Measurement fluorescence

Fluorescence measurements have much greater sensitivity than absorption measurements. Therefore, the experimenter must take special precautions in making fluorescence measurements because any contaminant or impurity in the system can lead to inaccurate results. The following factors must be considered when preparing for a fluorescence experiment. 

Another related phenomenon that results in a lower quantum yield than expected is called concentration quenching. This can occur when a macromolecule, such as an antibody, is heavily labeled with a fluorophore, such as fluorescein isothiocyanate. When this compound is excited, the fluorescence labels are in such close proximity that radiationless energy transfer occurs. Thus, the resulting fluorescence is much lower than expected for the concentration of the label. This is a common problem in flow cytometry and laser-induced fluorescence when attempting to enhance detection sensitivity by increasing the density of the fluorescing label. 

Certain quartz glass and plastic materials that contain ultraviolet absorbers will fluoresce. Some solvents, such as ethanol, are also known to cause appreciable fluorescence. It is therefore important when developing a fluorescence assay to assess the background fluorescence of all components of the reaction mixture. Fluorescence grade solvents and cuvets with minimum fluorescence emission, which minimize these types of fluorescence background problems, are commercially available. 

A serum or urine sample contains many compounds that fluoresce. Thus the sample matrix is a potential source of unwanted background fluorescence and must be examined when new methods are developed. The most serious contributors to unwanted fluorescence are proteins and bilirubin. However, because protein excitation maxima are in the spectral region of 260 to 290 nm, their contribution to overall background fluorescence is minor when excitation occurs above 300 nm. 

In conventional fluorometry, excitation of weakly fluorescing or dilute solutions with intense fight sources wfll cause photochemical decomposition of the analyte (photobleaching). 

The following steps help to minimize photodecomposition effects  

Even derivative spectra do not produce an increase in information compared to zero order absorption spectra. Differentiation can visualise the wavelength regions in which spectral changes are exceptional. For this reason derivative spectroscopy has been applied to kinetic analysis [87]. Fig. 4.20 demonstrates an example of a derivative reaction spectrum given for the above mentioned photoreaction of stilbene. The second derivatives are shown. The advantage in selection of characteristic wavelengths improves the kinetic evaluation as discussed in Chapter 5. However, in each application the optimum between increased noise and extracted information has to be found. 

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Altkorn R and Zare R N 1984 Effects of saturation on laser-induced fluorescence measurements of population and polarization Annual Review of Physical Chemistry ed B S Rabinovitch, J M Schurr and H L Strauss (Palo Alto, CA Annual Reviews)... 

The sensitivities of particular spectroscopic teclmiques to specific chemical features are described more fully in tire next section. Perhaps tire most common and versatile probes of reaction dynamics are time-resolved UV-vis absorjDtion and fluorescence measurements. Wlren molecules contain cliromophores which change tlieir stmcture directly or experience a change of environment during a reaction, changes in absorjDtion or fluorescence spectra can be expected and may be used to monitor tire reaction dynamics. Altliough absorjDtion measurements are less sensitive tlian fluorescence measurements, tliey are more versatile in tliat one need not rely on a substantial fluorescence yield for tire reactants, products or intennediates to be studied. 

Fluorescence. The fluorescence detection technique is often used in clinical chemistry analyzers for analyte concentrations that are too low for the simpler absorbance method to be appHed. Fluorescence measurements can be categorized into steady-state and dynamic techniques. Included in the former are the conventional simultaneous excitation-emission method and fluorescence polarization. 

In situ quantitation The in situ fluorescence measurement was carried out at =... 

Figure 6-21. Site-selective fluorescence measurements of a T thin film. Starting from the top spectrum the excitation energies were at 17860. 17700, 17390, 17240, 17095, and 16950 cur, respectively. The reminiscence of the exciting laser lines is shown by the sharp peak on the left.

Note. It is good practice to make the fluorescence measurements for samples and standards as close together as possible to minimise any drift in instrument response. 

Within the confines of the present volume it is not possible to provide a detailed discussion of instrumentation for atomic fluorescence spectroscopy. An instrument for simultaneous multi-element determination described by Mitchell and Johansson53 has been developed commercially. Many atomic absorption spectrophotometers can be adapted for fluorescence measurements and details are available from the manufacturers. Detailed descriptions of atomic fluorescence spectroscopy are to be found in many of the volumes listed in the Bibliography (Section 21.27). 

Deprotonation in Pu sulfate systems 260-61 Detector, IR fluorescence measurements of PuF6(g). 162... 

Fluorescence measurement at 365 nm after derivatization with primulin... 

Figure 2. The binding and dissociation of FLPEP and receptor on intact neutrophils at 37 C The data are plotted as the specific binding of FLPEP (pmoles/10 cells) on a log plot versus time. Experimental details 10 cells/mL were exposed at time 0 to 1 nAf FLPEP. At 15, 30, 60, or 120 s, antibody to fluorescein is added to each sample. Fluorescence is monitored continuously during the additions. The data ate derived from a point-by-point comparison of the fluorescence measured under conditions of receptor binding and receptor blockade. Data are representative of observations in more than 10 separate experiments. (Reproduced with permission from reference 22. Copyright 19S7 Journal of Biological Chemistry.)...

