Two-photon excitation fluorescence pH detection using 2,3-dicyanohydroquinone: a spectral ratiometric approach

The use of 2,3-dicyanohydroquinone (DCHQ) as an emission ratiometric probe of pH in vitro and in fibroblast cells was evaluated using two-photon excitation fluorescence microscopy (TPEFM). In addition, methods for spectrally calibrating the Zeiss LSM510 META spectroscopy system for TPEFM were also developed. The emissions of both the acid and base forms of DCHQ were detectable when using an 800-nm excitation in TPEFM, thereby allowing ratiometric determination of pH. These data suggest that, in contrast to most other emission ratiometric probes, both acid and base forms of DCHQ have similar two-photon cross-sectional areas at 800 nm. Acid (maximum at ∼457 nm) and base (maximum at ∼489 nm) DCHQ TPEFM emission spectra were similar to previously reported one-photon excitation emission spectra. Calibration curves for pH were successfully constructed using the ratio of DCHQ emission difference maxima at 460 nm and 512 nm in vitro and in cells. To our knowledge, DCHQ is currently the only effective emission ratiometric pH indicator for two-photon microscopy and may serve as a useful starting point for the development of other TPEFM ratiometric dyes for quantitative measurement of other cell parameters such as Ca²⁺, Mg²⁺ or Na⁺.     

BioPhotonics workstation: a versatile setup for simultaneous optical manipulation, heat stress, and intracellular pH measurements of a live yeast cell

In this study we have modified the BioPhotonics workstation (BWS), which allows for using long working distance objective for optical trapping, to include traditional epi-fluorescence microscopy, using the trapping objectives. We have also added temperature regulation of sample stage, allowing for fast temperature variations while trapping. Using this modified BWS setup, we investigated the internal pH (pH(i)) response and membrane integrity of an optically trapped Saccharomyces cerevisiae cell at 5 mW subject to increasing temperatures. The pH(i) of the cell is obtained from the emission of 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, at 435 and 485 nm wavelengths, while the permeability is indicated by the fluorescence of propidium iodide. We present images mapping the pH(i) and permeability of the cell at different temperatures and with enough spatial resolution to localize these attributes within the cell. The combined capability of optical trapping, fluorescence microscopy and temperature regulation offers a versatile tool for biological research.     

CaO-B2O3-2SiO2干凝胶制备及其生物模拟矿化现象

采用溶胶一凝胶和常压干燥法制备了CaO-B2O3-2SiO2干凝胶。首次研究了干凝胶在37℃,初始pH=9的K2HPO40．25M水溶液中的生物模拟矿化现象,采用XRD、FTIR和SEM分析了干凝胶及其矿化产物的微观结构和形貌。研究表明,CaO-B2O3-2SiO2干凝胶具有硼硅二元复合氧化物的网络骨架,经矿化反应10d后可转变成呈交叉形式排列的片状碳酸羟基磷灰石晶体,并具有多孔状微观形貌结构。     

Scanning tunnelling microscopy studies of heteroepitaxy: CaF2 on Si(111)

We apply scanning tunnelling microscopy and tunnelling spectroscopy to study the initial stages of CaF₂ epitaxy on Si(111)-(7×7) for a range of deposition temperatures. We find that deposition at surface temperatures <700°C leads to CaF₂ clustering. At temperatures between 750 and 800°C two new structures, a 2×3 and a (√3 X √3***)R30°, are observed. Tunnelling spectra and topographs obtained at different energies allow us to measure interface band gaps, and determine the valence state of Ca bonded directly to silicon and the valence state of Ca in the second layer. More importantly, we demonstrate that STM can be used to image thick insulating layers of CaF₂ by tunnelling into the conduction band of the films. One of our goals in this study has been to test the ability of STM to image insulating multilayers. It is generally believed that STM cannot be applied to insulators. We find, however, that despite the large band gap of ～12 eV, the CaF₂ conduction band is accessible, and that insulating films can be imaged by STM.     

超临界CO_2微乳液法制备纳米氧化锆

采用超临界CO2微乳液法制备了纳米ZrO2颗粒,利用XRD、TEM、BET、TG-DSC、激光粒度分析等方法对所得的样品进行表征。结果表明:经超临界CO2微乳液过程可制得非晶相ZrO2前躯体,前躯体经550℃煅烧后转变为四方相ZrO2,900℃煅烧后转变为单斜相ZrO2,ZrO2颗粒分布均匀,粒径为40 nm左右。     

Propagation of intercellular calcium waves in C6 glioma cells transfected with connexins 43 or 32

