Determination of protein with Thioguanine (6-TG) as a probe by synchronous fluorescence technique

The synchronous fluorescence technique was developed for the determination of human serum albumin (HSA) in human body fluids with Thioguanine (6-TG) as a molecular probe. Under the optimal experimental conditions, the synchronous fluorescence peak of HSA–6-TG system is located at about 301 nm and the enhancement synchronous intensity was proportion to the concentration of HSA. The calibration graphs are linear over the range of 0.69–552.0 μg mL⁻¹ with a correlation coefficient (R) of 0.9994. The detection limit was 0.133 μg mL⁻¹. It was successfully applied to determine the protein in human body fluids including serum, urine and saliva samples with 6-TG as probe with a satisfying result. In the analysis of human body liquids samples, the relative standard deviations (RSDs) were 0.55–3.31%, which obtained from 6 replicate determinations and recoveries were in the range of 97.0–104.1%.     

基于PCA的时间分辨油荧光光谱分析及优化

激光诱导荧光技术可广泛应用于油污染的监测中,然而普通的油荧光光谱技术只能实现油污染监测的粗分类,无法区分原油与燃料油的荧光特征。本文基于主成分分析方法(PCA)的时间分辨油荧光分类方法,实验测量了20种油样本的时间分辨荧光光谱特征,给出了对应的荧光寿命和时间分辨油荧光光谱的时序特征。在此基础上,利用前三个主成分构成的三维特征矢量空间,通过分析不同采集时刻下油样本矢量间相关距离的变化,对油样本的时间分辨荧光光谱进行聚类分析。为了体现油荧光变化的时序性,引入矢量距离的离散度参量,提出基于PCA进行时间分辨油荧光光谱分析的优化方法。实验结果表明,基于时间分辨油荧光光谱识别可实现原油与燃料油的光谱时序特征区分,具备良好的油荧光分类效果。     

A GREEN METHOD FOR THE DETERMINATION OF CHROMIUM(VI) AND IRON(III) IN WATER BY SEQUENTIAL INJECTION ANALYSIS AND SPECTROPHOTOMETRIC DETECTION

In this article, an organic-reagent-free method was described for the determination of Cr(VI) and Fe(III) by double-system double-wavelength sequential injection technique with a single sample injection. In this approach, the determination of Cr(VI) was based on the detection of a blue unstable intermediate compound resulting from the reaction of Cr(VI) with hydrogen peroxide, and the determination of Fe(III) was based on the color reaction of Fe(III) with thiocyanate in acidic medium. Sequential injection analysis (SIA) parameters, including spacer solution volume, aspiration order, aspiration volumes, flow rate, acid medium, solution acidity, and reagent concentrations, were optimized. The linear range for the determination was 3.0–60.0 μg mL^?1 for Cr(VI) and 1.0–40.0 μg mL^?1 for Fe(III), respectively. The limit of detection (LOD) was 0.6 μg mL^?1 for Cr(VI) and 0.2 μg mL^?1 for Fe(III), and the limit of quantitation (LOQ) was 2.0 μg mL^?1 for Cr(VI) and 0.67 μg mL^?1 for Fe(III). The total volume of the reagent consumed in each determination was only 0.11 mL. The proposed method was applied to the simultaneous determination of Cr(VI) and Fe(III) in electroplating wastewater and environmental waters. The results were in good agreement with those obtained by atomic absorption spectrometry. The recoveries were in the range of 97.5–101.1%.     

荧光显微镜的使用与校正方法

荧光显微镜是最常用的医用光学仪器之一,在各大医院实验室和临床部门得到了广泛的应用。叙述荧光显微镜的原理、使用和校正方法。     

Morphology, fluorescence properties, and their photothermal changes of poly(substituted thiophene) films revealed by near-field fluorescence microspectroscopy

The morphology and fluorescence spectrum of poly{3-[2-(N-dodecylcarbamoyloxy)ethyl]thiophene-2,5-diyl} film were examined with spatial resolution of 100 nm using near-field fluorescence microspectroscopy. Fluorescence spectra observed at protruding domains were blue-shifted compared with flat areas, and further blue-shift was observed there more appreciably by long-time irradiation via a near-field scanning optical microscope probe. It is considered that the polymer chains at the protruding domains take disordered conformations, in which conjugated lengths are shorter and further disordering can be induced more easily by irradiation compared with those in the flat areas.     

