The source of ascitic fluid in experimental cirrhosis in the rat.

In order to disclose the source of ascitic fluid in liver cirrhosis, normal and cirrhotic rats were injected with fluorescein into the paracecal vein. The green fluorescence was then evaluated on the surface of the liver, the intestine and the peritoneum. Among healthy rats and in those with anascitic cirrhosis a very slight fluorescence was detected on the liver capsule whereas among rats with ascitic cirrhosis a distinct fluorescence was shown on the liver surface, the small intestine and the peritoneum. Therefore, the peritoneum is a source of ascitic fluid in cirrhosis of the rat.     

Oxidized LDL induces mitochondrially associated reactive oxygen/nitrogen species formation in endothelial cells

Exposure of cells to complex mixtures of oxidized lipids such as those found in oxidized low-density lipoprotein (oxLDL) induce reactive oxygen and nitrogen species (ROS/RNS) formation. The source of the ROS/RNS within cells is unknown; it is thought they may be involved in redox cell signaling. Although this possibility was initially overlooked, it is becoming clear that mitochondria, which are a source of superoxide and hydrogen peroxide, may play a critical role in the response of cells on exposure to oxidized lipids. In this study, we tested the possibility that mitochondria are a potential source of oxLDL-dependent formation of ROS/RNS in endothelial cells. Using confocal microscopy, we demonstrated that a significant proportion of oxLDL-dependent dichlorodihydrofluorescein (DCF) fluorescence is colocalized to mitochondria. In support of this concept, rho0 endothelial cells showed a substantial decrease in ROS/RNS formation stimulated by oxLDL. In contrast, mostly nonmitochondrial DCF fluorescence was detected in cells exposed to an extracellular source of hydrogen peroxide. The exposure of cells to a nitric oxide synthase inhibitor and urate resulted in a decrease in oxLDL-induced DCF fluorescence that was restored by addition of nitric oxide donors to the medium. Taken together, these results suggest that oxLDL-dependent DCF fluorescence is mitochondrially associated and may be due to the formation of peroxynitrite.     

The application of fluorescence spectroscopy to organic matter characterisation in drinking water treatment

Key to effective disinfection byproduct (DBP) management is source water control and management, and more specifically, organic matter (OM) control and management. However, the content and character of OM in source waters is spatially and temporally variable, and the prediction of its composition is challenging. Water treatment companies require adequate analytical techniques for OM characterisation to maintain the operation of the water supply and treatment systems adjusted to constantly changing environmental conditions. There is a requirement, therefore, for an improved understanding of OM composition and character in source water, how that composition and character varies with flow conditions, and how this impacts on drinking water treatment. This paper demonstrates that fluorescence spectroscopy offers a potential alternative to other analytical methods of OM characterisation. The advantages of fluorescence include rapid, sensitive and selective characterisation of OM, no sample pre-treatment, small sample volume, and the potential for on-line monitoring incorporation. Fluorescence can provide useful information on OM reactivity and treatability together with an indication of the OM sources (allochthonous or autochthonous). The paper discusses a body of literature which has identified relationships between fluorescence spectra and OM physico-chemical properties (i.e. degree of hydrophobicity, microbial content), has applied fluorescence spectroscopy to characterise the changes in OM upon disinfection, and has related the fluorescence properties to DBP formation. Further work is required in the robust management of data arising from fluorescence spectroscopy analysis and, in particular, Excitation Emission Matrices. Consideration must be given as to how the data might best be employed to greatest effect on a routine basis at WTW.     

Use of Tunable, Pulsed Dye Laser for Quantitative Fluorescence in Syphilis Serology (FTA-ABS Test)

A pulsed dye laser was used as an excitation source in a fluorescent treponemal antibody absorption (FTA-ABS) test. A high precision in quantitative fluorescence was obtained with this high-power excitation source coupled to an electronic detection system and a storage oscilloscope by standardization of fluorescence evaluation and through elimination of human error. One 0.4-mus pulse exposure was sufficient to record fluorescence intensity data on the oscilloscope. Absence of fading of fluorescence after repeated excitation permitted multiple readings of the same microscope field. Almost 100% reproducible results were obtained for the FTA-ABS test with 40 samples. Electronic detection of fluorescence and the high sensitivity obtained with laser excitation raise doubts about the relative value of quantitative immunofluorescence in the FTA-ABS test.     

