Fluorometer

The sample cells for molecular fluorescence are similar to those for optical molecular absorption. Remote sensing with fiber-optic probes (see Figure 10.30) also can be adapted for use with either a fluorometer or spectrofluorometer. An analyte that is fluorescent can be monitored directly. For analytes that are not fluorescent, a suitable fluorescent probe molecule can be incorporated into the tip of the fiber-optic probe. The analyte s reaction with the probe molecule leads to an increase or decrease in fluorescence. 

Time, Cost, and Equipment As with other optical spectroscopic methods, fluorescent and phosphorescent methods provide a rapid means of analysis and are capable of automation. Fluorometers are relatively inexpensive, ranging from several... 

A sensitive method for the flow injection analysis of Cu + is based on its ability to catalyze the oxidation of di-2-pyridyl ketone hydrazone (DPKH) by atmospheric oxygen. The product of the reaction is fluorescent and can be used to generate a signal when using a fluorometer as a detector. The yield of the reaction is at a maximum when the solution is made basic with NaOH. The fluorescence, however, is greatest in the presence of HCl. Sketch an FIA manifold that will be appropriate for this analysis. 

The most used EIA reagents conjugate a fluotophote such as fluorescein-isothiocyanate (EITC) or thodarnine—isothiocyanate to antibody (or antigen) free amino groups. Examples of other commonly used fluotophotes for EIA and their spectral characteristics ate presented in Table 3. EIA assays ate available in sandwich and competitive formats similar to EIAs. Unlike EIA kits which can be used directly with visual color deterrnination, EIAs require a fluorometer, and thus ate primarily laboratory-based. 

Fluorescenz, Fluoreszenz, /. fluorescence, fluoreszenzerzeugend, a. fluorogenic. Fluoreszenz-farbe, /. fluorescence color luminous paint, -messer, m. fluorometer. fluorescieren, fluoreszieren, v.t. fluoresce. — fluoreszierend, p.a. fluorescent. 

At the top end of the program monitoring scale is the use of online fluorescence tracing systems, whereby tracer dye polymers form part of the water treatment program and their concentration can be measured online at various locations throughout the boiler plant system. Much less expensive, handheld fluorometers are now available to conduct the same type of analysis at the laboratory bench or on the boiler house firing-floor. These tracer dye polymers can be used to determine ... 

Jablonski (48-49) developed a theory in 1935 in which he presented the now standard Jablonski diagram" of singlet and triplet state energy levels that is used to explain excitation and emission processes in luminescence. He also related the fluorescence lifetimes of the perpendicular and parallel polarization components of emission to the fluorophore emission lifetime and rate of rotation. In the same year, Szymanowski (50) measured apparent lifetimes for the perpendicular and parallel polarization components of fluorescein in viscous solutions with a phase fluorometer. It was shown later by Spencer and Weber (51) that phase shift methods do not give correct values for polarized lifetimes because the theory does not include the dependence on modulation frequency. 

The use of phase sensitive detection with the phase fluorometer to analyze multicomponent systems was first described in 1970 by Veselova and coworkers (76). 

We will first summarize the fluorescence and spectroscopic assays that have been developed for the fluorometer and then describe their applications using flow cytometry. We will summarize research which exemplifies the utility of simultaneous measurement of responses and shows how these methods have provided Information about the signal transduction pathways and activation in neutrophils. 

Transmittance can be used to monitor aggregation. As the cells aggregate, there are fewer total particles to scatter light and, hence, more light is transmitted. This is the same principle behind aggregometers. We monitor this transmittance change on the SLM fluorometer (28). 

In the presence of cytochrome C, changes in transmittance at 550 nm reflect oxidant production as superoxide reduces the cytochrome C. This is usually done as an absorbance assay in a spectrophotometer, but it can be performed as a transmittance assay on the SLM fluorometer (20). 

Figure 2. Schematic of the SLM 8000 fluorometer. Excitation occurs through the excitation monochromator, and light emitted from the sample is observed in as many as four different positions. Photomultiplier tubes (PMTs) A, B, and C can be used to monitor fluorescence or right-angle light scatter through the monochromator (PMT A) or through filters (PUT B and C), and position D measures transmittance. Three channels can be monitored simultaneously with measurements being acquired at intervals of 1 s or less. The data are stored by the computer for subsequent manipulation.

