Luminescence Emission Fluorescence

Fig. 4.1.15 Comparison of the luminescence and fluorescence emission spectra of natural aequorin (left panel) and recombinant e-aequorin (right panel) the luminescence spectra of Ca2+ -triggered reaction (dark solid lines), the fluorescence emission spectra of the spent solution containing 2 mM Ca2+ (dashed lines), and the luminescence spectra of the spent solution after addition of coelenterazine (light solid lines). Reproduced with permission, from Shimomura, 1995d. the Biochemical Society.

According to the Kuwabara-Wassink paper, the purified luciferin in aqueous neutral buffer solution showed an absorption maximum at 320 nm, and a fluorescence emission peak at 490 nm. The luminescence emission maximum measured with Airth s fungal luciferase system was 524 nm at pH 6.5, whereas the chemiluminescence emission maximum of the luciferin with H2O2 plus a droplet of strong NaOH plus ferrous sulfate was 542 nm. No information was reported on the chemical nature of the luciferin. 

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... 

The physical basis of spectroscopy is the interaction of light with matter. The main types of interaction of electromagnetic radiation with matter are absorption, reflection, excitation-emission (fluorescence, phosphorescence, luminescence), scattering, diffraction, and photochemical reaction (absorbance and bond breaking). Radiation damage may occur. Traditionally, spectroscopy is the measurement of light intensity... 

More fluorescence features than just the emission intensity can be used to develop luminescent optosensors with enhanced selectivity and longer operational lifetime. The wavelength dependence of the luminescence (emission spectmm) and of the luminophore absorption (excitation spectrum) is a source of specificity. For instance, the excitation-emission matrix has shown to be a powerful tool to analyze complex mixtures of fluorescent species and fiber-optic devices for in-situ measurements (e.g. 

An ingenious variation on the standard fluorescence methods was proposed by Red kin et al. [50]. Water samples were extracted with non-polar solvents, transferred into hexane and the hexane solution frozen at 77 K. At that temperature the normally diffuse luminescence emission bands are present as sharp emission lines, making identification of fluorescing compounds considerably simpler. In the case of a complex mixture, some separation by column or thin layer chromatography might be necessary. 

Molecular emission is referred to as luminescence or fluorescence and sometimes phosphorescence. While atomic emission is generally instantaneous on a time scale that is sub-picoseconds, molecular emission can involve excited states with finite, lifetimes on the order of nanoseconds to seconds. Similar molecules can have quite different excited state lifetimes and thus it should be possible to use both emission wavelength and emission apparent lifetime to characterize molecules. The instrumental requirements will be different from measurements of emission, only in detail but not in principles, shared by all emission techniques. 

The much larger energy difference between Si and S0 than between any successive excited states means that, generally speaking, internal conversion between Si and S0 occurs more slowly than that between excited states. Therefore, irrespective of which upper excited state is initially produced by photon absorption, rapid internal conversion and vibrational relaxation processes mean that the excited-state molecule quickly relaxes to the Si(v0) state from which fluorescence and intersystem crossing compete effectively with internal conversion from Si. This is the basis of Kasha s rule, which states that because of the very rapid rate of deactivation to the lowest vibrational level of Si (or Td, luminescence emission and chemical reaction by excited molecules will always originate from the lowest vibrational level of Si or T ... 

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. 

The fluorescence lifetime of the /2 metastable state of Nd + ions in LaBGeOs (a solid state laser) is 280 /u.s and its quantum efficiency is 0.9. (a) Calculate the radiative and nonradiative rates from this excited state, (b) If the effective phonons responsible for the nonradiative rate have an energy of 1100 cm, use the Dieke diagram to determine the number of emitted effective phonons from the F3/2 excited state, (c) From which three excited states of the Nd + ions in LaBGeOs do you expect the most intense luminescence emissions to be generated ... 

Luminescence is a well-established class of analytical spectroscopic techniques where a species emits light after excitation. Emission is an elecnonic nansition from an excited state as opposed to the ground state as is the case in most other spectroscopies. Photoluminescence, or light-induced fluorescence (LIE), is the most common route to induce emission where sufficient incident photons of a particular energy excite the target species via absorption. Although less common, nomadiative excitation can also occur via a chemical reaction termed chemiluminescence. Unless otherwise stated, the terms luminescence and fluorescence within this review infers excitation by light induction. 

It should be self-evident that the time dependencies of the fluorescent emissions may well be very complicated, because they must reflect the simultaneous operation of a large number of relaxation mechanisms. A complete understanding of the transient luminescent emissions must therefore involve an intimate knowledge of a large number of factors. 

