Luminescence standards, requirements

Requirements of Standards. The general requirements for luminescence standards have been discussed extensively (3,7-9) and include stability, purity, no overlap between excitation and emission spectra, no oxygen quenching, and a high, constant qtiantum yield independent of excitation wavelength. Specific system parameters--such as the broad or narrow excitation and emission spectra, isotropic or anisotropic emission, solubility in a specific solvent, stability (standard relative to sample), and concentration--almost require the standard to be in the same chemical and physical environment as the sample. 

Because of the good agreement between the predicted and measured cooling effects, the fluorescence quantum yield of Rhodamine 6G in mono-deuterated ethanol has to be very close to the value 0.99 measured by the thermal leasing method. This indicates the high accuracy of this method, which does not require a luminescence standard. In fact, using the thermal... 

Cormier and Dure (1963) found another type of luciferin and called it protein-free luciferin. Protein-free luciferin was found in the vapor condensate of freeze-drying whole animals, and also in the 3 5-56 % ammonium sulfate fraction of the crude extract noted above. The protein-free luciferin behaved like an aromatic or heterocyclic compound and it was strongly adsorbed onto Sephadex and other chromatography media, requiring a considerable amount of solvent to elute it. The luminescence reaction of protein-free luciferin in the presence of luciferase required a 500-times higher concentration of H2O2 compared with the standard luciferin preparation. Both types of the luciferin preparation had a strong odor of iodoform. 

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. 

Requirements for standards used In macro- and microspectrofluorometry differ, depending on whether they are used for Instrument calibration, standardization, or assessment of method accuracy. Specific examples are given of standards for quantum yield, number of quanta, and decay time, and for calibration of Instrument parameters. Including wavelength, spectral responslvlty (determining correction factors for luminescence spectra), stability, and linearity. Differences In requirements for macro- and micro-standards are considered, and specific materials used for each are compared. Pure compounds and matrix-matched standards are listed for standardization and assessment of method accuracy, and existing Standard Reference Materials are discussed. 

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... 

Due to the modifications of the electronic cloud induced by complexation, the quantum yield and the excitation spectrum are also modified. As the direct determination of the absolute quantum yield is very difficult to achieve, one usually finds in the literature quantum yield values determined by comparison to well-known standards, such as quinine sulfate. For example, some values can be found in Georges (1993) or in Klink et al. (2000) for some europium complexes but may be found also in many other papers on lanthanide luminescence. Studies on the correlations between the photophysical properties of a given type of europium complexes and the energy levels can be found in Latva et al. (1997), Klink et al. (2000). A correlation has been found between the excitation properties and the stoichiometry of various Eu(III) complexes (Choppin and Wang, 1997). Note that the changes in the excitation maximum induced by complexation usually amount to a few tenths of nanometers, which requires high resolution for detection. In the case of Eu(III), a correlation has been found between the frequency... 

Toxicity screening follows the conventional TLC development and analysis process, which has to be performed in advance to separate possible toxic substances on the HPTLC plate. The developed and dried plate is dipped into a suspension of bioluminescent bacteria. Vibrio fischeri, thereby exposing any separated bioactive compounds to the test orgartisms. The luminescent activity is reduced or stopped by substances toxic to the bacteria, which may also be toxic to humans. Healthy Vibrio fischeri, emit very weak tight of greenish color, which can not be detected with standard systems using daylight cameras. Thus, a specific bioluminescence detection system is required. 

It is very important to select promptly the most effective antibiotic for successful therapy of infectious diseases, and wound and post-surgical infections. The duration of standard microbiology assays applied in clinical practice exceeds 3-5 days since preliminary isolation of the pathogen from the clinical sample is required. In the present study we optimized a rapid bio luminescent antibiotic susceptibility assay based on comparison of bacterial ATP concentrations (bioluminescent signals) in a control (aliquot of the sample, free of the antibiotic) and probe (aliquot of the sample, supplied with antibiotic examined) after short-time incubation. For validation of the proposed assay, bacteria strains isolated from clinical samples were analyzed in parallel by the Bioluminescent Assay and Standard Microbiology Assay - Disk Method or Serial Dilutions Method. 

An aqueous sample may be added to the cocktail directly, after minor prior processing, or at the end of a radiochemical separation procedure. Direct addition is the equivalent of gross activity counting discussed in Section 7.2.4 except that some spectral analysis may be possible. Alpha particles can be differentiated from beta particles by deposited energy, pulse shape, and decay time. Self-absorption is of no concern. Quenching and luminescence, discussed in Section 8.3.2, often occur. Identification by maximum beta-particle energy is approximate, and requires comparison to radionuclide standards. 

CMOS detectors have been compared with the laboratory standard devices such as PMTs and cooled CCD cameras. One study indicated that CMOS detectors can be sufficient for applications that do not need the sensitivity of a cooled CCD, and require low power consumption and simple read-out. CMOS detectors also have been combined with fluorescent probes sequestered in a porous glass called a xerogel [12]. The benefits of xerogels are that their pore dimensions are controllable, they have good thermal and photo-stability, and they can be used for many spectroscopic tools [31, 32, 54]. This sensor system is demonstrated to detect oxygen concentration using a luminescent probe tris(4,7-diphenyl-1,10-phenanthroline)mthenium(ll)dication... 

Measurements. Absorption spectra measurements on the film and solutions of the metal complexes were measured on a modified Cary 14 spectrometer. Luminescence spectra were recorded with a custom photon counting spectrometer or a PTI (Deer Park Drive, South Brunswick, NJ 08852) luminescence spectrometer system. The FT-IR spectra were recorded with a PE-1600 spectrometer or a Bio-Rad FTS-40 spectrometer (Professor R. Crooks, Chemistry Department, University of New Mexico). RA spectra for LB films were measured on the FTS-40 with a Hanick gra2dng angle attachment and MCT detection system. Typically 256 scans were requir to obtain adequate intensity for the monolayer films. The scanning tunneling microscopy measurements employed a Nanoscope II system (Digital Instruments, Inc.). Etched Pt-Ir tips were used with the standard, 0.6 micron head. The system can be operated in either the constant current or constant height mode but the film contours were similar for either. 

It was not possible to determine quantitatively the two-photon cross-seclion of the [Tb(DPA)3] because of its low efficiency and because the use of ns-pulsed laser induced important error in the ct2pa determination. Using a fs Ti Sa laser as source and an integration sphere, Btinzli et al. [62] determined the three-photon cross-section by comparison of the 3PA and 1 PA luminescence. This absolute method does not require any standard and the [Pg.208]

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