Luminescence, measurement

When an intense pulse of monochromatic laser light is focussed on a transparent liquid or solid, there is an emission of white light over a wide continuous spectral range. This process is known as self-phase modulation . We will not consider its physics. For our purpose it is important to note its photochemical implications. On the one hand, this pulse of white light can be used to provide a probe light in ps and fs flash photolysis (sections 8.1 and 8.2). On the other hand, it can be a source of stray light in some luminescence measurements. This comes as a surprise to many users of lasers for luminescence kinetics measurements, but it is an unavoidable problem. 

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Fig. 2.4 The spectrum of bacterial luminescence measured with B. harveyi luciferase, FMN, tetradecanal and NADH, in 50 mM phosphate buffer, pH 7.0, at 0°C (dashed line from Matheson et al., 1981) and the absorption and fluorescence emission spectra of LumP (solid lines) and Rf-LumP (dotted lines) obtained from P. leiog-natbi, in 25 mM phosphate buffer, pH 7.0, containing 1 mM EDTA and 10 mM 2-mercaptoethanol, at room temperature (from Petushkov et al, 2000, with permission from Elsevier). LumP is a lumazine protein, and Rf-LumP contains riboflavin instead of lumazine in the lumazine protein. Fluorescence emission curves are at the right side of the absorption curves.

Fig. 4.1.5 The time course of aequorin luminescence measured with various concentrations of Ca2+. Calcium acetate solution (5 ml) was added to 10 pi of aequorin solution to give the final Ca2+ concentrations of 10 2 M (A), 10-4 M (B), 10-5 M (C), 10 6 M (D), and 10 7 M (E) at 25°C. The dashed line (F) represents the light emitted following the addition of deionized distilled water that had been redistilled in quartz. The concentration of EDTA derived from the aequorin sample was 10 7 M (final cone.). From Shimomura et al., 1963b, with permission from John Wiley Sons Ltd.

The immense growth in the luminescence literature during the period between these two reviews had little to do with developments in fundamental theory. It was mainly due to the availability of new instrumentation, such as the photomultiplier (around 1950), the laser (around 1960), transistor and microcircuit electronics (around 1970), and ready access to laboratory computers (around 1975). All aspects of luminescence theory now being used to interpret luminescence measurements have been known since the early 1900 s and nearly all of the types of measurements now being made had been initiated with cruder techniques by 1930. We discuss here many of the latest techniques in luminescence analysis with selected highlights from the historical development of luminescence and a look at several recent developments in luminescence applications that appear likely to be important to future research. 

Luminescence measurements on proteins occupy a large part of the biochemical literature. In what surely was one of the earliest scientific reports of protein photoluminescence uncomplicated by concurrent insect or microorganism luminescence, Beccari (64), in 1746, detected a visible blue phosphorescence from chilled hands when they were brought into a dark room after exposure to sunlight. Stokes (10) remarked that the dark (ultraviolet) portion of the solar spectrum was most efficient in generating fluorescent emission and identified fluorescence from animal matter in 1852. In general, intrinsic protein fluorescence predominantly occurs between 300 nm and 400 nm and is very difficult to detect visually. The first... 

Even the fact that some species do not fluoresce may be used as a selectivity tool. Computers and modem electronics have made multidimensional measurements possible on a hitherto unprecedented scale and this approach has been one of the most effective ways to achieve high selectivity in luminescence measurements. Many ingenious techniques have recently become available for utilizing the multiple luminescence variables and these have made luminescence measurements a routine and valuable tool in almost all areas of experimental science. 

Many current multidimensional methods are based on instruments that combine measurements of several luminescence variables and present a multiparameter data set. The challenge of analyzing such complex data has stimulated the application of special mathematical methods (80-85) that are made practical only with the aid of computers. It is to be expected that future analytical strategies will rely heavily on computerized pattern recognition methods (79, 86) applied to libraries of standardized multidimensional spectra, a development that will require that published luminescence spectra be routinely corrected for instrumental artifacts. Warner et al, (84) have discussed the multiparameter nature of luminescence measurements in detail and list fourteen different parameters that can be combined in various combinations for simultaneous measurement, thereby maximizing luminescence selectivity with multidimensional measurements. Table II is adapted from their paper with the inclusion of a few additional parameters. 

In general, luminescence measurements are relative rather than absolute, since the Instrument characteristics and sample properties that determine the fluorescence Intensities are often not well defined. Absolute luminescence measurements are difficult to perform and require time and Instrumentation not available In most laboratories. Thus, luminescence measurements rely heavily on standards to determine Instrument responses and parameters, the chemical composition of samples, and the characteristics of chemical systems. To... 

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. 

Temperature-dependent luminescence measurements in the range from 77 to 300 K show quenching of the peak luminescence by a factor of about 15. Similar behavior is observed in the lifetime quenching [665, 666], As the band gap of the PECVD a-Si H is about 1.6 eV, nonradiative deexcitation of Er may occur at elevated temperatures. The amount of quenching lies in between that of c-Si and LPCVD a-Si H, just like the bandgap. 

Ultraviolet absorption spectra were obtained from a Cary 118C Spectrophotometer. Luminescence measurements were obtained from a Perkin-Elmer Model MPF-3 Fluorescence Spectrophotometer equipped with Corrected Spectra, Phosphorescence and Front Surface Accessories. A Tektronix Model 510N Storage Oscilloscope was used for luminescence lifetime measurements. Fiber irradiation photolyses were carried out in a Rayonet Type RS Model RPR-208 Preparative Photochemical Reactor equipped with a MGR-100 Merry-go-Round assembly. 

