Fluorescence intensity detection has a much broader range of applications than absorbance detection. For fluorescence intensity measurements, an optical system (the excitation system) illuminates the sample using a specific wavelength (selected by an optical filter, or a monochromator), thereby exciting the sample. The excitation causes the sample to emit light (i.e. fluoresce) at a different wavelength. The emitted light is collected by a second optical system (emission system) and the signal is measured by a light detector such as a photomultiplier tube (PMT).
Samples are chemically bound with fluorescent tag
Light at a specific wavelength excites the sample
Excited samples emit light at a different wavelength
The advantages of fluorescence detection over absorbance detection are increased sensitivity and a broader application range, given the wide selection of fluorescent labels available today. For example, a technique known as calcium imaging measures fluorescence intensity of calcium-sensitive dyes to assess intracellular calcium levels.