Fluorescent Parameters Research Articles

基于线性方法的时域荧光扩散光层析实验验证

We present a full three-dimensional, featured-data algorithm for time-domain fluorescence diffuse optical tomography that inverts the Laplace-transformed time-domain coupled diffusion equations and employs a pair of appropriate transform-factors to effectively separate the fluorescent yield and lifetime parameters. By use of a time-correlation single-photon counting system and the normalized Born formulation, we experimentally validate that the proposed scheme can achieve simultaneous reconstruction of the fluorescent yield and lifetime distributions with a reasonable accuracy.

Topography Studies on the Membrane Interaction Mechanism of the Eosinophil Cationic Protein

The eosinophil cationic protein (ECP) is an antipathogen protein involved in the host defense system. ECP displays bactericidal and membrane lytic capacities [Carreras et al. (2003) Biochemistry 42, 6636-6644]. We have now characterized in detail the protein-membrane interaction process. All observed fluorescent parameters of the wild type and single-tryptophan-containing mutants, as well as the results of decomposition analysis of protein fluorescence, suggest that W10 and W35 belong to two distinct spectral classes I and III, respectively. Tryptophan residues were classified and assigned to distinct structural classes using statistical approaches based on the analysis of tryptophan microenvironment structural properties. W10 belongs to class I and is buried in a relative nonpolar, nonflexible protein environment, while W35 (class III) is fully exposed to free water molecules. Tryptophan solvent exposure and the depth of the protein insertion in the lipid bilayer were monitored by the degree of protein fluorescence quenching by KI and brominated phospholipids, respectively. Results indicate that W35 partially inserts into the lipid bilayer, whereas W10 does not. Further analysis by electron microscopy and dynamic light scattering indicates that ECP can destabilize and trigger lipid vesicle aggregation at a nanomolar concentration range, corresponding to about 1:1000 protein/lipid ratio. No significant leakage of the vesicle aqueous content takes place below that protein concentration threshold. The data are consistent with a membrane destabilization "carpet-like" mechanism.

High‐throughput liquid‐chromatography method with fluorescence detection for reciprocal determination of furosemide or norfloxacin in human plasma

A simple, high-throughput, highly selective and sensitive HPLC-FLD method for isolation and determination of furosemide and/or norfloxacin in human plasma samples following a simple organic solvent deproteinization step with acetonitrile as sample 'clean-up' procedure is reported. One of the two drug substances plays the internal standard role for the determination of the other. Separation of analyte and internal standard was achieved in less than 5.3 min (injection to injection) on a Chromolith Performance RP-18e column, using an aqueous component containing 0.015 mol/L sodium heptane-sulfonate and 0.2% triethylamine brought to pH = 2.5 with H(3)PO(4). The composition of the mobile phase was: acetonitrile-methanol-aqueous component = 70:15:15 (v/v/v) and the flow-rate was set up to 3 mL/min. The chromatographic method applied to the determination of furosemide relies on fluorescent detection parameters of 235 nm for the excitation wavelength, and 402 nm for the emission wavelength. In case of norfloxacin, the excitation wavelength is set up to 268 nm and the emission wavelength is set up to 445 nm. The overall method leads to quantitation limits of about 27 ng/mL for furosemide, and 19.5 ng/mL for norfloxacin, using an injection volume of 250 microL. The method was applied to the bioequivalence study of two furosemide-containing formulations.

