Photobleaching-Based Quantitative Analysis of Fluorescence Resonance Energy Transfer inside Single Living Cell

The current advances of fluorescence microscopy and new fluorescent probes make fluorescence resonance energy transfer (FRET) a powerful technique for studying protein-protein interactions inside living cells. It is very hard to quantitatively analyze FRET efficiency using intensity-based FRET imaging microscopy due to the presence of autofluorescence and spectral crosstalks. In this study, we for the first time developed a novel photobleaching-based method to quantitatively detect FRET efficiency (Pb-FRET) by selectively photobleaching acceptor. The Pb-FRET method requires two fluorescence detection channels: a donor channel (CH ₁ ) to selectively detect the fluorescence from donor, and a FRET channel (CH ₂ ) which normally includes the fluorescence from both acceptor and donor due to emission spectral crosstalk. We used the Pb-FRET method to quantitatively measure the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside single 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 Pb-FRET inside living cells was verified by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM). The temporal resolution of Pb-FRET method is in second time-scale for ROI photobleaching, even in microsecond time-scale for spot photobleaching. Our results demonstrate that the Pb-FRET method is independent of photobleaching degree, and is very useful for quantitatively monitoring protein-protein interactions inside single living cell.     

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.     

消癌平诱导人肺腺癌(ASTC-a-1)细胞内caspase-3活化的荧光光谱分析

采用CCK-8技术检测发现传统中药消癌平（XAP）对人肺腺癌（ASTC-a-1）细胞的增殖活性具有显著的抑制作用。利用共聚焦扫描荧光显微成像、荧光共振能量转移（FRET）及其受体光漂白技术证实了基于FRET技术构建的SCAT3质粒在ASTC-a-1细胞中的稳定表达。将消癌平加入细胞培养液中培育细胞,并在不同的时间检测活细胞内SCAT3的荧光发射光谱,从而监测细胞中caspase-3的活化状态。实验结果表明：(1)消癌平可以有效抑制人肺腺癌（ASTC-a-1）细胞的增殖活性并诱导细胞的死亡,消癌平对细胞的抑制作用具有剂量依赖性。(2)消癌平处理细胞72 h后,细胞内大量的SCAT3被切割,表明细胞内caspase-3的活化水平明显升高。(3)将含消癌平的细胞培养液与细胞共同培养24 h,然后采用没有消癌平的细胞培养液培养细胞48和72 h后,细胞内SCAT3的光谱没有明显改变,表明消癌平作用细胞24 h内没有显著激活细胞内的caspase-3。     

Application of the “electronic bridge” theory to some highly converted nuclear transitions

The influence of the atomic electron cloud on the gamma decay constantλ _γ was evaluated in third-order approximation according to the theory of Krutov for some highly converted nuclear transitions (resonance case only). The results areλ _γ ⁽³⁾ =1.5×10⁵ λ _γ ⁽³⁾ for the 73 eV transition of U-235m andλ _γ ⁽³⁾ =1.01λ _γ ⁽¹⁾ for the 2.17 keV transition of Tc-99m. Values calculated forZ=80,E _γ =2 keV are in good agreement with those of Krutov. Experimental evidence of the calculated effects does not seem to be obtainable at present because of the extremely low gamma intensity of the transitions in question.     

Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells

3-cube-based Förster resonance energy transfer (FRET) microscopy, a sensitized acceptor FRET quantification method, has been widely used to visualize dynamic protein–protein interaction in living cells. Determining the FRET sensitized-quenching transition factor (G factor) of a particular donor-acceptor pair and optical system is crucial for 3-cube FRET quantification. We here improved the acceptor photobleaching-based G factor determination method (termed as mPb-G) and the two-plasmid-based G factor determination method (termed as mTP-G) for rapid and reliable measurement of the G factor. mTP-G method determines G factor by simultaneously detecting three images of cells exclusively expressing each of two tandem constructs with multiple donors and multiple acceptors. This method circumvents switchover of the cells exclusively expressing each of the two constructs. mPb-G method images G factor by detecting three images of cells expressing a donor-acceptor tandem FRET construct before and after partially photobleaching acceptor. We performed the two methods on our dual-channel wide-field FRET microscope to obtain reliable G factor, and also measured the FRET efficiency and acceptor-to-donor concentration ratio of tandem constructs with different acceptor-donor stoichiometries in living HepG2 cells. mTP-G and mPb-G methods provide two simple and reliable tools for determining the G factor, in turn, quantitatively measuring FRET signal and monitoring dynamic biochemical processes in living cells.     