The data were collected using fluorescence measurements, which allow both identification and quantitation of the fluorophore in solvent extraction. Important experimental considerations such as solvent choice, temperature, and concentrations of the modifier and the analytes are discussed. The utility of this method as a means of simplifying complex PAH mixtures is also evaluated. In addition, the coupling of cyclodextrin-modified solvent extraction with luminescence measurements for qualitative evaluation of components in mixtures will be discussed briefly. 

Reagents. Perylene was obtained from Sigma Chemical Company (St. Louis, Missouri). All other PAHs were supplied by Aldrich Chemical Company (Milwaukee, Wisconsin) and were reported to contain less that 3% impurities. All PAHs were used without further purification. Isopropyl ether (99%) for extraction work was also purchased from Aldrich. Hydroquinone, a fluorescent stabilizer present in the ether, was removed prior to solution preparation by rotary evaporation. Fluorometric-grade 1-butanol was supplied by Fisher Scientific Company (Fair Lawn, New Jersey). All solutions for extractions of PAHs were prepared by evaporating portions of a stock cyclohexane solution and diluting to the appropriate volume with isopropyl ether. Fluorescence measurements were performed on 1 10 dilutions of the stock and final organic phase solutions. The effect of dissolved CDx on the fluorescence intensity of the organic phase PAH was minimized by dilution with isopropyl ether. 

Samples for studies of CDx effects on fluorescence enhancement in organic solution were prepared using pyrene, because pyrene possesses a long lifetime and is very susceptible to quenching and enhancement in solution (23). An aliquot of pyrene stock solution in cyclohexane was placed under a nitrogen purge to evaporate the cyclohexane. Samples were redissolved in a 1 A mixture of Isopropyl ether and 1-butanol, which was saturated with aqueous CDx solution. Pyrene samples were also prepared in which the organic solvent was not saturated with CDx solution. The mixed solvent was used in order to minimize the effects of ether evaporation and thus allow more accurate quantitation. Fluorescence measurements were made on diluted samples of these solutions. The solvent used to make up the... 

In this chapter, we present the theory and results of measurements on humic acid fractions using fluorescence techniques. The fluorescence techniques are attractive for this application because of the natural fluorescence of humic materials, the hi sensitivity of fluorescence detection, and the ability to directly observe the morphology of the molecule in aqueous solutions without the need for drying or applying harsh chemical conditions. Several interesting types of information are obtained from fluorescence measurements ... 

Quimtua Yields of Fluorescence Measurements. All of the quantum yields of fluorescence were measured by the relative fluorescence measurement technique of Parker and Rees (24). This method compares the fluorescence of the compound of interest to the fluorescence of some known compound. All of the fluorescence quantum yields were measured using I as a reference. Compound I had previously been measured by this same method using rhodamine B as a standard. 

Laboratory work involved making calibration curves which show the response of the system for various concentrations of pollutant, e.g., phenol. Typically, remote laser-induced fluorescence measurements from both the laboratory apparatus and the mobile unit are made on... 

As is well known the difficulty of analysis of a sample increases as its complexity increases. Analysis usually commences with a rather nonspecific clean-up step and requires that the separation step that follows be highly selective and depends on a detection step that is as specific as possible. As the selectivity of detection increases there is also an increase in the reliability of the identification and it is possible to reduce the demands made on the selectivity of the preceding separation method. This is the case for radiometric and enzymatic methods and also explains the popularity of fluorescence measurements. The latter obtain their selectivity from the freedom to choose excitation and measurement wavelengths. 

Such layer structure does not allow ns to say a priori that hybridization of DNA will be possible, for it is protected by the octadecylamine layer. In order to control for this possibility, fluorescence measurements were performed. The first indication that hybridization was successful is that after the process, the sample surface became wettable, while before it and after cold hybridization it was not wettable at all. The results of the fluorescence measurements are summarized in Table 10. The results of the specific hybridization are three times more with respect to unspecific hybridization and one order of magnitude more with respect to cold hybridization. Thus, it appears that during a normal hybridization (100% homology) some structural changes and redistribution of the layer takes place. As a result, DNA becomes available for the specific reaction. Such a model also explains why the fluorescence level after unspecific hybridization (10% homology) is higher with respect to cold hybridization. Because the molecules have some mobility when the film is warmed, some DNA from the film could be hybridized on itself, while during cold hybridization this is impossible. 

Figure 4.7 Changes in intraceiiuiar calcium in cultured rat ventricular myocytes exposed to oxidant stress. Calcium was measured using the fluorescent probe Fura>2. The ratio of the Fura-2 fluorescence measured at 340 and 380 nm excitation is shown and this is proportional to the intracellular calcium concentration. The fast-speed traces shown (note the 3.5 s time-scale) were recorded after various durations of oxidant stress. Myocytes under control conditions (before t = 0) show spontaneous calcium transients. These transients decreased in frequency with oxidant stress until cells failed to show spontaneous activity but continued to maintain a low intracellular calcium.

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