Complex NAPL Site Characterization Using Fluorescence Part 3: Detection Capabilities for Specific Excitation Sources

Non-aqueous phase liquid (NAPL) contaminants comprised of polycyclic aromatic hy drocarbons (PAHs) can be detected using fluorescence spectroscopic methods. Dense non-aqueous phase liquid (DNAPL) contaminant source zones can be delineated us ing commercially available cone penetrometer (CPT)devices by detecting commingled oils fuels, and naturally occurring organic materials entrained by DNAPLs and carried to depths below the water table. It has been demonstrated that commercially avail able CPT based fluorescence detection systems can be ranked based on how effectively their excitation source wavelengths induce fluorescence using excitation emission ma trices (EEMs). Several neat NAPLs and dilutions with selected DNAPLs were analyzed for specific fluorescence characteristics to determine the optimal excitation source for site characterization efforts. A comprehensive spectral library and corresponding opti mization matrix were generated for complex petroleum mixtures. Based onfield results documenting successful indirect CPT fluorescence detection of a DNAPL source zone, aviation and dieselfuels were selected from this library, diluted with chlorinated sol vents, and evaluated for fluorescence characteristics. Dilution of these complex NAPL mixtures led to changes in the corresponding EEMs. The optimal excitation source for aviationfuel remained relatively constantf or each dilution. However, sensitivityf or each of the commercially available CPT excitation sources was strongly dependent on diesel concentration, whereby higher energy (lowerwavelength) sources yielded improved sen-sitivityfor lower concentrations. Since field concentrations can be highly variable, these observations support the need for multiple wavelength excitation sources for optimal detection capabilities, particularly when diesel fuel ispresent.     

Coherent scattering in multi-harmonic light microscopy

By focusing a pulsed laser beam into a sample, harmonic up-conversion can be generated as well as multi-photon excited fluorescence. Whereas multi-photon excited fluorescence microscopy is well established, the use of multi-harmonic generation for three-dimensional image contrast is very recent. Both techniques can provide similar resolution and, for adequate radiating source density, comparable signal levels, allowing them to be combined in a single versatile instrument. However, harmonic generation differs fundamentally from fluorescence generation in that it is coherent and produces radiation patterns that are highly sensitive to phase. As such, multi-harmonic generation microscopy provides a unique window into molecular spatial organization that is inaccessible to fluorescence.     

Complex NAPL Site Characterization Using Fluorescence Part 2: Analysis of Soil Matrix Effects on the Excitation/Emission Matrix

Commercially available cone penetrometer (CPT)fluorescence based sensor platforms have been used to detect non-aqueous phase liquids (NAPLs), such as petroleum oils and lubricants, in situf or more than a decade. These approaches have also been used to detect dense non-aqueous phase liquid (DNAPL) source zones by detecting commingled oilsfuels, and naturally occurring organic materials entrained by DNAPLs and carried to depths below the water table. Several neat NAPLs and mixtureswere added to various soil types and analyzedfor specific fluorescence characteristics to determine the optimal excitation source for site characterization efforts. Using excitationlemission matrices (EEMs), we demonstrate that an optimized excitation wavelength can be determinedfor specific fiuowphores within the NAPL mixtures, and that available systems can be ranked based on the specific contaminant and site soil types. An optimal excitation wavelength yields the maximum fluorescence within an EEM spectrum. We ranked commercially available cone penetrometer fluorescence detection systems according to the potential for ease of detection based on maximum fluorescence response. When soils were added tocomplexNAPLmixtures,analytefluorescence emissionwasattenuatedinpreferential portions of the EEM, leading to differences in the optimal excitation source wavelength. Furthermore, impure silica-containing minerals impact the emission signal, potentially leading to incorrect conclusionsf or several commercially available systems. Our find ings suggest that afrequency-agile (e.g., tunable excitation source) probe system would be superior to any other system commercially available, provided the system would be relatively easy to operate and would have rapid in-situ EEM generating capabilitiesfo r optimization in the field.     