On spectral invariance of single scattering albedo for water droplets and ice crystals at weakly absorbing wavelengths

The single scattering albedo ω_0λ in atmospheric radiative transfer is the ratio of the scattering coefficient to the extinction coefficient. For cloud water droplets both the scattering and absorption coefficients, thus the single scattering albedo, are functions of wavelength λ and droplet size r. This note shows that for water droplets at weakly absorbing wavelengths, the ratio ω_0λ(r)/ω_0λ(r₀) of two single scattering albedo spectra is a linear function of ω_0λ(r). The slope and intercept of the linear function are wavelength independent and sum to unity. This relationship allows for a representation of any single scattering albedo spectrum ω_0λ(r) via one known spectrum ω_0λ(r₀). We provide a simple physical explanation of the discovered relationship. Similar linear relationships were found for the single scattering albedo spectra of non-spherical ice crystals.     

Effects of cross-correlation between the real and imaginary parts of the quantum noise in a two-mode saturation laser system

A two-mode saturation laser model with cross-correlation between the real and imaginary parts of the quantum noise is considered. The laser intensity Langevin equation and corresponding Fokker-Planck equation are derived by the phase-locking method. The effects of the cross-correlation strength λ between the real and imaginary parts of quantum noise and the cavity decay constant K on the steady-state intensity distribution Q(I₁,I₂), the mean light intensity 〈I〉, the normalization autocorrelation λ₁₁(0) and cross correlation λ₁₂(0) are studied by numerical calculation. The results show that as λ increases the Qs(I₁,I₂) show two extrema, and λ almost does not affect the 〈I〉, λ₁₁(0) and λ₁₂(0) when the laser system is operated far above threshold. Nevertheless, when the laser system is operated at and below threshold, λ makes the curves of Qs(I₁,I₂) have the higher peak and drop faster. Furthermore, it enhances the deviation of λ₁₁(0) and λ₁₂(0) and lessens the mean light intensity 〈I〉 when the laser system is operated at and below threshold.     

Coupling constant thresholds in nonrelativistic quantum mechanics. I. Short-range two-body case

Consider ?Δ + λV with V short range at a value λ₀ where some eigenvalue e(λ) → 0 as λ ↓ λ₀. We analyze two questions: (i) What is the leading order of e(λ), i.e., for what α does e(λ) ～ c(λ ? λ₀)^α? (ii) Is e(λ) analytic at λ = λ₀ and, if not, what is the natural expansion parameter? The results are highly dimension dependent.     

Photoluminescence characterization of a novel red-emitting phosphor In2(MoO4)3:Eu3+ for white light emitting diodes

The red-emitting phosphor In₂(MoO₄)₃:Eu³⁺ with cubic crystal structure was synthesized by a conventional solid-state reaction technique and its photoluminescence properties were investigated. The prepared phosphor can be efficiently excited by ultraviolet (395 nm) and blue (466 nm) light. The emission spectra of the phosphor manifest intensive red-emitting lines at 612 nm due to the electric dipole ⁵D₀→⁷F₂ transitions of Eu³⁺. The chromaticity coordinates of x=0.63, y=0.35 (λ_ex=395 nm) and x=0.60, y=0.38 (λ_ex=466 nm) are close to the standard of National Television Standard Committee values (NTSC) values. The concentration quenching of In₂(MoO₄)₃:Eu³⁺ is 40 mol% and the concentration self-quenching mechanism under 466 nm excitation was the dd intereaction. As a result of the strong emission intensity and good excitation, the phosphor In₂(MoO₄)₃:Eu³⁺ is regarded as a promising red-emitting conversion material for white LEDs.     