When pyridoxamine with a dipolar ionic ring structure (Fig. 14-9) and an absorption peak at 30,700 cm-1 (326 ran) is irradiated, fluorescence emission is observed at 25,000 cm 1 (400 ran). When basic pyridoxamine with an anionic ring structure and an absorption peak at 32,500 cm 1 (308 nm) is irradiated, fluorescence is observed at 27,000 cm-1 (370 nm), again shifted 5500 cm 1 from the absorption peak. However, when the same molecule is irradiated in acidic solution, where the absorption peak is at 34,000 cm 1 (294 nm), the luminescent emission at 25,000 cm 1 is the same as from the neutral dipolar ionic form and abnormally far shifted (9000 cm ) from the 34,000 cm-1 absorption peak.185186 The phenomenon, which is observed for most phenols, results from rapid dissociation of a proton from the phenolic group in the photoexcited state. A phenolic group is more acidic in the excited state than in the ground state, and the excited pyridoxamine cation in acid solution is rapidly converted to a dipolar ion. 

There is good evidence from 13C NMR and electronic spectra for an enzyme-bound reduced flavin hydroperoxide as in Eq. 15-31. While this hydroperoxide can decompose slowly to flavin and H202 in the dark, it can also carry out the oxidation of the aldehyde with emission of light.685/685a The luminescent emission spectrum resembles the fluorescence spectrum of the 4a -OH adduct (Eq. 23-49), which is probably the light-emitting species.686-688... 

Figure 3.10 The electronic transitions [absorption in (a)] of small molecules show vibrational and rotational lines in addition to the purely electronic spectrum, (b) Luminescence emission is resonance fluorescence (f), and chemical reactions (R) can originate from several excited states...

Another hydrazine derivative of fluorescein, 5-(((2-(Carbohydrazino)methyl)thio)-acetyl)-aminofluorescein, contains a longer spacer arm off its No. 5 carbon atom of its lower ring than fluorescein-5-thiosemicarbazide, described previously (Molecular Probes). The reagent can be used to react spontaneously with aldehyde- or ketone-containing molecules forming a hydrazone linkage (Fig. 209). It also can be used to label cytosine residues in DNA or RNA by use of the bisulfite activation procedure (Chapter 17, Section 2.1). The resulting fluorescent derivative exhibits a maximal excitation at 490 nm and a maximal luminescence emission peak at 516 nm when dissolved in buffer at pH 8. In the same buffered environment, the compound has an extinction coefficient of approximately 75,000 M-1cm 1 at 490 nm. 

Bimolecular deactivation (pathway vii, Fig. 1) of electronically excited species can compete with the other pathways available for decay of the energy, including emission of luminescent radiation. Quenching of this kind thus reduces the intensity of fluorescence or phosphorescence. Considerable information about the efficiencies of radiative and radiationless processes can be obtained from a study of the kinetic dependence of emission intensity on concentrations of emitting and quenching species. The intensity of emission corresponds closely to the quantum yield, a concept explored in Sect. 7. In the present section we shall concentrate on the kinetic aspects, and first consider the application of stationary-state methods to fluorescence (or phosphorescence) quenching, and then discuss the lifetimes of luminescent emission under nonstationary conditions. 

The electronic excited state is inherently unstable and can decay back to the ground state in various ways, some of which involve (re-)emission of a photon, which leads to luminescence phenomena (fluorescence, phosphorescence, and chemiluminescence) (22). Some biologic molecules are naturally fluorescent, and phosphorescence is a common property of many marine and other organisms. (Fluorescence is photon emission caused by an electronic transition to ground state from an excited singlet state and is usually quite rapid. Phosphorescence is a much longer-lived process that involves formally forbidden transitions from electronic triplet states of a molecule.) Fluorescence measurement techniques can be extremely sensitive, and the use of fluorescent probes or dyes is now widespread in biomolecular analysis. For example, the large increase in fluorescence... 

First, consider the simplest case in which the sensitizer-activator interaction is treated as equivalent to the sensitizer-sensitizer interaction so the exciton becomes trapped only when it happens to hop onto an activator site. Also let us use as an example a simple cubic lattice of sensitizers and assume an electric dipole-dipole interaction as the mechanism causing the energy transfer. The hopping time is represented by th and the probability of host fluorescence per time of one step is a. The fraction of lattice sites which are traps is Cj and the probability of luminescence emission from a trapped exciton per time of one step is (3. The probability of host luminescence at the nth step in the random walk is... 

Although it is not the purpose of this article to champion one technology over another, it is fair to say that chemiluminescence offers the sensitivity of fluorescence detection without some of the attendant problems of luminescent emission from analytical samples. Thus, chemiluminescence detection can be comparable to and, with the aid of enzyme amplification, even superior to that of I. This is not to say that there are no problems associated with chemiluminescence-based analytical techniques. As we shall see later (Section 2.5), the actual signal from chemiluminescent molecules is, in most cases, a transient flash, lasting... 

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