荧光寿命分析法在地沟油鉴别中的应用研究

通过时间相关单光子计数法检测含有杂质的植物油中的荧光光子的寿命,通过其荧光寿命与纯净植物油的荧光寿命对比,以此为依据可以鉴别出植物油中是否掺入其他油。此方法的优点在于利用荧光寿命检测方法不受光源波动、老化以及外界杂散光的影响,而且只要植物油中掺入了其他有杂质的油就能被检测出来,与掺入的油的浓度无关。因此荧光寿命测量对检测植物油的纯度表现出更好的检测精度及稳定性。实验结果也证明了荧光寿命法检测地沟油的方案具有可行性,且样品处理步骤简单,用于检测的用量少,检测结果准确可靠。     

鼻咽组织荧光寿命影响因素研究

本文在研究了离体人体鼻咽正常组织和癌变组织的荧光寿命的基础上,实验研究了生理盐水的浓度、组织光学特性参数、激发光源的偏振性对癌变和正常鼻咽组织的荧光寿命的影响。实验结果表明：组织的光学特性参数对组织的荧光寿命有不同程度的影响;而不同浓度的生理盐水和光源的偏振性对组织的荧光寿命没有显著的影响。荧光寿命与该发射荧光的强度没有关系,只决定于局部环境,受微环境的物理化学性质因素的影响,因此荧光寿命作为人体组织癌变的检测方法,有着很好的应用前景。     

Isolation and Characterization of Photosystem Ⅱ Reaction Center D1-D2-Cytochrome b-559 Complex from the Chloroplasts of Spinach

Photosystem Ⅱ reaction center D1-D2-cytochrome b-559 pigment-protein complex has been isolated and purified from chloroplasts of spinach and its properties have been studied. The Isotared photosystem II reaction center contains close to six chlorophyll a per two pheophytin a molecules. Analysis of fluorescence decaying by phase modulation fluorometry suggests that the reaction center has three components of fluorescence decaying with lifetimes of 1.5 nS, 6.23 nS, 36.26 nS in terms of fractions to total fluorescence yield as 0.06, 0.67, 0.27 respectively. The ,6.25 nS fluorescence component corresponds to chlorophyll a which is energetically uncoupled from the process of charge separation. The proportion of 1.51 nS component is very low, and its source is unclear. The 36.25 nS fluorescence component is attributed to the recombination of the primary radical pair, and so represents the activity of charge separation.     

A subnanosecond resolving spectrofluorimeter for the analysis of protein fluorescence kinetics

A spectrofluorometer is described consisting of an excitation source, optics, detector and time resolving electronics. The excitation source consists of a mode-locked Ar ion laser, synchronously pumps a dye laser, followed by a frequency doubling device. The repetition frequency of the U.V. pulses (FWHM some ps) has been reduced by an extra-cavity electro-optical modulator. Provisions have been made in the optical configuration to determine both time-resolved fluorescence spectra and fluorescence anisotropy decay curves. The commercially avialable electronics have been optimized for maximum time resolution. The spectral output of the excitation source is confined between 280 and 310 nm, which encompasses the region for eliciting protein fluorescence. The performance of the complete system has been tested with single lifetime standards line p-terphenyl in cyclohexane or with $\text{N-}\text{acetyl}\text{-}\text{L}\text{-}\text{tryptophanamide}$ in pH 7.5 buffer. Serum albumins from human and bovine sources have been employed as examples for time resolved fluorescence spectra and for the demonstration of anisotropy decay curves. Using these methods protein dynamics in the (sub)nanosecond time region can be directly explored.     

Evaluation of source leaf responses to water-deficit stresses in cotton using a novel stress bioassay

Water-deficit stresses preferentially reduce shoot growth, thereby disrupting the flow of carbohydrates from source leaves to the developing sinks. Here, we use a novel stress bioassay to dissect responses of field and greenhouse-grown cotton (Gossypium hirsutum) source leaves to water-deficit stresses. Fifth main stem leaf samples were harvested at sunrise and subjected to a prolonged elevated respiratory demand in the dark. Sucrose levels are lower in nonstressed cotton at sunrise compared to water-deficit stressed cotton, potentially predisposing the nonstressed tissue to succumb more rapidly. Tissue death was determined initially using the cell viability stain 2,3,5-triphenyltetrazolium chloride, but was determined in subsequent experiments by monitoring the decline in chlorophyll fluorescence yield. Fluorescence yield measurements were obtained within minutes of harvesting and individual samples were monitored over the time course of the treatment. Analyses of the time course and magnitude of chlorophyll fluorescence yield decline in samples from irrigated and dryland plots permitted the detection of stress responses within 24 h of the cessation of irrigation. The rate of fluorescence yield decline during the elevated respiratory demand treatment slowed as the water-deficit stress increased. Upon irrigation, the source leaves of the water-stressed plants recovered to prestress values within 4 d. Well-watered cotton overexpressing heat shock protein 101 had identical rates of fluorescence yield decline as nontransgenic cotton. These results suggest that the delayed decline in fluorescence yield of water-stressed tissue exposed to prolonged elevated respiratory demand can be used as a sensitive indicator of water-deficit stress responses.     

