What is fluorescence?
Fluorescence is the property of some atoms and molecules to absorb light at a particular wavelength (the excitation: Ex) followed by a short-lived emission (Em) of light at a longer wavelength (Figure 2). The distance between the excitation and emission peaks is known as the Stokes shift and is fluorophore dependent (Figure 1).
Fluorescence involves an external light source to excite the sample at a particular wavelength. When excited at the appropriate wavelength, the molecule is transformed from a ground to an excited state. As the molecule returns to the ground state, energy is released in the form of heat (loss of energy) and light at a different longer wavelength of lower energy (Figure 3).
How does fluorescence detection work?
A microplate reader with fluorescence intensity (FI) detection uses a light source, usually a Xenon flash lamp or LED, to excite a fluorophore (fluorescent molecule) at a particular wavelength. The wavelength required to excite the sample can be selected using either a filter of a specific wavelength or a monochromator tuned to the required wavelength.
The fluorophore then emits light of a different wavelength, selected by a second filter or monochromator. This emitted fluorescence is detected by a photomultiplier tube (PMT), and the fluorescence intensity of the sample is expressed as relative fluorescence units.