DETERMINATION OF PICOMOLE CONCENTRATIONS OF ALUMINUM (III) IN HUMAN SALIVA AND URINE BY A LUMINOL-CARBOXYMETHYL CHITOSAN CHEMILUMINESCENCE SYSTEM

A luminol–carboxymethyl chitosan (CMCS) system was established using flow injection chemiluminescence (CL) based on the enhancing effect of CMCS on the luminol–dissolved oxygen reaction. The CL intensity was linear with the CMCS concentration ranging from 0.01–30.0 µM. Al(III) was shown to quench the CL of the luminol–CMCS reaction, and the decrease of CL intensity was linear with the logarithm of Al(III) concentration over the range from 10–1000 pM with a detection limit of 3.5 pM (3σ). At a flow rate of 2.0 mL min^?1, the analysis was performed within 30 s. The proposed CL method was successfully applied to the determination of picomole levels Al(III) in human saliva and urine after oral intake of two aluminum hydroxide tablets, with recoveries from 90.6–108.7% and relative standard deviations <3% (n = 5). The results indicated that the excreted Al(III) in saliva and urine reached its maximum values at 3 hr and 2 hr, and the total excretive ratio were 1.24 × 10^?3% and 3.45% in 6 hr and 12 hr, respectively. The elimination rate constant k and the half–life time t _1/2 in human saliva and urine were 0.3747 hr^?1, 1.8495 hr, 0.7132 hr^?1, and 0.9717 hr, respectively. The possible CL mechanism of the luminol–CMCS–Al(III) reaction is discussed.     

时间分辨荧光光纤生物传感器的研制及应用

首次研制了一种完全利用光纤实现光耦合及传输的时间分辨荧光光纤生物传感器.该传感器以镧系离子(铕离子)作为被检测物质的荧光标记物,根据铕离子的荧光特性,利用时间分辨检测技术进行生物医学检测.光学部分采用光纤耦合器和石英光纤作生物样品激发光的传导光学元件,生物样品受激发产生的荧光也同样由光纤耦合器及光纤收集和传导,实现了该传感器的紧凑性和小型化,提高了检测灵敏度.经性能测试,该仪器不稳定性小于2.3%,重复测量的互相关高达99.9%.测铕离子标准液的检测限为7.3×10^-10g/L,达到国外有关文献报道的水平.     

激光诱导荧光水质监测系统的研制

提出以Nd：YAG激光器的三倍频355nm波长激光为激发光源,以单个光电倍增管（PMT）为探测器,以水体污染监测为目的的激光诱导荧光系统的研制。采用光电倍增管高速双脉冲门控与单次脉冲触发数据采样相结合新技术,有效减少了户外环境下背景光的影响,提高了系统的测量精确度。试验结果表明,该系统能接收水体表面受激发射的荧光信号,探测到的荧光信号经信号处理后与水体受污染程度有良好的相关性。     

二氧化硅包覆对纳米多孔金增强荧光特性的调制

为了减弱金属基底对表面增强荧光的淬灭效应,设计了增强效果更好的荧光增强基底。采用化学生长二氧化硅的方法对纳米多孔金(NPG)表面进行修饰,避免荧光分子和NPG表面直接接触引起的淬灭效应,在SiO_2@NPG表面分别组装上罗丹明6G(R6G)和辐射中心波长为700 nm的量子点(QD 700)。通过探测分析荧光光谱,可以得出:二氧化硅包覆的基底可以使表面增强荧光得到显著的增强,并且二氧化硅厚度对荧光强度有调节作用;在基底增强量子点荧光信号的同时,量子点和NPG之间还出现非辐射的能量转移现象,二氧化硅的厚度对能量转移同样有调节作用,厚度约为5 nm时能量转移现象最显著。本实验为基于荧光能量转移的检测以及设计更好的荧光增强基底提供了参考。     