Evaluation of intranuclear BrdU detection procedures for use in multicolor flow cytometry

Measurement of cell proliferation via BrdU incorporation in combination with multicolor cell surface staining would facilitate studies on cell subsets that require multiple markers for their identification. However, the extent to which the often harsh cell preparation procedures required affect the staining quality of more recently developed fluorescent dyes has not been assessed. Three cell preparation protocols for BrdU measurement were compared for their ability to maintain fluorescent surface staining and scatter parameters of in vivo BrdU-labeled cells by flow cytometry. A 10-color fluorescent panel was developed to test the quality of surface staining, following cell treatment and the ability to perform BrdU measurements on even small B lymphocyte subsets. All cell preparation procedures affected the quality of fluorescent and/or scatter parameters to varying degrees. Paraformaldehyde/saponin-based procedures preserved sufficient fluorescent surface staining to determine BrdU incorporation rates among all splenic B cell subsets, including B-1a cells, which constitute roughly 0.5% of cells. Turnover rates of B-1a cells were similar to immature B cells and higher than those of the other mature B cell subsets. Paraformaldehyde/saponin-based cell preparation procedures facilitate detailed cell turnover studies on small cell subsets in vivo, revealing new functional information on rare cell populations.

Ceramide-platform formation and -induced biophysical changes in a fluid phospholipid membrane

To understand the formation and properties of ceramide-enriched domains in cell membranes, the biophysical properties of the binary system palmitoyloleoylphosphatidylcholine (POPC)/palmitoylceramide were thoroughly characterized. Diverse fluorescent probes and parameters were necessary to unravel the complexity of this apparently simple system. For the first time, a complete phase diagram is reported, characterizing the lamellar phases of these mixtures, and providing a quantitative framework integrating biophysical and biological studies. The diagram suggests that in resting cells no ceramide domains exist, but upon apoptotic stimuli, platforms may form. Moreover, our data show that 2 mol% of Cer strongly affects the POPC fluid matrix, suggesting that a small increase in Cer levels can significantly affect cell membrane properties. In this work, we also show that Cer domains, formed in conditions similar to physiological, are extremely ordered and rigid. The domains composition is estimated from the phase diagram and their large size was concluded from fluorescence resonance energy transfer. Dynamic light scattering and electron microscopy were used to characterize the system morphology, which is highly dependent on ceramide content and includes vesiculation and tubular structure formation.

New ketocyanine-based fluorescent optodes for the development of integrated waveguide sensors

Twenty ketocyanine dyes are evaluated as potential pH fluoroionophores to be used in the development of new integrated waveguide fluorescence optodes (IWFOs). Its fluorescence characterization, as well as the pH response is first undertaken in ethanolic solution and then in plasticized PVC membranes with a conventional spectrofluorimeter. Analytical parameters such as basicity, reversibility, leaching and calibration curves are determined in both media and finally, photostability is also measured. Besides, some photophysical fluorescent parameters, such as the emission wavelength, are evaluated for both species, the acidic and the basic form, and quantum yields are calculated in ethanolic solution. Ketocyanine dyes have demonstrated their suitability for being used as acidochromic fluoroionophores in bulk optodes for the development of new IWFOs. They exhibit high fluorescence intensities in the far-visible region, an adequate photostability and an optimum pH response.

Comparative Studies on the Structure and Stability of Fluorescent Proteins EGFP, zFP506, mRFP1, “dimer2”, and DsRed1

To obtain more information about the structural properties and conformational stabilities of GFP-like fluorescent proteins, we have undertaken a systematic analysis of series of green and red fluorescent proteins with different association states. The list of studied proteins includes EGFP (green monomer), zFP506 (green tetramer), mRFP1 (red monomer), "dimer2" (red dimer), and DsRed1 (red tetramer). Fluorescent and absorbance parameters, near-UV and visible CD spectra, the accessibility of the chromophores and tryptophans to acrylamide quenching, and the resistance of these proteins to the guanidine hydrochloride unfolding and kinetics of the approaching of the unfolding equilibrium have been compared. Tetrameric zFP506 was shown to be dramatically more stable than the EGFP monomer, assuming that association might contribute to the protein conformational stability. This assumption is most likely valid even though the sequences OF GFP and zPF506 are only approximately 25% identical. Interestingly, red FPs possessed comparable conformational stabilities, where monomeric mRFP1 was the most stable species under the equilibrium conditions, whereas the tetrameric DsRed1 possessed the slowest unfolding kinetics. Furthermore, EGFP is shown to be considerably less stable than mRFP1, whereas tetrameric zFP506 is the most stable species analyzed in this study. This means that the quaternary structure, being an important stabilizing factor, does not represent the only circumstance dictating the dramatic variations between fluorescent proteins in their conformational stabilities.