Figure 7 Luminescent lanthanide complexes with representative luminescence lifetimes, and hydration states (derived from luminescence measurements) where appropriate.

Cells are lysed for Firefly and Renilla luciferase assays using the Dual-Luciferase Reporter Assay system (Promega), following the manufacturer s instructions. We use a multimode microplate reader with automatic injectors (FLUOROstar Optima from BMG Labtech, OfFenburg, Germany) for luminescence measurements. 

British scientists immobilized53 in a porous sol-gel glass aequorin - the bioluminescent protein found in the jellyfish Aequorea aequorea. The luminescence from this protein is specifically triggered by the presence of calcium ions. The intensity of the luminescence, measured at the peak... 

Ru(dpp)3]2+) sequestered within the xerogels. The results of SEM and luminescence measurements shown that certain ([Ru(dpp)3]2) doped Octyl-triEOS/TEOS composites form uniform, crack-free xerogel films that can be used to construct high-sensitivity O2 sensors that have linear calibration curves and excellent long-term stability (over a period of 11 months). 

Lanthanide chelates also can be used in FRET applications with other fluorescent probes and labels (Figure 9.51). In this application, the time-resolved (TR) nature of lanthanide luminescent measurements can be combined with the ability to tune the emission characteristics through energy transfer to an organic fluor (Comley, 2006). TR-FRET, as it is called, is a powerful method to develop rapid assays with low background fluorescence and high sensitivity, which can equal the detection capability of enzyme assays (Selvin, 2000). 

Luck, W.A.P. 1981. Structures of water in aqueous systems. In Water Activity Influences on Food Quality (L.B. Rockland and G.F. Stewart, eds), pp. 407 134. Academic Press, New York. Ludescher, R.D., Shah, N.K., McCaul, C.P., and Simon, K.V. 2001. Beyond Tg Optical luminescence measurements of molecular mobility in amorphous solid foods. Food Hydro colloids 15, 331-339. Ludwig, R. 2001. Water From cluster to the bulk. Angewandte Chem. Int. Ed. 40, 1808-1827. Maclnnes, W.M. 1993. Dynamic mechanical thermal analysis of sucrose solutions. In The Glassy State in Foods (J.M.V. Blanshard and PJ. Lillford, eds), pp. 223-248. Nottingham Univ. Press, Loughborough, Leicestershire. 

UV irradiation (335 nm) at pH 7.3 showed that some tetracyclines produced significant concentrations of the superoxide anion radical. Demeclocycline (122), oxytetracycline (123) and chlortetracycline (124) were the most active in this. These are all potent photosensitizers and probably give the observed exaggerated sunburn as a result of superoxide production in the skin [90]. Other photochemical experiments demonstrated the production of singlet oxygen. Yields obtained by luminescence measurements in alkaline ethanol were in the order (122) > (120) > (124) > (121) > (125). Chlortetracycline... 

Hamilton Umicon Lumicon chemi- and biolumium assay luminometer This equipment is used in test-tube scale luminescent immunoassays. With its sample compartment (thermostatted by means of Peltier elements, which allow the temperature to be set from 15°C to 40°C with a precision of 0.1°K) this instrument is suitable for the measurement of temperature-sensitive bioluminescence resulting from enzymic reactions and also in phagocyte-mediated luminescence measurements. 

Generally speaking, luminescence spectra (fluorescence and phosphorescence) provide more information about excited states than do absorption spectra. This is because luminescence measurements are much more sensitive than absorption measurements, and the two types of emission can be studied separately due to their widely differing lifetimes. 

D. H. Metcalf, S. W. Snyder, S. Wu, G. L. Hilmes, J. P. Riehl, J. N. Demas, and F. S. Richardson, Excited-state chiral discrimination observed by time-resolved circular polarized luminescence measurements, /. Am, Chem. Soc. 11, 3082-3083 (1989). 

The labelled polyacrylic acid PAA-M, used for polarized luminescence measurements, was prepared by grafting of 9-aminoacridine on the PAA-800 000 chain (15). The degree of derivatization of the polyacid (evaluated from the UV-vis spectra of PAA-M And 9-aminoa-cridine) is very low ... 

A. Azzi B. Chance (1969) Biochim. Biophys. Acta 189, 141. Selected entries from Methods in Enzymology [vol, page(s)] Calibration of calcium binding, 260, 425-427 luminescence measurement, 260, 424-425 mitochondria-targeted hybrid protein [calcium quantitation, 260, 418, 422, 424-428 stable expression, 260, 418-421 transient expression, 260, 420 intracellular localization, 260, 421-425 reconstitution with coelenterazine, 260, 422-422] structure, 260, 418. 

The optical setup for time-resolved micro-luminescence measurements is based around an Axiotech 100 HD Zeiss microscope, modified to allow laser injection and fluorescence collection. The sample is observed either under transmission or reflection of polarized white light, or under UV illumination (HBO lamp). A set-up consisting of a dichroic mirror, for the selection of the excitation wavelength, and an objective (Epiplan Neofiuar obj. > 350 nm Ealing/Coherent reflection obj. < 350 nm) is used to focus the laser beam on the sample (spatial resolution over 5 pm with a x50 objective). The lim-... 

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