In this study, gap junction-deficient C6 glioma cells, transfected with either connexin 43 (Cx43) or 32 (Cx32), have been used to evaluate the ability of these connexins to pass intercellular Ca2+ waves. Ca2+ waves, observed with fluorescence imaging using fura-2 or fluo-3, were initiated by mechanical stimulation in the presence of a supra-perfusion of the extracellular fluid or by the non-contact technique of flash photolysis of intracellular caged-IP3. Following manual mechanical stimulation, the parental C6 glioma cells and cells expressing Cx43 and Cx32 gap junctions all propagated intercellular Ca2+ waves. Ca2+ waves in cells expressing Cx43 traveled approximately twice the distance as compared to waves in cells expressing Cx32 or parental cells. The cells expressing Cx43 were also about twice as sensitive to ATP as cells expressing Cx32. In the presence of a supra-perfusion of extracellular fluid, the Ca2+ waves in parental cells were almost abolished while the mechanically induced Ca2+ waves in the cells expressing Cx43 and Cx32 propagate similar but limited distances of several cells in a direction opposite to the fluid flow. The photolytic release of IP3, but not Ca2+, in cells expressing Cx43 or Cx32 resulted in the propagation of Ca2+ waves that traveled distances similar to those observed in the presence of supra-perfusion. Parental C6 glioma cells did not initiate intercellular Ca2+ waves when stimulated by photolysis. From these studies we conclude that (1) both Cx43 and Cx32 based gap junctions are permeable to IP3 and can serve to communicate Ca2+ waves, (2) that Ca2+ wave propagation via gap junctions was dependent on the diffusion of IP3 but not Ca2+, (3) that an extracellular messenger capable of communicating waves is released from only the stimulated cell, and (4) that simultaneous intracellular and extracellular signaling can occur to enhance the propagation of intercellular Ca2+ waves.     

发光染料罗丹明B的荧光传感特性

本文对有机染料罗丹明B(RhB)的荧光光谱受温度、应力以及pH值等物理或化学量的影响进行了研究。实验采用波长为405nm的激光激发RhB/H610胶黏剂复合材料或RhB水溶液,得到其荧光发射谱,用谱峰频移及新型表征方法"谱带重心频移法"分析了环境参数对谱带位置的影响,并通过线性拟合得到了RhB荧光谱带重心λ_B传感温度、压应力以及pH值的传感方程。结果表明,谱带重心频移法有效抑制了光强涨落的白噪声,能够显著提高传感的精度和稳定性。在测量范围中间值附近,重心法对温度、压应力以及pH值传感的相对灵敏度分别为0.026%/℃,0.09%/MPa和0.34%。由于RhB具有很高的发光效率和优异的生物相容性,因此基于RhB荧光的传感方法在生物体系的检测表征方面具有较高的应用价值。     

An infra-red light-transmitting aperture controller for use in single-cell fluorescence photometry

Photometric techniques are commonly used to monitor the output from fluorescent indicators during the study of cellular signalling. At the single-cell level, the region of interest is normally set by a variable aperture placed within the microscope emission pathway. The present study reports an improved aperture controller which adjusts the area for fluorescence measurement, whilst allowing objects throughout the field of view to be continuously monitored using infra-red illumination. A rectangular aperture is selected by four 715-nm long-pass glass filters which block > 99.9% of the fluorescence emission at 480–600 nm. A 780-nm long-pass glass filter is used to provide infra-red illumination which does not interfere with the fluorescence signal, yet is detectable by a standard CCD camera. This allows detection of morphological events throughout the field of view and facilitates manipulation of extracellular pipettes, without interruption to a single-cell fluorescence recording. The infra-red light-transmitting controller is suitable for use with a range of other fluorescent indicators, including those routinely used to detect Ca²⁺, Cl^?, Na⁺ and pH. Data are presented which demonstrate the use of this controller to measure ADP-evoked [Ca²⁺]_i increases in single human erythroleukaemia cells loaded with the Ca²⁺ indicator fura-2.     

圆二色谱研究VP-16在溶液中的构型构象变化及与DNA的作用

本文测定了抗癌药物VP-16及其在体内的代谢产物苦VP-16,苦VP-16羟基酸的第一偶合圆二色谱。研究了在不同pH条件下VP-16在溶液中的异构化和水解现象。通过圆二色谱的改变讨论了不同浓度的药物VP-16对小牛胸腺DNA的作用。     