Phenylene dendrimers and novel hyperbranched polyphenylenes as light emissive materials for blue OLEDs

New materials based on low-generation polyphenylene dendrimers with the light emission in the blue spectrum range were synthesized and examined for an efficient organic light emitting diodes (OLED) application. It has been shown that the ratio of p-phenylene groups with high fluorescence parameters to 1,3,5-triphenylbenzene groups with low fluorescent parameters may be the possible reason for the experimental variations of relative quantum yield of photoluminescence in the compounds explored. The quantum yield value is increased with a number of dendrimer generations up to 50–70%. The role of bromine atoms as the luminescence quenchers have been demonstrated, which is important for synthesis route choice.

Effect of low-intensity (3.75-25 J/cm2) near-infrared (810 nm) laser radiation on red blood cell ATPase activities and membrane structure.

The biostimulation and therapeutic effects of low-power laser radiation of different wavelengths and light doses are well known, but the exact mechanism of action of the laser radiation with living cells is not yet understood. The aim of the present work was to investigate the effect of laser radiation (810 nm, radiant exposure 3.75-25 J/cm(2)) on the structure of protein and lipid components of red blood cell membranes and it functional properties. The role of membrane ATPases as possible targets of laser irradiation was analyzed. A variety of studies both in vivo and in vitro showed significant influence of laser irradiation on cell functional state. At the same time another group of works found no detectable effects of light exposure. Some different explanations based on the light absorption by primary endogenous chromophores (mitochondrial enzymes, cytochromes, flavins, porphyrins) have been proposed to describe biological effects of laser light. It was suggested that optimization of the structural-functional organization of the erythrocyte membrane as a result of laser irradiation may be the basis for improving the cardiac function in patients under a course of laser therapy. Human red blood cells or isolated cell membranes were irradiated with low-intensity laser light (810 nm) at different radiant exposures (3.75-25 J/cm(2)) and light powers (fluence rate; 10-400 mW) at 37 degrees C. As the parameters characterizing the structural and functional changes of cell membranes the activities of Na(+)-, K(+)-, and Mg(2+)-ATPases, tryptophan fluorescence of membrane proteins and fluorescence of pyrene incorporated into membrane lipid bilayer were used. It was found that near-infrared low-intensity laser radiation changes the ATPase activities of the membrane ion pumps in the dose- and fluence rate-dependent manner. At the same time no changes of such integral parameters as cell stability, membrane lipid peroxidation level, intracellular reduced glutathione or oxyhaemoglobin level were observed. At laser power of 10 mW, an increase of the ATPase activity was observed with maximal effect at 12-15 J/cm(2) of light dose (18-26% for the total ATPase activity). At laser power of 400 mW (fluence rate significantly increased), inhibition of ATPases activities mainly due to the inhibition of Na(+)-, K(+)-ATPase was observed with maximal effect at the same light dose of 12-15 J/cm(2) (18-23% for the total ATPase activity). Fractionation of the light dose significantly changed the membrane response to laser radiation. Changes in tryptophan fluorescent parameters of erythrocyte membrane proteins and the increase in lipid bilayer fluidity measured by pyrene monomer/excimer fluorescence ratio were observed. Near-infrared laser light radiation (810 nm) induced long-term conformational transitions of red blood cell membrane which were related to the changes in the structural states of both erythrocyte membrane proteins and lipid bilayer and which manifested themselves as changes in fluorescent parameters of erythrocyte membranes and lipid bilayer fluidity. This resulted in the modulation of membrane functional properties: changes in the activity of membrane ion pumps and, thus, changes in membrane ion flows.