Figure 5B. Correlation of right-angle light scatter measured by fluorometry and flow cytometry. The top panel shows flow-cytometric data of side scatter of fixed, stained cells during the time course of stimulation by 1-nM (solid line, solid circles) or 0.01-nH (dashed line, open circle) FLPEP. The bottom panel shows the corresponding right-angle light-scatter data acquired pseudo-simultaneously on live cells in the fluorometer. The flow-cytometric data have been averaged, but the fluorometry data are plotted for both duplicates from one donor. Reproduced with permission from Ref. 27. Copyright 1985 Rockefeller University Press.

Basis. The rotational mobility of a small ligand is relatively unrestricted in solution (anisotropy approaches 0). The mobility is restricted when the ligand binds to a large immobilized molecule such as the receptor. In a T-format fluorometer, the parallel and perpendicular components of the emission can be examined simultaneously. While precautions must be exercised in working with turbid suspensions, it is nonetheless practical to make continuous measurements of binding and dissociation. 

A similar analysis using a Farrand fluorometer fitted with a 10-pL flow cell gave limits of fluorescence detection of 1 pmol ... 

If the signal decay is a single-exponential curve, equations 16 and 17 result in values for X that are in agreement with each other. Dissimilar values indicate multiexponential decay, which usually means that the sample contains more than one fluorophore. Multiexponential decay can be resolved by using a phase fluorometer with phase sensitive detection. A time-independent, direct-current signal is produced that is proportional to the cosine of the difference between the phase angle of the detector ( D) and the phase angle of the fluorescence ( ) ... 

Schreiber, U. (1986). Detection of rapid induction kinetics with a new type of high frequency modulated chlorophyll fluorometer. Photosynthesis Research, 9, 261-72. 

C.N. Ho, G.D. Christian and E.R. Davidson, Application of the method of rank annihilation to quantitative analysis of multicomponent fluorescence data from the video fluorometer. Anal. Chem., 52 (1980) 1108-1113. 

Stabilisers are usually determined by a time-consuming extraction from the polymer, followed by an IR or UV spectrophotometric measurement on the extract. Most stabilisers are complex aromatic compounds which exhibit intense UV absorption and therefore should show luminescence in many cases. The fluorescence emission spectra of Irgafos 168 and its phosphate degradation product, recorded in hexane at an excitation wavelength of 270 nm, are not spectrally distinct. However, the fluorescence quantum yield of the phosphate greatly exceeds that of the phosphite and this difference may enable quantitation of the phosphate concentration [150]. The application of emission spectroscopy to additive analysis was illustrated for Nonox Cl (/V./V -di-/i-naphthyl-p-phcnylene-diamine) [149] with fluorescence ex/em peaks at 392/490 nm and phosphorescence ex/em at 382/516 nm. Parker and Barnes [151] have reported the use of fluorescence for the determination of V-phenyl-l-naphthylamine and N-phenyl-2-naphthylamine in extracted vulcanised rubber. While pine tar and other additives in the rubber seriously interfered with the absorption spectrophotometric method this was not the case with the fluoromet-ric method. 

Fig. 8. Dependence of (A) corrected diffusion coefficient (D), (B) steady-state fluorescence intensity, and (C) corrected number of particles in the observation volume (N) of Alexa488-coupled IFABP with urea concentration. The diffusion coefficient and number of particles data shown here are corrected for the effect of viscosity and refractive indices of the urea solutions as described in text. For steady-state fluorescence data the protein was excited at 488 nm using a PTI Alphascan fluorometer (Photon Technology International, South Brunswick, New Jersey). Emission spectra at different urea concentrations were recorded between 500 and 600 nm. A baseline control containing only buffer was subtracted from each spectrum. The area of the corrected spectrum was then plotted against denaturant concentrations to obtain the unfolding transition of the protein. Urea data monitored by steady-state fluorescence were fitted to a simple two-state model. Other experimental conditions are the same as in Figure 6.

Spencer, R. D. and Weber, G. (1969). Measurement of subnanosecond fluorescence lifetimes with a cross-correlation phase fluorometer. Ann. N. Y. Acad. Sci. 158, 361-76. 

Gratton, E. and Limkeman, M. (1983). A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution. Biophys. J. 44, 315-24. 

Lakowicz, J. R. and Maliwal, B. P. (1985). Construction and performance of a variable-frequency phase-modulation fluorometer. Biophys. Chem. 21, 61-78. 

Hedstrom, J., Sedarous, S. and Prendergast, F. G. (1988). Measurements of fluorescence lifetimes by use of a hybrid time-correlated and multifrequency phase fluorometer. Biochemistry 27, 6203-8. 

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