Reaction dependence of nuclear decay linewidths

Various light- and heavy-ion reactions, 20 < E < 100 MeV, have been used to study the reaction dependence of α-decay widths for ⁸Be¹(2 ⁺ , 2.9 MeV) and ¹⁶O¹(1^?, 9.6 MeV). Although slight differences (< 20 %) are found for the observed line shapes (Γ), the resonance widths inferred (Γ_R) are self-consistent and indicate little if any reaction dependence (< 10 %). Near a decay threshold one may expect Γ < Γ_R by 20 % or more, however, and thus care must be taken in comparing decay widths inferred from nuclear reactions with those from scattering resonances. Reduced formal α-decay widths of γ_λ² = 680 ± 100 keV (s = 4.8 fm) and γ_λ² = 350 ± 50 keV(s = 5.4 fm), corresponding to θ_λ² = 0.50 and θ_λ² = 0.49 are deduced for ⁸Be¹(2 ⁺ , 2.9 MeV) and ¹⁶O¹(1^?, 9.6 MeV) using the nuclear-reaction Γ_R values and a particular set of α-nucleus potentials.     

Magnetization,magnetocrystalline anisotropy,magnetostriction and elastic constants of the Heusler alloy: Cu2Mn Al

Three kg of stoichiometric single crystals of Cu₂Mn Al have been grown by the Bridgman method. The thermal variations of their magnetization, anisotropy, magnetostriction and elastic constants have been measured between 4.2 and 300 K. This alloy exhibits a small magnetocrystalline anisotropy, but a large elastic anisotropy $\text{(}\text{C}{}^{\mathrm{?}}\text{C}{}^{\mathrm{\gamma }}\text{= 4.9)}$ which explains the strong anisotropy of the magnetostriction $\text{(}\text{λ}{}^{\mathrm{\gamma ,2}}\text{λ}{}^{\mathrm{?,2}}\text{= 10)}$. The magnetic moment per Mn atom at 0 K is 3.6 μ_B.     

Optical and electrical properties of thin NiO films deposited by reactive magnetron sputtering and spray pyrolysis

Thin NiO films are deposited by reactive magnetron sputtering and spray pyrolysis. The main optical constants, i.e., refractive index n(λ), absorption coefficient α(λ), extinction coefficient k(λ), and thickness d, are determined. The temperature dependence of the resistance of thin films is found, and the activation energy of films deposited by different methods is determined.     

Multi-band nature of superconducting anisotropy in PrFeAsO1−δ single crystal

We investigated the magnetic torque of superconducting PrFeAsO_1−δ single crystal using the torque magnetometer at temperatures from 2 K to 200 K in magnetic field of 30 kG. The torque curve tends to have a singular shape compared to other superconductors. The analyses of the PrFeAsO_1−δ torque curves by the single-band and the multi-band Kogan model yield 3 < γ_λ < 5 in 30 kG. With the aid of the multi-band Kogan model, however, we obtain γ_ξ = 0.79 ± 0.01 and γ_λ = 19 ± 3 in 30 kG at 20.5 K. The smallness of γ_ξ(<1) thus obtained is due to the multi-band nature of the iron pnictide.     

Influence of magnetic field on exciton luminescence

The magnetic field dependence of the exciton emission intensity I_ex(H) has been investigated in Ge crystals stressed along the direction near 〈100〉. In the low field limit the magnetic field correction has been evaluated to the wave functions of the ground and some excited states of an isotropic exciton. The calculated dependence I_ex(H) in the case of Ge is in a good agreement with the experimental one at H ? 0.5 T.     

IR optical absorption and reflection spectra in narrow gap semiconductors

Light transmission T(λ) and reflection R(λ) spectra were measured for the crystals Cd_xHg_1−xTe (x = 0.26). InSb and Mn_xHg_1−xTe (x = 0.12) with low impurity content at T = 300 K in the wavelength range 2.5 μm ⩽ λ ⩽40 μm. From the measured T(λ) and R(λ) data a dependence of the absorption coefficient x(λ) for the free-carriers light absorption (FCLA) was derived, which is caused by the intrabund electron and hole transitions and the intcrband transitions of the heavy holes from the band V₁ to the band V₂ of the light holes: x(λ) = x_intraband(λ) + x_interband(λ).It is concluded from the quantum mechanical approach that the intraband FCLA is directly related with the carriers scattering mechanisms. Calculations of x_intraband(λ) within the framework of this approach demonstrated that under room temperature the major contribution was from the scattering by polar optical phonons. In the case of less perfect Mn_xHg_1−xTe however, one should take into account the scattering by the short-range potentials of the defects.     