High-performance liquid chromatography-based protease detection at the picogram level

Highly sensitive detection of proteolytic activity canbe achieved by means of fluorophore-tagged artificialsubstrates. Protease-catalyzed cleavage of the substrateleads to a quantitative release of an uncoupled fluoro-phore and a corresponding increase in fluorescence [1–7]. Typically, the protease/substrate mixture in an assaybuffer solution is incubated at 25–37 C with fluores-cence intensity of the reaction product measured in asuitable fluorometer. The procedure, however, can be-come quite tedious when protease detection requires alarge number of samples and prolonged incubation time.In addition, the fluorescent intensity parameter alonemay not be significant enough to provide statisticallyvalid and reproducible data in the lower, close to thebackground, detection range.In this report we describe a new procedure for pro-tease detection utilizing a semiautomated, HPLC-basedtechnique.Trypsinservedasamodelproteolyticenzyme.The rhodamine-conjugated substrate was chosen for itsstability, low fluorescence background, and high sensi-tivity[5,6].Thedevelopedprocedurerequiredonlyabout5min per analysis and minimal labor and allowed de-tection ofa protease concentration at the picogram level.Reagents and solvents were of the highest availablepurity. The rhodamine 110, bis-(Cbz)-

Fluorescent behavior of B-phycoerythrin in microemulsions of aerosol OT/water/isooctane

Taking advantage of its unusual fluorescent properties, the incorporation of B-phycoerythrin (B-PE) in aerosol OT (AOT, sodium bis-(2-ethylhexyl) sulphosuccinate)/water/isooctane microemulsions was investigated by following their steady-state and time-resolved fluorescence as a function of the water-to-surfactant molar ratio, w 0. The fluorescent intensity at 575 nm increased continuously with increasing water content, saturating at a w 0 around 35 and staying practically constant at w 0⩾40. The steady-state anisotropy showed an initial increase with increasing water content until w 0=23 and then decreased strongly, staying practically constant when w 0⩾40. The values of the fluorescent parameters, anisotropy and fluorescent intensity, were unchanged when the water content of the system increased in the range between w 0=40 to 50. This implies the effective incorporation of B-PE in the microemulsion droplets with w 0⩾40, as well as the equilibrium of the dispersion at these water/surfactant ratios, since higher water content does not affect the main surrounding microenvironment of the protein. The overall incorporation in the microemulsion droplets caused minor spectroscopic changes with respect to biliprotein in aqueous solution of 20 mM sodium phosphate buffer, pH 7.0, such as a blue absorption shift of 3 nm and an emission shift of 1.5 nm, as well as a slight increase in excitation anisotropy spectrum mainly caused by a decrease in protein mobility. Therefore, there are no important interactions between the chromophores and the AOT sulfonate head groups. Emission intensity decays followed complex kinetics in both aqueous and dispersion media. The stability with time and temperature of the biliprotein in the microemulsion was higher than in the aqueous solution. All the results can be explained in terms of B-PE inclusion in the water droplets of AOT microemulsions where the protein has similar configuration and conformation to that in aqueous solution but with the chromophores more protected.

Sol–gel derived urease-based optical biosensor for the rapid determination of heavy metals

A urease optical biosensor for the determination of heavy metals based on sol–gel immobilization technique was developed. A fluorescent dye, FITC-dextran, was encapsulated and parameters including optical properties of the probe, relative enzyme activity, initial pH value and the buffer concentration for substrate preparation were investigated. In sol–gel immobilization, 1 mM Tris–HCl at pH 7.1 provided a sufficient buffer capacity for metal ion analysis as well as the enzyme activity maintenance. Also, two analytical procedures, incubated and un-incubated systems, were compared to understand the sensitivity and applicability to heavy metal analysis. The developed optical biosensor showed high reproducibility and the relative standard deviation (R.S.D.) of 5.1% ( n=10) was obtained. Also, eight measurements can be completed automatically within 36 min. The biosensor has high sensitivity to Cu(II) and Cd(II) and an analytical range of 10–230 μM with a detection limit of 10 μM was achieved. Moreover, biological and environmental samples were examined to evaluate the applicability of the developed biosensor. A 19–82% of inhibition was observed when 20–45 μM metal ions were amended into tested samples, revealing that the developed system has the potential for the determination of heavy metals in real samples.