真空紫外Al/MgF_2反射镜

采用三步热舟蒸发制作法研制了真空紫外Al/MgF2反射镜,研究了改善制备工艺有效提升反射率的方法。在两层Al/MgF2反射镜制备过程中,第一步在室温石英基板上快速蒸发厚约70nm的铝膜;第二步在铝膜表面迅速蒸发厚约10nm的MgF2;第三步先对基板加热到一定温度后,再在Al+MgF2的表面上蒸发15~20nm厚的MgF2。通过调整基板温度(室温、100℃、200℃和300℃),研究了基板温度对Al/MgF2反射率的影响。真空紫外反射率计测试结果表明:第二步蒸镀MgF2之后增加基板温度有利于提高反射镜的反射率;MgF2薄膜的厚度对反射镜的反射率起到一定的调制作用,MgF2厚为26.7nm的反射镜在122nm处的反射率达85%。在实验室环境下存放1个月和5个月后,反射镜的反射率没有变化。研究结果为真空紫外光学系统需求的高性能光学元件的研制提供了技术基础。     

高温真空分解V2O5制备VO2

尝试采用真空分解V2O5熔体的方法制备高纯度的VO2,通过XRD和SEM等方法进行了分析。结果表明,该方法可以直接获得高纯度的VO2的沉积层,沉积层为连续单相多晶组织。当熔体以小于10℃/h的升温速率加热到1350℃,真空度控制为10-6atm,所获得的沉积层的电阻突变量级达到最大为4~5,相变温度为68℃,相变温度滞后为1℃~2℃。     

荧光光谱法研究葛根素与牛血清白蛋白的相互作用

在不同温度下,采用荧光猝灭光谱法和同步荧光光谱法研究了葛根素与牛血清白蛋白(BSA)相互作用的光谱学行为。根据在292K和311K温度下葛根素对BSA的荧光猝灭作用,利用Stern-Volmer方程及双倒数方程对实验数据进行处理,结果表明葛根素对BSA的荧光猝灭作用属于静态猝灭过程。根据Frster非辐射能量转移理论计算出了葛根素与BSA间的结合距离r=2.84nm,结合常数(Kb)分别为1.334×105mol/L(292K)和4.513×105mol/L(311K)。热力学数据表明,二者主要靠静电引力结合。采用同步荧光光谱法分析了葛根素对BSA构象的影响。     

高频电磁在水处理中阻垢作用

应用静态挂片试验研究了高频电磁在处理水中的阻垢作用,讨论了电压、水中钙镁离子总浓度、pH、处理后放置时间及Ca^2＋／Mg^2＋比值等因素对阻垢效率的影响,并分析了其阻垢机理。结果表明,电压、水中钙镁离子总浓度、pH、处理后放置时间及Ca^2＋／Mg^2＋比值等因素对电子水处理器的阻垢效率的影响不同。当水加热时,水中盐类在显微镜下观察到点状结晶比杆状结晶多,同时其结晶总数亦明显增多。     

紫外-真空紫外波段的Al+MgF2膜

Al+MgF₂膜是真空紫外波段常用的一种反射膜。根据薄膜光学的电磁场理论计算了正入射条件下Al+MgF₂膜在真空紫外波段的反射率随氟化镁膜厚度的变化规律。研究了Al+MgF₂膜的制备工艺,利用Seya-Namioka紫外-真空紫外反射率计测得Al+MgF₂膜的反射率在150nm～340nm的波段上高于80%。Al+MgF₂膜制备一年后,其真空紫外波段的反射率未有明显变化。     

高效液相色谱法分析小麦芽粉中的氨酪酸

建立一种分析小麦麦芽粉中氨酪酸的离子交换色谱法;本方法以邻苯二甲醛-巯基乙醇为柱后衍生试剂,固定相为R-SO3-Na+,流动相为pH=3.15的柠檬酸三钠和pH=9.70的硝酸钠缓冲液,梯度程序洗脱,柱后衍生,荧光检测器检测;方法稳定、快速、准确,氨酪酸的平均回收率(n=3)为96.0%。     

不同氧化剂对6H-SiC化学机械抛光的影响

选用胶体SiO₂纳米颗粒为磨粒,研究不同pH值条件下高锰酸钾和双氧水两种氧化剂对6H-SiC晶片化学机械抛光的影响,并使用原子力显微镜观察抛光后表面质量。采用Zeta电位分析仪分析溶液中胶体SiO₂颗粒的Zeta电位,采用X射线光电子能谱分析SiC抛光表面元素及其化学状态。结果表明：SiC晶片的材料去除率随pH值变化而变化,采用高猛酸钾抛光液抛光时,材料去除率在pH 6时达到峰值185 nm/h,R_a为0.25 nm;采用双氧水抛光液抛光时,材料去除率在pH 8时达到峰值110 nm/h,R_a为0.32 nm。pH值低于5时,电负性的SiO₂颗粒会通过静电作用吸附在带正电的SiC表面,抑制SiC晶片表面原子的氧化及去除,降低材料去除率;pH值高于5时,SiO₂颗粒在双氧水抛光液中的静电排斥力弱于高锰酸钾抛光液中静电排斥力,从而影响了SiO₂颗粒的分散性能,降低了抛光效果。采用高锰酸钾抛光液抛光后,SiC晶片表面的Si-C氧化产物含量（Si-C-O、Si₄C_4-xO₂和Si₄C₄O₄）较高,高锰酸钾抛光液的氧化能力较强。     

