyanobacterial phycobilisomes oriented in polyvinyl alcohol (PVA) films

Polarized absorption (at 296 and 85 K), fluorescence, and photoacoustic (at 296 and 85 K) spectra of antenna complexes—phycobilisomes isolated from cyanobacteriaTolypothrix tenuis andOscillatoria and embedded in isotropic and anisotropic polyvinyl alcohol films—were measured. From the sets of polarized components of emission, the anisotropy of fluorescence for the pools of differently oriented molecules was calculated. On the basis of polarized photoacoustic and emission spectra, the competition between the process of thermal deactivation of excitation and excitation energy transfer in a chain of excitation donor and acceptor chromophores of phycobilisomes is discussed.     

Effect of a protein on the light energy conversion in dual fluorophore-nitroxide probes studied by ESR and fluorescence spectroscopy

Probing biological environment with dual fluorophore-nitroxide (FN) molecules in which fluorophore is tethered with nitroxide, a fluorescence quencher, opens unique opportunities to study molecular dynamics and micropolarity of the medium which affect intramolecular fluorescence quenching (IFQ), electron transfer, photoreduction and light energy conversion. In such molecules, the excited fragment of the chromophore can serve as an electron donor, and the nitroxide fragment as an electron acceptor. The same groups allow monitoring of molecular dynamics and also make it possible to measure micropolarity of the medium in the vicinity of the donor (by fluorescence technique) and acceptor (by electron spin resonance [ESR]) moieties. In the present work, two dual dansyl-nitroxide probes were incorporated in a binding site of bovine serum albumin. Their interactions with the protein, mobility, and photoreduction, as well as micropolarity of the media, have been studied by ESR and fluorescence methods. IFQ and spectral relaxation shift of the dansyl fragment have been monitored by time-resolved fluorescence technique. In parallel, computational studies on intramolecular dynamics of the FN probe were performed. On the basis of the Marcus model of the electron transfer between the excited dansyl fluorophore (donor) and nitroxide group (acceptor) and our experimental data, the mechanism of the electron transfer in the dual molecules incorporated into bovine serum albumin was established. It was shown that dual FN molecules in the protein meet main requirements for an efficient light energy conversion system.     

Effects of Internal Rotations on the Time-Resolved Fluorescence in a Bichromophoric Protein System Under the Energy Transfer Interaction

Effects of internal rotations of chromophores under the energy transfer interaction in proteins on the time-resolved fluorescence were examined by numerical calculations. Expressions used for the calculations are based on the approximations that the energy transfer takes place according to Foöurster's mechanism and the rotational motions of the energy donor and acceptor along the surfaces of cones are described by a set of rotational diffusion equations. The intensity decay of the donor depended a little on the rotational diffusion coefficient of the donor in some cases, while that of the acceptor did very little. Anisotropy of the donor decayed faster as the diffusion coefficient of the donor increased. Anisotropy decay of the acceptor markedly depended not only on the mutual configuration of the pair in the protein, but also on the diffusion coefficient of the donor. The dependence of the time-resolved fluorescence on the diffusion coefficient of the acceptor was not as great as that of the donor.     

绿荧光蛋白的双光子激发的荧光特性研究

利用双光子激发方式研究了重组绿荧光蛋白（recombinant green fluorescent protein,简称rGFP)的光转换特性,研究结果表明rGFP具有较强的双光子激发荧光,双光子激发的荧光偏振光谱表明rGFP在辐照前质子态和去质态之间存在着有效的能量转移过程,rGFP辐照后导致生色团构象的变化,部分阻断了rGFP内源的氨基酸与生色团之间的能量转移过程,导致rGFP的双光子激发的荧光强度下降,观察到rGFP的三光子激发的荧光特性,这种三光子激发的荧光主要来源于rGFP内源的氨基酸（色氨酸,酪氨酸等）的吸收。研究结果对在实际使用定量的光显微镜时具有一定的指导意义。     

Nature of the Electron Excited States and Energy Transfer in Bichromophore Coumarin Molecules

Results of experimental and theoretical research for three bichromophore molecules, trans-stilbene-CH₂-coumarin 120 (I), 4-methylumbelliferone-CH₂-UC 17, and 4-(3-fluoro)-methylumbelliferone-CH₂-UC 17 (II, III), are presented. Schemes of photophysical processes in the bichromophore molecules based on quantum chemical calculations by the INDO method and theory of radiationless transitions in polyatomic organic molecules are suggested. After optical excitation to the strong donor absorption band, the fast internal conversion processes develop there. As a result, the molecule is found in the S ₁ ^* -state localized on the acceptor moiety. It is shown that a mechanism of intramolecular transfer energy in bichromophores different from that proposed by Förster may be realized. Excitation energy, initially located on D, will be transferred from the donor moiety to the acceptor chromophore in convenience of the internal conversion process. The intramolecular electronic energy transfer from energy donor to energy acceptor may be interpreted as the internal conversion process. The rate constants of internal conversion are calculated.     