偏最小二乘法荧光光谱预测啶虫脒农药残留

为满足农药残留多组分含量测定的要求,对荧光光谱法测量农药残留得到的混合光谱进行分离,基于偏最小二乘法建立荧光光谱测量系统校正模型,并预测啶虫脒残留量。选择20个特征波长,采用交互验证方法,以预测残差平方和为评价指标,确定最优主成份数,获得了最佳分析模型。通过对预测集进行测试,滤纸带和西红柿表面啶虫脒残留浓度为100,220,450mg/kg的预测值分别是101.45,222.91,440.08mg/kg和98.67,208.56,419.22mg/kg,预测值和真实值的相关系数分别达到0.996和0.988。实验显示,采用偏最小二乘法结合荧光光谱测定啶虫脒农药残留,具有快速、无损、测量精度高等特点,并表明该方法用于定量分析复杂多组分体系是有效的。     

Time-resolved fluorescence microscopy using an improved europium chelate BHHST for the in situ detection of Cryptosporidium and Giardia

Fluorescent immunoconjugates prepared with the europium chelate BHHCT (4,4'-bis(1',1',1',2',2',3',3'-heptafluoro-4',6'-hexanedion-6'-yl)-chlorosulfo-o-terphenyl) have previously been reported as suitable labels for time-resolved fluorescence applications. BHHCT is limited by a tendency to destabilize immunoglobulins when covalently bound to the protein at moderate to high fluorophore to protein ratios (F/P). We report a new derivative of BHHCT prepared by appending a short hydrophylic tether to the chlorosulfonate activating group on BHHCT. The new derivative, BHHST (4,4'-bis-(1',1',1',2',2',3',3'-heptafluoro-4',6'-hexanedion-6'-yl)sulfonylamino-propyl-ester-N-succinimide-ester-o-terphenyl), was activated to bind at the tether terminus with a succinimide leaving group that displayed less aggressive coupling activity and improved storage stability. BHHST has been used to prepare a stable and useful immunoconjugate with the anti-Cryptosporidium monoclonal antibody CRY104. The BHHST immunoconjugate provides more than a 10-fold enhancement in the signal to noise ratio (SNR) of labeled oocyst fluorescence over background when observed using TRFM techniques. An immunoconjugate was also prepared with BHHST and (goat) anti-mouse that effectively labeled Giardia cysts in situ. Detection of cysts with the TRFM was achieved with an 11-fold increase in SNR when a gate-delay of 60 micros was employed. The storage half-life of both immunoconjugates is extended more than 20-fold when compared to immunoconjugates prepared with BHHCT.     

同步荧光光谱结合CARS变量优选预测猪肉中四环素残留含量

为快速检测猪肉中的四环素残留含量,采用同步荧光法结合竞争适应重加权采样(CARS)变量优选法建立了预测猪肉中四环素残留含量的支持向量回归(SVR)模型.从样本的三维同步荧光光谱中确定了最佳波长差为65 nm,采用CARS方法从中挑选出与四环素相关的特征波长变量,并与连续投影算法(SPA)及遗传算法(GA)进行比较.最后,应用SVR算法对优选出的16个波长变量建立猪肉中四环素含量的预测模型.分析发现,多元散射校正(MSC)光谱预处理后的CARS方法优于SPA及GA变量选择方法,可以有效地筛选出全光谱中的特征波长变量.CARS-SVR建立的四环素预测模型优于原始光谱的SVR模型,其预测集的决定系数(R2)和预测均方根误差(RMSEP)分别为0.961 2和10.94 mg/kg.研究结果表明,采用同步荧光法结合CARS-SVR模型可以预测猪肉中的四环素残留含量,且CARS-SVR能有效地简化模型并提高预测精度.     

