Fluorescence application

The application of fluorescence measurements to a large variety of chemical, biochemical, biological and physical problems is extensive. The majority of fluorescence applications involve the use of extrinsic fluorescence probes. These are chromophoric molecules that are attached to or adsorbed onto another molecule and their fluorescence is measured. These molecules probe the properties of the substances to which they are attached. The catalogue and manual published by Molecular Probes (9) is an excellent collection of a large number of different examples of the use of fluorescence, especially as they relate to bio- 

1 A standard material is selected firom a list of quantum yield standards (6. 7 and rofde T). Hie standard should have an absorption spectrum in the same range as that of the sample. The fluorescence spectra of the standard and sample should span a similar i range. 

1 Selea Aex for the sample and if there is a sin e chromophoric species in the sample then this wavelmigth should be one where the absorbance does not change rapidly. The standard should also absorb at this X. 

2 Measure and record the absorbance of the sample and standard at this Aex. subtracting any solvent blank contribution, using the 5 m c indiical cuvette. Adjust the concentration of the sample and the standard so that A(5 cm) 0.25. The absorption spectrophotometer bandpass in these measurements should be nearly the same as the bandpass of ExM, that is typically 4 nm. 

3 Transfer the solutions to separate 1-cm rectangular quartz fluorescence cuvettes. Two cuvettes should also contain the respective blanks for the sample and standard. 

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McDermott F, Mattey DP, Hawkesworth CJ (2001) Centennial-scale Holocene climate variability revealed by a high-resolntion speleotherm record from SW Ireland. Science 294 1328-1331 McGarry SF, Baker A (2000) Organic acid fluorescence applications to speleothem paleoenvironmental reconstraction. Qnat Sci Rev 19 1087-1101... 

DsRed (Fig. 5.3E) is a bright RFP with excitation and emission maxima at 558 and 583 nm, respectively. Despite the bright red fluorescence, application of DsRed has been restricted, because of slow and inefficient maturation and its tetrameric structure [70, 71], The poor maturation efficiency has been overcome by random mutagenesis, which resulted in the fast maturing variant DsRedTl [72]. However, DsRedTl remains tetrameric. 

There can be little doubt there is still much progress to be made in the application of solid-state detectors to the study of near-IR fluorescence. However, solid-state photomultipliers capable of replacing the conventional photomultiplier tube in mainstream fluorescence applications would still seem to be a long way away. 

The laser beam can be directed along one of several optical paths available in either the upright or inverted microscope configurations. For fluorescence applications, it is often convenient to make use of the standard epi-illumination... 

There are many fluorescent indicators for detection of [Mg +] [319] and fluori-nated NMR reporters have been proposed. The simplest is fluorocitrate [313], which shows a change in chemical shift upon binding Mg +. However, it is critical that the reporter molecule be used as the + isomer only, which has relatively little toxicity [320], Levy et al. [8,321] developed the o-aminophenol-A/,A/,0-triacetic acid (APTRA) structure both for fluorescent application and by incorporation of fluorine atoms for F NMR, which have been used in the perfused rat heart [322]. 

N. L. Thompson and B. C. Lagerholm, Total internal reflection fluorescence Applications in cellular biophysics, Curr. Opin. Biotech. 8, 58-64 (1997). 

The mobile phase plays an important part in the fluorescence of a molecule. Unless chosen with care, the mobile phase can quench the fluorescence of the molecule of interest. Most of the non-halogen-containing solvents used in HPLC can be used with fluorescence detection. However, dissolved oxygen or other impurities in the eluent can cause quenching. The solvent polarity and the pH of the mobile phase can also affect the fluorescent process if they influence the charge status of the chromophore. For example, aniline fluoresces at pH 7 and at pH 12, but at pH 2, where it is cationic, it does not fluoresce. Table 3.5 shows wavelength selections for some common LC-fluorescence applications.30... 

Benninghoff, L., von Czarnowski, D., Denkhaus, E., and Lemke, K., Analysis of human tissues by total reflection x-ray fluorescence. Application of chemometrics for diagnostic cancer recognition, Spectrochim. Acta, Part B, 52B (17), 1039-1046, 1997. 

Dzubay, T. G., X-ray Fluorescence Applications to Environmental Analysis, Ann Arbor Press, 1976. 

Fluorescence detectors measure the intensity of the fluorescence of the eluate, stimulated either by monochromatic fight or a laser. As most polymers and colloids do not exhibit fluorescence, applications of this detector have been very limited. 

Aslan, K., Zhang, Y., Hibbs, S., Baillie, L., Previte, M. J., and Geddes, C. D. (2007). Microwave-accelerated metal-enhanced fluorescence application to detection of genomic and exosporium anthrax DNA in <30 seconds Analyst 132 1130-1138. 

McGarry, S.F. and Baker, A., 2000, Organic acid fluorescence applications to speleothem... 

Inorganic extractables/leachables would include metals and other trace elements such as silica, sodium, potassium, aluminum, calcium, and zinc associated with glass packaging systems. Analytical techniques for the trace analysis of these elements are well established and include inductively coupled plasma—atomic emission spectroscopy (ICP-AES), ICP-MS, graphite furnace atomic absorption spectroscopy (GFAAS), electron microprobe, and X-ray fluorescence. Applications of these techniques have been reviewed by Jenke. " An example of an extractables study for certain glass containers is presented by Borchert et al. ". ... 

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