Electronic excitation energy transfer between two single molecules embedded in a polymer host

Unidirectional electronic excitation energy transfer from a photoexcited donor chromophore to a ground state acceptor chromophore - both linked by a rigid bridge - has been investigated by low temperature high-resolution single molecule spectroscopy. Our approach allows for accurately accessing static disorder in the donor and acceptor electronic transitions and to calculate the spectral overlap for each couple. By plotting the experimentally determined transfer rates against the spectral overlap, we can distinguish and quantify F?rster- and non-F?rster-type contributions to the energy transfer.     

Dynamics of fluorescence of solid solutions of quantum dots and the organic dye DODCI: Experiment and numerical simulation

The fluorescence of solid solutions of CdSe/ZnS quantum dots and the organic dye DODCI is investigated. It is shown that nonradiative transfer of electronic excitation energy to dye molecules, which with some probability lose their acceptor properties as a result of photoisomerization or photodegradation, is responsible for a significant increase in the fluorescence intensity of a donor. The degree of polarization of the donor fluorescence attains values exceeding 0.5, which is due to the difference in the fluorescence quantum yields of donors with different orientations of the oscillator with respect to the electric vector of an excitation light wave. A numerical simulation of the experimentally observed dependences is performed.     

Molecular nanocluster fluorescence in microwave infrared-radiation fields

The paper considers a donor-acceptor nanocluster fluorescing in a microwave infrared radiation field. It is assumed that the nanocluster consists of two dipole-dipole interacting organic molecules. It is shown that the fluorescence process of the nanocluster occurs if the donor molecule contains a substructure of identical diatomic pairwise interacting bonds of dipoles (an IR antenna). This antenna is capable of accumulating vibrational energy as a sum of collective vibrational quanta (excimols). The acceptor molecule has no permanent dipole moment and cannot be excited by microwave IR radiation. This molecule is polarized in the dipole moment field of the donor IR antenna and can receive energy accumulated in the IR antenna of the donor molecule. If the acceptor molecule has an electronically excited state in the long-wavelength visible region of its absorption spectrum, then after receiving energy equal to the energy of this state from the donor antenna, the electronic excitation of the acceptor molecule and its fluorescence is possible. As an example, the fluorescence of a nanocluster is considered whose donor molecule has a C _n H_2n IR antenna. The acceptor molecule is aromatic and the external infrared frequency, 1.1 × 10¹⁴ s^?1, is equal to the frequency of the excimol in the donor infrared antenna.     

金属卟啉分子组装体系中的三线态能量传递

利用稳态和瞬态光谱技术研究了人工组装锌卟啉(ZnP)-苯桥(BB)-铁卟啉(Fe(Cl)P)超分子体系中给体三线态到受体的能量传递及其机理。结果表明体系中存在着由给体ZnP三线态向受体Fe(Cl)P的超快能量传递过程,在室温和低温下通过激发给体ZnP,其单线态的激发能经由系间窜越过程使其三线态布居,在受体Fe(Cl)P存在的情况下,位于给体三线态的激发能经由桥联分子B传递到受体Fe(Cl)P,室温下传递速率为7.2×105s-1。由于体系中给体到受体之间的空间距离约为2.5nm,由给体-受体直接耦合引起的传递机理可以排除,由桥联分子媒介的超交换机理是该能量传递过程的主要物理机理。     

具有电子推拉结构的多支化分子的光物理特性的研究

利用稳态光谱和飞秒时间分辨荧光亏蚀的技术,研究了不同溶剂中一系列有分子内电荷转移特性的分子的结构与光物理性质的关系,研究体系为三苯胺作为电子给体,2,1,3-苯并噻二唑作为受体的单支分子及其对应的两支和三支分子. 并结合TD-DFT计算进一步解释了实验中所观察到的现象. 三个分子相似的吸收和荧光光谱以及强的溶剂依赖光谱特性表明两支与三支分子激发态与单支分子相似,表明激发态都定域在其中一支上. 激发时多支分子内发生多维电荷转移,然后快速地定域到某一支上发射. 另一方面多支分子相对于单支分子吸收和发射光谱的红     

Energy transfer by singlet and triplet excitons in carbazole-containing polymers

Quantum yields of pyrene fluorescence and bis[2-(2′-benzothienyl)-pyridinato-N,C^3′](acetylacetonate)iridium [Btp₂Ir(acac)] phosphorescence upon excitation via a matrix of poly-N-epoxypropylcarbazole (PEPC) or poly-N-epoxypropyl-3,6-dibromocarbazole (DBrPEPC), respectively, were found to be lower than those for the compounds directly excited in a polystyrene (PS) matrix. It was established that the energy in PEPC was transferred to an acceptor by both singlet excitons (by migration and long-range dipole–dipole interaction) and triplet excitons (through migration and short-range exchange electron interaction); however, only by triplet excitons in DBrPEPC, which did not show any fluorescence. The energy-transfer efficiency in PEPC by singlet excitons was higher than by triplet excitons. The observed effects were explained by the fact that energy transfer to the acceptor competed with such processes as localization of the excitons in the “tail” energy states, dissociation of singlet excitons into geminal electron–hole pairs (EHP), and capture of triplet excitons by polymer oxidation products.     