Operators in a translation invariant two-dimensional bag model

Operator matrix elements are studied in a translation invariant version of the bag model in two-dimensional space time. In particular we calculate the matrix elements of quark fields Q(x), currents J_μ(x), and bilocal currents Q(x)γ_μλ^aQ(0) whose Taylor expansion yields the twist two operators of leptoproduction. The structure functions of this model are related to those of a naive cavity approximation by a particularly simple transformation.     

Measurement of the f0(1270) polarization in the J/ψ → f0γ decay

The polarization of the f⁰(1270) and ?⁰(770) produced in the J/ψ decays into f⁰γ and ?⁰π⁰ are measured in e⁺e^? collisions at 3.1 GeV. A fit to the f⁰ production and decay angular distributions yields the values A₁/A₀ = 0.6 ± 0.3 and A₂/A₀ = 0.3 _?1.6^+0.6, where A_λ are the f⁰ helicity amplitudes. These results are in good agreement with the values predicted from a QCD two-gluon-exchange model. In addition an upper limit of 2.3 × 10^?4 is obtained for the J/ψ → f′⁰(1514) γ decay branching ratio which implies Γ(J/ψ → f′γ)/Γ(J/ψ → f⁰γ) ? 0.12 ± 0.05.     

Large deviations and Lifshitz singularity of the integrated density of states of random Hamiltonians

We consider the integrated density of states (IDS) ρ_∞(λ) of random Hamiltonian H_ω=?Δ+V_ω, V_ω being a random field on ? ^d which satisfies a mixing condition. We prove that the probability of large fluctuations of the finite volume IDS |Λ|^?1ρ(λ, H_Λ(ω)), Λ ? ? ^d , around the thermodynamic limit ρ_∞(λ) is bounded from above by exp {?k|Λ|},k>0. In this case ρ_∞(λ) can be recovered from a variational principle. Furthermore we show the existence of a Lifshitztype of singularity of ρ_∞(λ) as λ → 0⁺ in the case where V_ω is non-negative. More precisely we prove the following bound: ρ_∞(λ)≦exp(?kλ^?d/2) as λ → 0⁺ k>0. This last result is then discussed in some examples.     

Angular correlation measurement in the B11(d,pγ)B12 reaction

Thep-γ angular correlations in B¹¹(d, p)B¹² reaction have been measured in both reaction and azimuthal planes for proton angles 35° at E_D=1.60 and 2.40 MeV. The correlation coefficient A ₂ ⁰ and the distortion parameterλ were calculated. The obtained values of the shift of the symmetry axis from the recoil direction, Φ₀ and the parameterλ are far from there plane wave limits. The anisotropy of the angular correlation gives two possible values, 1⁺ and 2⁺, for the spin of the first excited state of B¹².     

The correlation function for a quantum oscillator in a low-temperature heat bath

The momentum autocorrelation function c(t) for a quantum oscillator coupled with harmonic forces to a heat bath of oscillators is calculated at low temperatures. It is found that c(t) contains two distinct terms: one, the zero-point contribution c₀(t), is temperature independent, and the other, c₁(t), does depend on temperature. We concentrate our attention on the low-temperature case. An expression for c₁(t) is obtained, which is valid for arbitrary strenghts of the coupling and for arbitrary times. It is shown that c₁(t) is governed by the low-frequency behaviour of F(λ) = A²(λ)ϱ(λ), where ϱ(λ) is the density of normal modes and A(λ) is the central-oscillator component of the λth normal mode; other details of the problem are irrelevant. It is found that c₁(t) decays in time as an inverse-power law, with a relaxation time t_q ≈ ħ/kT.