Luminescent emission spectra, corrections

Luminescence of Pyrosoma. All species of the genus Pyrosoma (about 10 species) are bioluminescent. Pyrosoma is one of the few organisms reported to luminesce in response to light (Bowlby et al., 1990). The luminescence emission spectrum of Pyrosoma atlantica is bimodal according to Kampa and Boden (1957), with the primary peak near 482 nm, and the secondary near 525 nm. Swift et al. (1977) reported the emission maxima of two Pyrosoma species at 485 and 493 nm, respectively, and Bowlby et al. (1990) found an emission peak at 475 nm with P. atlantica. A corrected bioluminescence spectrum of P. atlantica (A.max 485 nm) reported by Herring (1983) is shown in Fig. 10.5.2. 

The luminescence emission spectrum of a specimen is a plot of luminescence intensity, measured in relative numbers of quanta per unit frequency interval, against frequency. When the luminescence monochromator is scanned at constant slit width and constant amplifier sensitivity, the curve obtained is the apparent emission spectrum. To determine the true spectrum the apparent curve has to be corrected for changes of the sensitivity of the photomultiplier, the bandwidth of the monochromator, and the transmission of the monochromator with fre-... 

Fig. 4.2.2 Left panel-. Uncorrected Ca2+-triggered bioluminescence spectrum of W92F obelin derived from O. longissima. Right panel Corrected bioluminescence spectrum of the same obelin (dotted line), and the fluorescence emission spectrum of the spent solution after luminescence (solid line). From Deng et al., 2001, with permission of the Federation of the European Biochemical Societies.

Photophysical Processes in Pol,y(ethy1eneterephthalate-co-4,4 -biphenyldicarboxyl ate) (PET-co-4,4 -BPDC). The absorption and luminescence properties of PET are summarized above. At room temperature the absorption spectrum of PET-co-4,4 -BPDC copolymers, with concentrations of 4,4 -BPDC ranging from 0.5 -5.0 mole percent, showed UV absorption spectra similar to that of PET in HFIP. The corrected fluorescence spectra of the copolymers in HFIP exhibited excitation maxima at 255 and 290 nm. The emission spectrum displayed emission from the terephthalate portion of the polymer, when excited by 255 nm radiation, and emission from the 4,4 -biphenyldicarboxylate portion of the polymer when excited with 290 nm radiation. 

When a luminescence spectrum is obtained on an instrument such as that used to produce the spectra in Figure 7.23, it will depend on the characteristics of the emission monochromator and the detector. The transmission of the monochromator and the quantum efficiency of the detector are both wavelength dependent and these would yield only an instrumental spectrum. Correction is made by reference to some absolute spectra. Comparison of the absolute and instrumental spectra then yields the correction function which is stored in a computer memory and can be used to multiply automatically new instrumental spectra to obtain the corrected spectra. The calibration must of course be repeated if the monochromator or the detector is changed. 

In order to obtain true emission and excitation spectra it is uaially necessary to apply conections for variations in excitation intentity and the wavelength sensitivity of the detection system. The correction needed may be calculated by comparing the instmment response for a standard compound of known corrected ectral characteristics with that of the sample under study, although q)ectrofluorimeters have been described which fully electronically compensate for intensity and wavelength response of the system Comparison of the area under the corrected emission spectrum with that of various standard fluorescence compounds allows the quantum yield of the luminescence process to be calculated ... 

Luminescence decay curves may be observed by displaying the output of the photomultiplier on an oscilloscope. Precautions must be taken to correct for instrumental distortion of fast decay curves (D13). In multicomponent systems with differing decay times, electronic gating may be used to isolate the signal due to one component (time resolved phosphorimetry) (SI). A complete emission spectrum can be observed using a spectrograph with a photographic plate or television camera tube, but these systems are as yet only of specialist interest. 

Luminescent standards have been established for use in calibrating fluorescence spectrometers and have been suggested for Raman spectroscopy in the past (18). The standard is a luminescent material, usually a solid or liquid, that emits a broad reproducible luminescence spectrum when excited by a laser. Once the standard is calibrated for a particular laser wavelength, its emission spectrum is known, and it can provide the real standard output , d)i(AF) depicted in Figure 10.8. In practice, a spectrum of the standard is acquired with the same conditions as an unknown then the unknown spectrum is corrected for instrument response function using the known standard... 

Analytical absorption spectroscopy in the ultraviolet and visible regions of the elechomagnetic spectrum has been widely used in pharmaceutical and biomedical analysis for quantitative purposes and, with certain limitations, for the characterisation of drugs, impurities, metabolites, and related substances. By contrast, luminescence methods, and fluorescence spectroscopy in particular, have been less widely exploited, despite the undoubted advantages of greater specificity and sensitivity commonly observed for fluorescent species. However, the wider availability of spectrofluorimeters capable of presenting corrected excitation and emission spectra, coupled with the fact that reliable fluorogenic reactions now permit non-fluorescent species to be examined fluorimetrically, has led to a renaissance of interest in fluorimetric methods in biomedical analysis. 

Figure 10.11. Observed and corrected emission curves for two luminescent standards. Raw curves were recorded for coumarin 540a soiution excited by 514.5 nm light, and for Kopp 2412 glass excited by 785 nm light. Raman shift is stated relative to the appropriate laser wavelength. Corrected output was calculated by comparison to a standard tungsten source. All curves are normalized to their maximum output. See Reference 20 for details. Spectrum A was determined on a Dilor X-Y spectrometer, B was acquired with a Chromex 2000.

It should be noticed that to calculate K it is sufficient to measure the luminescence quenching of only one component, because the concentration of pseudorotaxane C j 2 2+can be evaluated independently as both (CJ2+ —, 2+) and (C2 — C2). In the present case both components of the complex are luminescent, thereby enabling the determination of K from two independent series of emission measurements. The comparison of the results of these two experiments, which of course must be consistent with one another, can be useful for checking the validity of the corrections that have been made. As one can see from Fig. 5.15, the absorption spectrum of the solution containing [1 2] and its free components 1 " and 2 is rather complex in particular, it is impossible to excite 2 selectively, and luminescence of both 1 and 2 (Fig. 5.15, inset) is reabsorbed by the solution. Therefore, considerable corrections of the observed luminescence intensities are necessary. 

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