Characterization of defects in semiconductors by combined application of SEM(EBIC) and SDLTS*

Besides the characterization of the geometrical structure of defects in semiconductors by TEM the estimation of their electrical activity is of importance. SEM(EBIC) and SDLTS (scanning deep level transient spectroscopy) are especially suitable for this purpose; they allow the inspection of electronic properties with a spatial resolution in the micron-range. On the one hand, SEM(EBIC) yields information on the recombination efficiency of defects in the crystal volume adjacent to a pn junction or a Schottky barrier; on the other hand, SDLTS enables the detection to be carried out of the distribution and the energetic levels of deep level defects lying in the space charge region. Accordingly, the combined application of these techniques is very promising for investigating physical processes implying an inhomogeneous incorporation of deep level defects in semiconductor crystals. In comparison to the widely used SEM(EBIC) technique SDLTS has only rarely been applied, a fact that is due to the high detection sensitivity necessary for measuring capacity transients. The application of a highly sensitive (10^—6 pF) micro-computer-controlled SDLTS system in combination with a conventional EBIC system allows a reliable inspection of semiconductor materials and devices, based on A₃B₅ compounds and on silicon. A typical application of the above technique is the investigation of the impurity distribution around extended crystal defects, like dislocations and precipitates, to study their gettering activity.     

Multiphoton microscopic imaging of in vivo hair mouse skin based on two-photon excited fluorescence and second harmonic generation

Mouse is an important animal model to investigate skin physiological and pathological states. In this article, multiphoton microscopic imaging of in vivo hair mouse skin based on two-photon excited fluorescence and second harmonic generation was examined. Our results show that multiphoton microscopy can clearly display microstructure of stratum corneum, stratum spinosum, and dermis of in vivo mouse skin. The main components of epidermis and dermis such as corneocytes, spinosum cell, collagen fibers, and hair follicles can be distinctly identified in MPM images. Using the optional HRZ 200 fine focusing stage, thickness of different layers can be easily assessed. The results demonstrate that MPM can be regarded as an efficient method for in vivo investigation of skin physiological and pathological states by using hair mouse animal model.     

Quantitative measurement of the resolution and sensitivity of confocal microscopes using line-scanning fluorescence correlation spectroscopy

Spatial resolution and the sensitivity to detect a fluorophore are the two most important optical parameters that characterize a confocal microscope. However, these are rather difficult to estimate quantitatively. We show that fluorescence correlation spectroscopy (FCS) provides an easy and reliable measure of these quantities. We modify existing schemes for performing FCS on a commercial confocal microscope to carry out these measurements, and provide an analysis routine that can yield the relevant quantities. Our method does not require any modification of the confocal microscope, yet it yields a robust measure of the resolution and sensitivity of the instrument.     

Dual colorimetric and fluorescent determination of iron (III) using a novel squaraine dye

A novel squaraine dye, 6-carboxy-2-[[3-[1,3-dihydro-3,3-dimethy-1-ethyl-2H-indol-2-ylidene)methyl]-2-hydroxy-4-oxo-2-cyclobuten-1-ylidene]methyl]-3,3-trimethy-3H-indolium, has been synthesized. The squaraine dye was found to be a dual colorimetric and fluorescent probe for the determination of iron (III) in CH₃CH₂OH/H₂O (4:1, v/v) exhibiting high selectivity and sensitivity. The binding of squaraine dye +Fe³⁺ was studied by a Job’s plot and Fourier transform infrared and ¹H nuclear magnetic resonance spectroscopies. The result indicates that the squaraine dye may be utilized as a naked-eye and real-time probe for the efficient determination of Fe^3+.     

