Absorption- and emission spectra of fluorescent dyes
The absorbance of a dye quantifies how much of visible or UV light is absorbed by it. The emission value shows at which wavelength a fluorescent dye sends light respective radiation out. Each fluorescent dye has its own absorption- and emission value (see schedule below). The choice of the right absorption-/ emission spectra of a dye depends on the individual assay as well on the type of instrument that is used.
Stokes shift
The difference in energy between the excitation and emission maximum is known as the Stokes shift. With fluorescent dyes, a large Stokes shift is often desirable when optical filters are used to separate exciting light and fluorescence emission.
Extinction coefficient
This value is a direct measurement of the dye's ability to absorb light. The ability to absorb light will clearly have an effect on the amount of light it is able to emit.
pH sensitivity
Some fluorophores are more sensitive to alkaline or acid pH conditions. For example, in alkaline solution above pH 9 some dye molecules could degrade. Other fluorophores are stable in alkaline as well as in acid pH ranges. One of the reasons for this is the structural characteristic of the molecules.
Stability to photobleaching
Photobleaching is the photochemical destruction of a fluorophore, which may impact the observation of fluorescent molecules, since they will eventually be destroyed by a constant light exposure. The loss of dye intensity respective of the quantum yield during experiments can produce erroneous results. However, it is important to consider your application and the instrument as well. For instance, stability to photobleaching is not as important for DNA sequencers and flow cytrometry as it is for fluorescence microscopy.
Fluorescence quantum yield
The quantum yield of a radiation induced process is the number of times that a defined event occurs per photon absorbed by the system. The fluorescence quantum yield (also quantum efficiency) gives the efficiency of the fluorescence process. It is defined as the ratio of the number of photons emitted to the number of photons absorbed. Generally, the maximum fluorescence quantum yield is 1.0 (100%).
The quantum yield of fluorescent labels strongly depends on the current existing microenvironment such as pH-value, type of solution, concentration or temperature. Therefore specific quantum yields of single fluorescent dyes can not be revealed.