Determination of the water droplet size distribution of fat spreads using confocal scanning laser microscopy.

Knowledge of the water droplet size distribution of fat spreads is necessary for the development and production of high quality microbiological safe products. Fat spreads are water-in-oil emulsions. The water droplet size distribution can be determined by confocal scanning laser microscopy (CSLM) after staining the fat with Nile Red. The profiles of the non-fluorescent water droplets in the 2D images are identified and measured using image analysis. The 'true' water droplet size distribution is calculated from the distribution of the measured profile diameters using a Wicksell transformation of log-normal distributions. The influence of the fluorescent staining and CSLM parameters on the information were studied. The CSLM method was tested on fat spreads with a fat content ranging from 40% to 80%. The results were compared with those obtained by nuclear magnetic resonance spectroscopy (NMR). The distribution parameters [volume weighed geometric mean diameter (D3,3) and the standard deviation (sigma) of the logarithm of the droplet diameter] calculated for 80% fat spreads are in good agreement with those obtained by NMR (within +/- 7% relative). Small differences were found for 65% fat spreads and large differences were identified for 40% fat spreads. The precision for the determination of the D3,3 value by CSLM is worse than that of NMR, even when three images were used to calculate this parameter [3-11% and 1-6% relative standard deviation (RSD), respectively]. The precision for the determination of exp(sigma) by CSLM is comparable or better than that of NMR (1-5% and 3-6% RSD, respectively). CSLM proved to be a reliable method for the determination of the water droplet size distribution of margarines (80% fat). The advantage of CSLM compared to NMR is that visual information is given about the water droplet size distribution in the sample.

Nine color eleven parameter immunophenotyping using three laser flow cytometry.

This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in analog hardware, in combination with final software compensation. An application characterizing low frequency murine B cell subpopulations is given. The fluorochromes used are: fluorescein (FITC), phycoerythrin (PE), Cy5PE and Cy7PE, excited at 488 nm by an argon laser; Texas Red (TR), allophycocyanin (APC), and Cy7APC excited at 595 nm by a pumped dye laser; and cascade blue (CB) and cascade yellow (CY) excited at 407 nm by a violet-enhanced krypton laser. Custom additions to commercial electronics and an extended optical bench allow the measurement of these nine parameters plus forward and side scatter light signals. We find the use of partial analog compensation reduces the variation in the background staining levels introduced by the compensation process. Novel B cell populations with frequencies below 1% are characterized. Nine color flow cytometry is capable of providing measurements with high information content. The choice of reagent-dye combinations and the ability to compensate in multi-parameter measurement space are crucial to obtaining satisfactory results.

Nine color eleven parameter immunophenotyping using three laser flow cytometry

This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in analog hardware, in combination with final software compensation. An application characterizing low frequency murine B cell subpopulations is given.The fluorochromes used are: fluorescein (FITC), phycoerythrin (PE), Cy5PE and Cy7PE, excited at 488 nm by an argon laser; Texas Red (TR), allophycocyanin (APC), and Cy7APC excited at 595 nm by a pumped dye laser; and cascade blue (CB) and cascade yellow (CY) excited at 407 nm by a violet-enhanced krypton laser. Custom additions to commercial electronics and an extended optical bench allow the measurement of these nine parameters plus forward and side scatter light signals.We find the use of partial analog compensation reduces the variation in the background staining levels introduced by the compensation process. Novel B cell populations with frequencies below 1% are characterized.Nine color flow cytometry is capable of providing measurements with high information content. The choice of reagent-dye combinations and the ability to compensate in multi-parameter measurement space are crucial to obtaining satisfactory results.