微流控实时荧光聚合酶链式反应成像非均匀性的校正

综合参考标样法和定标校正法的思想,提出了一种用于校正微流控实时荧光聚合酶链式反应(PCR)系统荧光成像非均匀性的”多目标像素区域定标线性校正”算法,以提高其检测结果的准确性.以与PCR荧光标记物SYBR Green光谱特性相似的荧光素钠溶液为样本,检测了11种不同浓度的均匀荧光素钠溶液受激发射荧光信号的强度,分析了各个目标像素区域荧光强度和荧光素钠溶液浓度之间的线性响应关系,采用两点定标校正方法计算了CCD各个目标像素区域的校正系数矩阵.实验表明,3种浓度荧光素钠溶液的成像均匀度分别从校正前的71.28％、72.01％、70.73％提高到校正后的77.49％、80.07％、90.64％;微流控四腔芯片中相同浓度的DNA样品在PCR扩增阶段的Ct值相对标准偏差由校正前的4.38％、1.94％、3.31％减小到校正后的2.44％、0.79％、1.31％,显著提高了微流控实时荧光PCR检测结果的准确性.     

Identification of powdered Chinese herbal medicines by fluorescence microscopy,Part 1: Fluorescent characteristics of mechanical tissues,conducting tissues,and ergastic substances

The light microscope has been successfully used in identification of Chinese herbal medicines (CHMs) for more than a century. However, positive identification is not always possible. Given the popularity of fluorescence microscopy in bioanalysis, researchers dedicated to finding new ways to identify CHMs more effectively are now turning to fluorescence microscopy for authentication purposes. Some studies on distinguishing confused species from the same genus and on exploring distributions of chemicals in tissues of CHMs by fluorescence microscopy have been reported; however, no systematic investigations on fluorescent characteristics of powdered CHMs have been reported. Here, 46 samples of 16 CHMs were investigated. Specifically, the mechanical tissues including stone cells and fibers, the conducting tissues including three types of vessels, and ergastic substances including crystals of calcium oxalate and secretions, in various powdered CHMs were investigated by both light microscope and fluorescence microscope. The results showed many microscopic features emit fluorescence that makes them easily observed, even against complex backgrounds. Under the fluorescence microscope, different microscopic features from the same powdered CHM or some same features from different powdered CHMs emitted the different fluorescence, making this information very helpful for the authentication of CHMs in powder form. Moreover, secretions with unique chemical profiles from different powdered CHMs showed different fluorescent characteristics. Hence, fluorescence microscopy could be a useful additional method for the authentication of powdered CHMs if the fluorescent characteristics of specific CHMs are known. Microsc. Res. Tech. 2010. © 2010 Wiley‐Liss, Inc.     

发光二极管用于光度测量的表征和颜色增强

发光二极管(LED)光源和发光体的光度测量表征的新标准要求光度和色度量的测量值.本文研究了不同光谱和不同色温下的一些LED光源在光度计和色度应用中的表现.这项研究依赖于这些不同类型的LED混合光谱来增强显色指数(CRI),以尽可能接近标准源.LED光源表征需要一些光度和色度的评价数量,如光通量(?),色度坐标(x,y)和(u′,v′),相关色温(CCT)和显色指数(CRI).     

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device

A novel compact illumination device in variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) is described. This device replaces the standard condensor of an upright microscope. Light from different laser sources is delivered via a monomode fibre and focused onto identical parts of a sample under variable angles of total internal reflection. Thus, fluorophores in close proximity to a cell–substrate interface are excited by an evanescent wave with variable penetration depth, and localized with high (nanometre) axial resolution. In addition to quantitative measurements in solution, fluorescence markers of the cytoplasm and the plasma membrane, i.e. calcein and laurdan, were examined using cultivated endothelial cells. Distances between the glass substrate and the plasma membrane were determined using the mathematical algorithm of a four‐layer model, as well as a Gaussian‐shaped intensity profile of the illumination spot on the samples. Distances between 0 and 30 nm in focal contacts and between 100 and 300 nm in other parts of the cell were thus determined. In addition to measurements of cell–substrate topology, the illumination device appears appropriate for numerous applications in which high axial resolution is required, e.g. experiments on endocytosis or exocytosis, as well as measurements of ion concentrations proximal to the plasma membrane. The compact illumination device is also suitable for combining TIRFM with further innovative techniques, e.g. time‐resolved fluorescence spectroscopy, fluorescence lifetime imaging (FLIM) or fluorescence resonance energy transfer (FRET).