Interaction of ribonuclease T1 with DNA,mononucleotides and oligonucleotides

The interaction of ribonuclease T₁ with DNA and nucleotides was investigated by fluorescence titration to establish whether or not this enzyme is a helix-destabilizing protein. Binding of the enzyme to DNA, ribonucleotides and oligodeoxyribonucleotides of chain length ten or more leads to enhancement of fluorescence emission of the enzyme as a function of increasing nucleotide/protein ratio. For deoxyribonucleotides of chain length less than ten, only quenching is observed. Energy transfer from the bases is postulated to be the source of the enhancement of fluorescence, while the decrease can be ascribed to changes in the distribution of charged groups in or near the binding site.     

An integrated portable apparatus for the simultaneous field measurement of photosynthetic CO2 and water vapour exchange, light absorption and chlorophyll fluorescence emission of attached leaves

Abstract. A portable apparatus has been constructed to measure simultaneously the quantum yield of CO₂ assimilation, light absorption, chlorophyll fluorescence emission and water vapour exchange of attached intact leaves in the field. The core of the instrument is a light-integrating spherical leaf chamber which includes ports for a light source, photosynthetically active radiation sensor, fluorescence probes and gas inlet and outlet manifolds. Measurement of the quantum flux inside the empty chamber and with a leaf present allows determination of leaf absorptance. An open gas exchange system is employed using an infra-red analyser to measure leaf CO₂ exchange. Using a DC white light source the quantum yield of CO₂ assimilation based on absorbed light (φ_abs) may be determined rapidly in either ambient air or artificial gas mixtures. Inclusion of capacitance humidity probes into the gas inlet and outlet ports allows simultaneous determination of water vapour exchange and subsequent estimation of stomatal conductance to CO₂ and intercellular CO₂ concentration. Measurement of fluorescence emission by the sample leaf exposed to white light is achieved by a modulated fluorescence detection system. In addition to determination of the minimal, maximal and variable fluorescence levels, a further analysis allows the photochemical and non-photochemical components of fluorescence quenching, to be estimated. The theory and design of this apparatus is described in detail. The use of the apparatus in the field is demonstrated through a study of the photosynthetic performance of a maize and bean crop during the growing season and by analysis of the photosynthetic performance of crops subjected to nitrogen-stress and a herbicide treatment.     

Comparison of X-ray fluorescence and optical fluorescence measured behind scattering layers as a basis for X-ray fluorescence endoscopy.

Recent developments in the technology of capillary-fiber optics suitable for X-rays in the range of approximately 4-10keV point to the possible realization of endoscopes applicable in X-ray fluorescence analysis. A general problem is the determination of scattering and absorption processes with consideration to tissue optics, X-ray scattering and X-ray absorption in a diagnostic partial volume. Therefore comparative investigations were performed in order to answer these questions. Zinc-oxide nanoparticles configured as single particles and ZnO clusters provided the fluorescence source in cell layers. An artificial scattering material was employed, which closely approximated the tissue optical conditions and the X-ray optical application conditions in possible diagnostic situations. As a result imaging of spatially resolved X-ray contrasts was better than adequate optical fluorescence imaging by approximately one magnitude. Hence a very important precondition for realizing X-ray fluorescence endoscopy is fulfilled.     