Resonance Rayleigh Scattering of K2Zn3[Fe(CN)6]2 Nanoparticles and its Application for the Determination of Vitamin C

Abstract

In Britton-Robinson buffer medium, (pH 4.43), potassium ferricyanide (K₃[Fe(CN)₆]) could react with vitamin C (VC) to produce potassium ferrocyanide (K₄[Fe(CN)₆]), which further reacted with Zn²⁺ to form potassium zinc hexacyanoferate K₂Zn₃[Fe(CN)₆]₂ nanoparticles. The shapes and diameters of the K₂Zn₃[Fe(CN)₆]₂ nanoparticles have been observed with transmission electron microscopy, which showed the shapes of these nanoparticles was cubic and their average sizes were about 50 nm in the presence of 2.0 × 10^?5 mol L^?1 VC. The characteristics of resonance Rayleigh scattering (RRS) spectra of this reaction have been studied. The optimum reaction condition for the determination of VC has been investigated. It was found that the RRS intensity of the system at the RRS peak of 363.4 nm was proportional to the VC concentration in the range of 4.0?80.0 µmol L^?1, and the detection limit (3σ) for VC was 0.075 µmol L^?1. A novel and simple RRS method for the determination of VC based on the formation of K₂Zn₃[Fe(CN)₆]₂ nanoparticles has been established.     

基于二阶校正法的多环芳烃污染物荧光检测

为了对复杂体系的多环芳烃进行定性识别和定量分析,构建了基于二阶校正法的荧光检测系统。利用二阶校正法在三维荧光数据处理中的优势,达到多种混合多环芳烃分离和鉴别的目的。采用FS920荧光光谱仪测量并分析了萘(NAP)、苊(ANA)及两者混合物的荧光光谱特性,发现NAP溶液有一个荧光峰λex/λem=290/322nm,ANA溶液存在两个荧光峰分别为λex/λem=290/322nm和λex/λem=290/336nm,NAP和ANA荧光光谱重叠严重,并且不同浓度配比混合物的荧光光谱具有差异性。通过将二阶校正法与三维荧光光谱法相结合,实现对多环芳烃混合物的浓度检测。分别采用平行因子(PARAFAC)算法和自加权交替三线性分解(SWATLD)算法对光谱数据进行分解。结果表明:两种算法对NAP和ANA混合物均有较高的分辨能力,预测平均回收率均在95%~99%、均方根误差均小于0.2μg/L。相比之下,SWATLD算法的检测效果更好。     

可用于自倍频激光器的Cr3+:YAB和Cr3+:YGAB晶体光学特性的研究

Cr³⁺:YAB和Cr^3-:YGAB晶体具有较宽的荧光光谱,从红光到近红外光。Cr³⁺:YAB晶体在744nm到852.5nm之间被测到光学增益。最大单程增益为1.9,对应波长为820nm.Cr³⁺:YAB晶体的二次谐波产生(37nm)的转换效率是LBO晶体的1.5倍。     

Four-dimensional visualization and quantitative analysis of meiotic spindle movements in live mouse oocytes

This paper made a different attempt of real-time observation of the meiotic spindle movements in living mouse oocyte using a convenient method. This method was based on an experimental phenomenon discovered in our work. In living mouse oocytes, a high concentration of calcium ions (Ca(2+) ) was observed throughout the region occupied by the initial meiotic spindle. After Ca(2+) labelling with Fura-2, a weakly fluorescent area (WFA) appeared on each side of the chromosomes. The activities of the WFAs changed during spindle development. By real-time tracking of WFAs, we were able to indirectly observe the meiotic spindle movements. Occasionally, it was observed that the first meiotic spindle rotated from an orientation parallel to the cortex to become perpendicular, instead of migrating from the oocyte centre to the cortex along its axis. Moreover, we analysed this uncommon rotation of the first meiotic spindle and found that the whole rotation process can be divided into two phases: the early slow-speed rotation and the subsequent rapid-speed rotation. We further characterized this rotation with respect to rotational speed and acceleration at all the stages of development. By using a two-photon laser-scanning microscope in combination with Fura-2 dye that is nondamaging to oocytes, we provide a convenient method for indirect visualization and quantitative analysis of spindle movements by real-time tracking of WFAs. This method is easy to operate and master, and economical with time and effort.