Fluorescence light emission

The gas component, which is the most important form in which matter is found in outer space (up to 98-99%), is dominated by the element hydrogen, which makes up 70% of the mass and 90% of the particles. In ionized form (the H-II regions) the gas can be recognized by its recombination and fluorescence light emission. The hydrogen is mainly present in neutral form (H-I regions), at a mean density of 2x 107 particles per cubic metre and a mean temperature of about 80 K. 

Figure 7-32 Micrograph of a mouse embryo fibroblast was obtained using indirect immunofluorescence techniques.313 The cells were fixed with formaldehyde, dehydrated, and treated with antibodies (formed in a rabbit) to microtubule protein. The cells were then treated with fluorescent goat antibodies to rabbit /-globulins (see Chapter 31) and the photograph was taken by fluorescent light emission. Courtesy of Klaus Weber.

Identification. Chromatographic methods (interpretation of fluorescing spots in thin layer chromatography or of UV absorption peaks and fluorescence light emission peaks in high performance liquid chromatography) allow fairly rapid and certain identification of FWAs [158-164], The same methods can be used for qualitative and quantitative characterization of the FWA purity in terms of fluorescent or nonfluorescent byproducts. 

Figure 4.4 Jablonski-type energy diagrams for possible excited energy states when light interacts with matter, (a) Three possible transition pathways for return to ground state without radiation, (b) Two possible transition pathways with fluorescent light emission as final step on return to ground state, (c) Two possible transition pathways with phosphorescent light emission as final step on return to ground state.

The theory for the time course of phosphorescent or fluorescent light emission was described by Stern and Volmer [7]. The relationship is given by... 

Figure 8.1. Simple energy level diagram for the excitation of an electron from the ground state of a tiuorophore, nonracSative decay of the electron to an excited state of lower energy, and return of the electron to the ground state, which results in the observed fluorescent light (emission).

Luminescent Pigments. Luminescence is the abihty of matter to emit light after it absorbs energy (see Luminescent materials). Materials that have luminescent properties are known as phosphors, or luminescent pigments. If the light emission ceases shortly after the excitation source is removed (<10 s), the process is fluorescence. The process with longer decay times is referred to as phosphorescence. 

Energy transfer to fluorescent proteins. There are marked differences among the various bacterial species and strains in terms of the in vivo luminescence spectra. The emission maxima are spread mostly in a range from 472 to 505 nm (Seliger and Morton, 1968), but one of the strains, P. fischeri Y-l, shows a maximum at 545 nm (Ruby and Nealson, 1977), as shown in Fig. 2.3. However, the in vitro luminescence spectra measured with purified luciferases obtained from the various bacterial species and strains are all similar (Amax about 490 nm). The variation in the in vivo luminescence spectra may be due to the occurrence of an intermolecular energy transfer that increases the efficiency of light emission. 

Isolation of F and P. The first attempt to isolate and purify the substances responsible for the light emission of M. norvegica was made by Shimomura and Johnson (1967). They isolated two substances, a protein (P) and a fluorescent compound (F), which produce a blue light... 

The product coelenteramide is not noticeably fluorescent in aqueous solutions, but is highly fluorescent in organic solvents and also when the compound is in the hydrophobic environment of a protein. When coelenterazine is luminesced in the presence of Oplophorus luciferase, the solution after luminescence (the spent solution) is not fluorescent, presumably due to the dissociation of coelenteramide from the luciferase that provided a hydrophobic environment at the time of light emission. An analogous situation exists in the bioluminescence system of Renilla (Hori et al., 1973). 

Fig. 4.2.1 Luminescence spectra of the Ca2+-triggered light emission of recombinant obelins (dotted lines), and the fluorescence emission spectra of their spent solution after luminescence (solid lines). Left obelin derived from O. geniculata right obelin derived from O. longissima. Reproduced from Markova etal., 2002, with permission from the American Chemical Society.

Fig. 5.4 Chemical mechanism of light emission in the bio- and chemiluminescence reactions of coelenterazine. The bottom row shows some of the fluorescence emitters of coelenteramide. The fluorescence characteristics of the dianion are unknown.

Harvey (1952) demonstrated the luciferin-luciferase reaction with O. phosphorea collected at Nanaimo, British Columbia, Canada, and with O. enopla from Bermuda. McElroy (1960) partially purified the luciferin, and found that the luminescence spectrum of the luciferin-luciferase reaction of O. enopla is identical to the fluorescence spectrum of the luciferin (A.max 510 nm), and also that the luciferin is auto-oxidized by molecular oxygen without light emission. Further investigation on the bioluminescence of Odontosyllis has been made by Shimomura etal. (1963d, 1964) and Trainor (1979). Although the phenomenon is well known, the chemical structure of the luciferin and the mechanism of the luminescence reaction have not been elucidated. 

The photoprotein is non-fluorescent. The absorption spectrum of purified photoprotein shows a very small peak at 410 nm, in addition to the protein peak at 280 nm (Fig. 10.2.5). The peak height at 410 nm appears to be proportional to the luminescence activity of the protein. The protein also shows extremely weak absorption peaks at about 497, 550 and 587nm (not shown). These absorption peaks, except the 280 nm peak, might be due to the presence of a chromophore that is functional in the light emission. 

Daubner, S. C., Astorga, A. M., Leisman, G. B., and Balwin, T. O. (1987). Yellow light emission of Vibrio barveyi strain Y-l purification and characterization of the energy-accepting yellow fluorescent protein. Proc. Natl. Acad. Sci. USA 84 8912-8916. 

The luminescence of an excited state generally decays spontaneously along one or more separate pathways light emission (fluorescence or phosphorescence) and non-radiative decay. The collective rate constant is designated k° (lifetime r°). The excited state may also react with another entity in the solution. Such a species is called a quencher, Q. Each quencher has a characteristic bimolecular rate constant kq. The scheme and rate law are... 

To our surprise and satisfaction, the general approach worked the CBI derivatives did chemiluminescence, and the sensitivity enhancement was 30- to 50-fold over fluorescence With this success, we embarked on a more thorough study of chemiluminescence with the goal of optimizing the method. Identifiable parameters that affected the efficiency of light emission from a chemically generated fluorescent molecule included ... 

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