Identification and removal of contaminating fluorescence from commercial and in-house printed DNA microarrays

Microarray analysis is a critically important technology for genome-enabled biology, therefore it is essential that the data obtained be reliable. Current software and normalization techniques for microarray analysis rely on the assumption that fluorescent background within spots is essentially the same throughout the glass slide and can be measured by fluorescence surrounding the spots. This assumption is not valid if background fluorescence is spot-localized. Inaccurate estimates of background fluorescence under the spot create a source of error, especially for low expressed genes. We have identified spot-localized, contaminating fluorescence in the Cy3 channel on several commercial and in-house printed microarray slides. We determined through mock hybridizations (without labeled target) that pre-hybridization scans could not be used to predict the contribution of this contaminating fluorescence after hybridization because the change in spot-to-spot fluorescence after hybridization was too variable. Two solutions to this problem were identified. First, allowing 4 h of exposure to air prior to printing on to Corning UltraGAPS slides significantly reduced contaminating fluorescence intensities to approximately the value of the surrounding glass. Alternatively, application of a novel, hyperspectral imaging scanner and multivariate curve resolution algorithms, allowed the spectral contributions of Cy3 signal, glass, and contaminating fluorescence to be distinguished and quantified after hybridization.     

Laser induced fluorescence emission (LIFE)

The authors used an ultraviolet laser as an excitation source to obtain fluorescence spectra from 4 μl of solution, or 0.1 μl equivalent of powder. A sensitivity of 0.1 part per trillion quinine sulfate was obtained. The system was sufficiently sensitive to detect Raman shifts. A measurement of the degree of fluorescence polarization was made.     

Long-range interactions of <Emphasis Type="Italic">Chara</Emphasis> chloroplasts are sensitive to plasma-membrane H<Superscript>+</Superscript> flows and comprise separate photo- and dark-operated pathways

Local illumination of the characean internode with a 30-s pulse of white light was found to induce the delayed transient increase of modulated chlorophyll fluorescence in shaded cell parts, provided the analyzed region is located downstream in the cytoplasmic flow at millimeter distances from the light spot. The fluorescence response to photostimulation of a remote cell region indicates that the metabolites produced by source chloroplasts in an illuminated region are carried downstream with the cytoplasmic flow, thus ensuring long-distance communications between anchored plastids in giant internodal cells. The properties of individual stages of metabolite signaling are not yet well known. We show here that the export of assimilates and/or reducing equivalents from the source chloroplasts into the flowing cytoplasm is largely insensitive to the direction of plasma-membrane H⁺ flows, whereas the events in sink regions where these metabolites are delivered to the acceptor chloroplasts under dim light are controlled by H⁺ fluxes across the plasma membrane. The fluorescence response to local illumination of remote cell regions was best pronounced under weak background light and was also observed in a modified form within 1–2 min after the transfer of cell to darkness. The fluorescence transients in darkened cells were suppressed by antimycin A, an inhibitor of electron transfer from ferredoxin to plastoquinone, whereas the fluorescence response under background light was insensitive to this inhibitor. We conclude that the accumulation of reduced metabolites in the stroma leads to the reduction of photosystem II primary quinone acceptor (Q_A) via two separate (photochemical and non-photochemical) pathways.     

激光荧光技术及其在生物医学研究中的应用

激光荧光技术是研究生物体系结构与功能的重要手段,发展迅速,应用广泛。我们研制了用氩离子激光器作光源的多参数流式细胞光度计,荧光漂白恢复装置以及毫微秒荧光谱仪。本文对其原理、结构及其在生物医学研究中的应用作了详细的介绍。     

One-step hydrothermal preparation of chiral carbon quantum dots and enantioselective sensing of glutamine enantiomeric isomers

A novel method for chiral identification of glutamine enantiomers based on chiral carbon quantum dots (cCQDs) fluorescent probes. cCQDs were prepared using a one-step hydrothermal method with L-tryptophan as the carbon source and chiral source, producing spherical nanoparticles exhibiting a blue colour luminescence. The fluorescence intensity (F) of cCQDs was enhanced or quenched following the addition of chiral enantiomeric glutamine (L/D-Gln), and therefore cCQDs, as a fluorescence probe, could be used for enantioselective sensing of the L/D-Gln. The fluorescence enhancement value (∆F_E) exhibited good linearity with L-Gln concentration in the range 0.23–10.00 mM, and the limit of detection was 0.14 mM. The fluorescence quenching value (∆F_Q) showed a good linear relationship with D-Gln concentration in the range 0.29–10.00 mM, and the detection limit was 0.18 mM. The mechanism of fluorescence enhancement/quenching was explored by molecular modelling and the type of quenching. The method was applied to the determination of L-Gln content in real samples, and the recovery rate was satisfactory. This study provided a novel approach for the synthesis of cCQDs and the recognition of amino acid enantiomers.