Five-color immunophenotyping plus DNA content analysis by laser scanning cytometry

Laser scanning cytometry is a new laboratory technology similar to flow cytometry but with advantages for certain clinical and research applications. To date, laser scanning cytometry has been successfully used to perform three-color immunophenotypic analysis of hematologic specimens, single-color immunophenotyping plus DNA content analysis of numerous specimen types, and automated analysis of fluorescence in situ hybridization specimens. Several other interesting applications are also in development. In general, advantages of laser scanning cytometry include reduced specimen size requirements, simplified methodologies, and the ability to microscopically examine individual cells-allowing for the direct correlation of cytologic morphology with objective fluorescence measurements. In this report, we describe a method which more fully takes advantage of the laser scanning cytometer's capabilities for immunophenotypic analysis of hematologic specimens. Specifically, we have devised a method to increase the number of fluorescent parameters from three to a total of six, five representing binding of immunofluorescent antibodies and one for stoichiometric measurements of DNA content. As with most laser scanning cytometric applications, this technique can be utilized on extremely small specimens and enables direct correlation of all of the measured fluorescent parameters with light microscopic cytologic morphology.

Five‐color immunophenotyping plus DNA content analysis by laser scanning cytometry

Laser scanning cytometry is a new laboratory technology similar to flow cytometry but with advantages for certain clinical and research applications (7). To date, laser scanning cytometry has been successfully used to perform three-color immunophenotypic analysis of hematologic specimens, single-color immunophenotyping plus DNA content analysis of numerous specimen types, and automated analysis of fluorescence in situ hybridization specimens (1–5,9,10). Several other interesting applications are also in development (6). In general, advantages of laser scanning cytometry include reduced specimen size requirements, simplified methodologies, and the ability to microscopically examine individual cells—allowing for the direct correlation of cytologic morphology with objective fluorescence measurements. In this report, we describe a method which more fully takes advantage of the laser scanning cytometer's capabilities for immunophenotypic analysis of hematologic specimens. Specifically, we have devised a method to increase the number of fluorescent parameters from three to a total of six, five representing binding of immunofluorescent antibodies and one for stoichiometric measurements of DNA content. As with most laser scanning cytometric applications, this technique can be utilized on extremely small specimens and enables direct correlation of all of the measured fluorescent parameters with light microscopic cytologic morphology. Cytometry (Comm. Clin. Cytometry) 34:36–38, 1998. © 1998 Wiley-Liss, Inc.

Excited state dynamics of 4(1 H-pyrrole 1-yl) benzoic acid and different environmental effects

The absorption and fluorescence characteristics of 4(1 H-pyrrole 1-yl) benzoic acid (PBA) in solvents of different polarities, acidities, glass matrices and also in β-cyclodextrin cavity were studied with regard to the influence of flexibility of the molecule in both the ground and excited sates. The study of dynamics of excited state reveals the presence of two competitive fluorescence emission from a common excited state of PBA. Of these two fluorescence bands one originates from delocalized excited (DE) state [i.e., Franck-Condon (F-C) excited state] and the other from twisted intramolecular charge transfer (TICT) state. The excitation of ground state of PBA leads to the immediate formation of F-C excited state and the TICT state is formed within 220 ps from the parent F-C excited state. The 220 ps time has also been interpreted as the time required for the molecule to relax by rotational motion along the C-N bond of PBA to achieve the TICT state. The steady state photophysical measurements show that the molecule is very weakly fluorescent and nonradiative rate parameters get prominence compared to radiative rate parameters. The fluorescence and phosphorescence studies at 77 K have also been reported. Quantum chemical calculations of the energies and dipole moments were performed for planar and different twisted conformations to confirm experimental findings qualitatively.

Basics of flow cytometry.

