聚二甲基硅氧烷微流道中光流控荧光共振能量转移激光

将单一折射率的石英裸光纤植入由聚二甲基硅氧烷构成的基片微流道中,以低折射率的罗丹明B(RhB)和吡啶821(LDS821)乙醇溶液构成的供体和受体对作为激光增益介质.采用沿光纤轴向消逝波抽运方式,首先以波长为532nm的连续波激光器作为激励光,对荧光共振能量转移特性参数进行了研究.然后以波长为532nm的脉冲激光器作为抽运光,通过直接激励供体分子RhB,并将其能量转移给临近的受体分子LDS821,在不改变抽运光波长的条件下,实现了较低阈值(1.26μJ/mm~2)的受体LDS821激光辐射.     

Efficiency of energy transfer by resonance in quenching of protein fluorescence by dyes

Considering the quenching of tryptophan fluorescence of bovine serum albumin by various dyes as an example, it is shown that overlap of radiation and absorption spectra does not necessarily lead to energy transfer by resonance. No correlation is revealed between the limiting quenching and the Forster overlap integral. Quenching can occur even in the absence of overlap. The magnitude of energy transfer is markedly lower than that of quenching owing to competing processes, namely, excitation deactivation by the dye and, probably, by the protein itself which undergoes conformation upon sorption of the dye. Negatively charged and neutral dyes posses, on the average, a higher quenching activity relative to albumin than do positively charged dyes. Institute of the Biophysics of Cells of the Russian Academy of Sciences, Pushchino, 142292, Moscow Region, Russia. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 65, No. 2, pp. 290–293, March–April, 1998.     

脂质体包裹荧光受体方法研究a-突触核蛋白在磷脂膜上的结构和动态特征

α-突触核蛋白(α-synuclein,α-syn)在帕金森病的发病机理中起着关键的作用,因而近年来受到了越来越广泛的关注.α-syn在膜上的动力学过程对理解其功能至关重要.本文使用基于脂质体的单分子荧光衰减方法——LipoFRET,首次对较高浓度下α-syn与磷脂膜的相互作用的动态过程进行了单分子层面上的研究.研究发现,在溶液中α-syn浓度升高时,其中央NAC区域可离开磷脂膜表面进入水相中;而N端部分位于膜表面内的位置变浅,并有更高的概率脱离膜表面进入溶液中.利用单分子荧光成像对α-syn解离的观察则发现,随着溶液中的α-syn浓度升高,脂质体上的α-syn解离速率加快.因此高浓度下,α-syn在膜上各区域垂直位置变化促进了蛋白从膜上的解离.结合LipoFRET的实验结果可以推断,α-syn的解离可能是由于不同的α-syn分子膜作用位点互相竞争而导致的解离.这样的特征,可能是体内环境中影响α-syn控制其聚集的重要性质.     

The use of the isotropic orientation factor in fluorescence resonance energy transfer (FRET) studies of the actin filament

A Dale-Eisinger style analysis (R. E. Daleet al., Biophys. J. 26, 161, 1979) is used to produce three-dimensional plots that display the limits on the average orientation factor k ² that is required to calculate molecular distances in F-actin from fluorescence resonance energy transfer measurements. Maxima and minima plots are generated for the transfer of energy from a donor to a single acceptor and for transfer to multiple acceptors that are related by F-actin helical symmetry. The analysis is performed in terms of dipole cone half-angles rather than depolarization factors, in order to facilitate the modeling of the multiple acceptor problem. Calculations are carried out under the restrictive condition of a single electric dipole moment per fluorophore. In addition, both surface and volume averaging of the donor and acceptor dipoles are considered. Comparisons between the plots show that for the multiple acceptor cases with F-actin symmetry, there is a great reduction in the range for maxima and minima limits on k ². The calculations also suggest guidelines for the choice of fluorescence label that will result in an average orientation factor occurring within acceptable limits, i.e., inside the limits for which k ²=2/3 may be employed. Thus, without having detailed knowledge of the mean donor or acceptor dipole relative orientations, the use of k ²=2/3 in radial coordinate studies of F-actin is more than reasonable and is fairly assured of being correct.     

Intramolecular energy transfer in compounds with two 1-pyrenoate groups separated by methylene spacers

Steady-state fluorescence anisotropy (r) and fluorescence lifetime () measurements have been used to study the efficiency of nonradiative singlet energy transfer as a function of alkane size in 1-pyrenecarboxylic acid alkanediyl esters (as a function ofm in Py-COO-(CH₂) _m -OOC-Py, where Py denotes pyrene substituted in the 1-position, andm=2–6). Experiments were performed in media of different viscosity, , obtained by changing the temperature (from –20 to 40°C) of dilute solutions in ethylene glycol and by examination of the compounds in a solid matrix of poly(methyl methacrylate) (PMMA) at ambient temperature. The Py-COO-(CH₂)_m-OOC-Py exhibit intramolecular excimer emission in ethylene glycol at these temperatures, but the intensity of this emission is much lower than when these compounds are placed in common solvents of lower . The values of indicate that excitation hopping or intramolecular energy transfer takes place between the chromophores attached to the ends of the alkane bridges. Values ofr obtained by the extrapolationsT/0 orT/0 in ethylene glycol, as well as the values obtained in the rigid matrix of PMMA, show very little dependence onm. A theoretical conformational analysis, using the rotational isomeric state (RIS) model, was also performed. The combination of the experimental results forr in the media of high with the theoretical (RIS) analysis produces an estimated value of 21 ±2 Å for the Förster radius in Py-COO-(CH₂)_m-OOC-Py.