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For the biomedical researcher, it comes the time when a fluorescent dye must be selected, to fit a particular experiment. Fluorescent dyes by nature absorb and emit light at different wavelengths and the greater the difference between these two values, the greater the quantum yield (Stoke-Shift) and generally the more useful the dye (due to its higher extinction coefficient). Therefore one has to take into account the spectral values of a given dye to match with the instrument available in the lab. Other factors are: pH sensitivity, light sensitivity, and instrumentation availability
The selection of the correct absorption/emission spectra of a dye depends on the particular assay to be carried out, as well as the instrumentation available. Each dye has a “signature” absorption(amount of visible/UV light absorbed)- emission ( amount of light given out), that is they do so at specific wavelengths.
The Stoke's shift is the difference in nanometers between the peak excitation and emission wavelengths of a fluorescent species. Some fluorescent dyes, such as FITC, exhibit a small Stoke's shift, whereas others, such as fura-2, exhibit a large Stoke's shift. The following graph, exhibits hypothetical fluorescence spectra of a dye which exhibits a small Stoke's shift. The Stoke's shift in this example is only 30nm.
This is defined by the Lambda Beer law; the higher the dye’s ability to absorb light , the higher the EC and generally greater sensitivity can be
Some fluorophores are more sensitive to alkaline or acid pH conditions. For example, in alkaline solution over pH 9 some dye molecules such as IRD700/800, Cy5, Cy5.5 or TAMRA could degrade. Other fluophores like Fam, Hex or Tet are stable in alkaline as well as in acid pH ranges. One of the reasons for this is the structural characteristic of the molecules.
Photolability refers to the destruction of tluorophore upon light exposure; samples must be kept covered with foil and protected from light when not in use. Photolability is most important in in fluorescent microscopy and perhaps of less consequences in DNA sequencing/ flow cytometry applications, where signal detection occurs rapidly. Quantum Yield
Quantum Yield can be defined by the equation: Q=photons emitted/photons absorbed. Quantum yield is essentially the emission efficiency of a given fluorochrome
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