In summary, a beginner requires fundamental knowledge about flow cytometric instrumentation in order to effectively use this technology. It is important to remember that flow cytometers are very complex instruments that are composed of four closely related systems. The fluidic system transports particles from a suspension through the cytometer for interrogation by an illumination system. The resulting light scattering and fluorescence is collected, filtered, and converted into electrical signals by the optical and electronics system. The data storage and computer control system saves acquired data and is also the user interface for controlling most instrument functions. These four systems provide a very unique and powerful analytical tool for researchers and clinicians. This is because they analyze the properties of individual particles, and thousands of particles can be analyzed in a matter of seconds. Thus, data for a flow cytometric sample are a collection of many measurements instead of a single bulk measurement. Basic knowledge of instrumentation is a tremendous aid to designing experiments that can be successfully analyzed using flow cytometry. For example, it is important to know the emission wavelength of the laser in the instrument that will be used for analysis. This wavelength is critical knowledge for selecting probes. It is also important to understand that a different range of wavelengths is detected for each fluorescent channel. This will aid selection of probes that are compatible with the flow cytometer. Understanding the complication that emission spectra overlap contributes to detection can be used to guide fluorochrome selections for multicolor analysis. All of these experiment design considerations that rely on knowledge of how flow cytometers work are a very practical and effective means of avoiding wasted time, energy, and costly reagents. Data analysis is a paramount issue in flow cytometry. Analysis includes interpreting as well as presenting data that has been stored in list-mode files. Data analysis is very graphically oriented. There are a number of types of graphic representation that are available to visually aid data analysis. Two standard types of displays are used. These data plots are one-parameter histograms and bivariate plots. A user must be familiar with these two fundamental types of display in order to effectively analyze data. Histograms are the most simple modes of data representation. Histograms allow visualization of a single acquired parameter. Mean fluorescence and distributional statistics can be obtained based on markers that the user can graphically set on the plot. Percentages of positively expressing particles relative to a control sample can also obtained in a similar manner. In addition, multiple histograms can be overlayed on one another to depict qualitative and quantitative differences in two or more samples. Two-parameter data plots are somewhat more complicated than histograms; however, they can yield more information. Two-parameter dot plots of FSC vs SSC allow visualization of both light-scattering parameters that are important for identifying populations of interest. Bivariate fluorescent plots allow discrimination of dual-labeled populations that might remain hidden if histograms were used to display fluorescent data. Two-parameter plots that combine one light-scattering parameter and a fluorescent parameter are useful for analyzing control samples to elucidate the origin of nonspecific binding. Data analysis is very graphically oriented. Experience and pattern recognition become important when using two-parameter data plots for qualitative as well as quantitative analysis. The technique of gating or drawing regions on dual parameter light-scatter plots allows one to exclude information and examine the population of interest by disallowing particles that might confound or interfere with analysis. This is one of the fundamental uses for gating. (ABSTRACT TRUNCATED)

Spectral identification and behaviour of dissolved organic fluorescent material during estuarine mixing processes

In most estuaries, a linear relationship between fluorescence intensity and salinity has been frequently reported. Accordingly, it is commonly accepted that the dissolved organic matter in an estuary is essentially conservative and is not substantially modified by dilution with sea water. In this paper we have tried to verify whether other fluorescent parameters, namely the position of the fluorescence maximum and the apparent fluorescence efficiency (A.Fl.E.), whose variations were measured during estuarine mixing of waters in different European estuaries are consistent with this conclusion. Field observations and laboratory simulation experiments show that upon mixing with sea water, an important blue shift (≅ 40 nm) of the fluorescence emission spectra as well a non-conservative evolution of the A.Fl.E. are observed in estuarine waters. These phenomena have been explained by a complex combination of processes involving mainly, the mixing of fresh water and marine water fluorophores having different fluorescence properties, but also, the loss of high molecular weight fluorescent matter from fresh waters possibly removed by flocculation.