Quantitative analysis of caspase-3 activation by fitting fluorescence emission spectra in living cells

Confocal fluorescence imaging and fluorescence resonance energy transfer (FRET) technology have been widely used to study protein–protein interactions in living cells. However, it is very difficult to quantitatively analyze FRET efficiency due to the excitation spectral crosstalk and emission spectral crosstalk between donor and acceptor. In this study, we developed a novel method to quantitatively obtain the FRET efficiency by fitting the emission spectra (FES) of donor–acceptor pair, and this method is free from both excitation and emission spectral crosstalk. We used the FES method to quantitatively monitor the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by FES are consistent with that by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM) in living cells stably expressing SCAT3. In this study, the FES was also used to analyze the caspase-3 activation in living cells during anti-cancer drug such as taxol, Artesunate (ART) or Dihydroartemisinin (DHA) treatment. Our results showed that ART or DHA induced apoptosis by a caspase-3-dependent manner, while caspase-3 was not involved in taxol-induced cell death.     

Enhanced Emission Induced by FRET from a Long-Lifetime, Low Quantum Yield Donor to a Long-Wavelength, High Quantum Yield Acceptor

We report observation of high quantum yield, long-lifetime fluorescence from a red dye BO-PRO-3 excited by resonance energy transfer (RET). The acceptor fluorescence was highly enhanced upon binding to the donor-labeled DNA. A ruthenium complex (Ru) was chosen as a donor in this system because of its long fluorescence lifetime. Both donor and acceptor were non-covalently bound to DNA. Emission from the donor-acceptor system (DA) at wavelengths exceeding 600 nm still preserves the long-lifetime component of the Ru donor, retaining average fluorescence lifetimes in the range of 30–50 ns. Despite the low quantum yield of the Ru donor in the absence of acceptor, its overall quantum yield of the DA pair was increased by energy transfer to the higher quantum yield acceptor BO-PRO-3. The wavelength-integrated intensity of donor and acceptor bound to DNA was many-fold greater than the intensity of the donor and acceptor separately bound to DNA. The origin of this effect is due to an efficient energy transfer from the donor, competing with non-radiative depopulation of the donor excited state. The distinctive features of DA complexes can be used in the development of a new class of engineered luminophores that display both long lifetime and long-wavelength emission. Similar DA complexes can be applied as proximity indicators, exhibiting strong fluorescence of adjacently located donors and acceptors over the relatively weak fluorescence of separated donors and acceptors.     

“Discrete shell model” for analysing time-resolved energy transfer in solids

We present a new mathematical method to describe the time-resolved fluorescence response of donors and acceptors for the case of energy transfer in crystals, taking into account energy diffusion within the donor system, direct donor-acceptor transfer with any arbitrary dependence of the transfer probability on distance, and the discrete crystal structure. For the first time we give the fluorescence responses for elevated temperatures including transfer of optical excitation energy from the acceptors back to the donors. Furthermore, the competition between neighbouring acceptors (excitation sinks), important for most real acceptor concentrations, is taken into account. The fluorescence responses of donors and acceptors to four commonly realized excitation conditions are given explicitly to any desired degree of precision as sums of exponential functions; they are based on the numerical solution of an eigenproblem. The method is designed to be used in computer analysis of time-resolved fluorescence data.     

Radiative energy transfer I. General equations

A set of equations is derived which makes possible to study the radiative energy transfer process whereby the photons emitted by the energy donor are absorbed by the energy acceptor and so increase the efficiency of the overall energy transfer. It is shown that the coefficients describing the radiative transfer which appear in the expressions for the intensities of the energy donor and the energy acceptor are not the same, due to the fact that part of the fluorescence absorbed by the acceptor comes from radiation which is not detected as donor emission when there is no acceptor present. The general equations derived are applied to two particular cases commonly considered: measurements in reflection, where the fluorescence emission is observed from the same face of the absorption and measurements in transmission where the fluorescence emission is observed from the opposite face of the cell.     

Electronic-Excitation Energy Transfer between Dye Molecules Adsorbed in One-Dimensional Photonic Crystals

Electronic-excitation energy transfer between molecules of Coumarin 7 (donor) and Rhodamine B (acceptor) have been experimentally investigated in one-dimensional photonic crystals based on oxidized mesoporous silicon and in a similar matrix, which does not possess the properties of a photonic crystal. The efficiencies of excitation transfer between donor and acceptor molecules have been determined based on the donor-fluorescence quenching and sensitized acceptor luminescence. It is established that the efficiency of electronic-excitation energy transfer in a photonic crystal increases in comparison with that for porous silicon.