Fluorescence microscopy is observed by the use of molecules called fluorophores.
Fluorphores are molecules that are capable of fluorescing. In a ground state or low energy state, fluorphores do not fluoresce. When an external light source is applied to the fluorophore, it raises it to a high energy or excited state. This process is known as excitation. There are many excited state levels that a fluorophore can obtain, dependent on the energy of the applied light source.
Because the fluorophore is unstable at an excited state, it quickly adopts the lowest excited energy level. As it drops to this lowest excited level, the energy that is lost is given off as heat.
Next, the fluorophore rearranges from the semi-stable excited state back to the ground state (Life
Technologies: Tutorials, 2013). This process is known as emission because the fluorophore will give off energy in the form of light as it goes from a level of high to low energy. The light given off during emission is of a lower energy, and therefore longer wavelength, than the light used by the external source, which is of a shorter wavelength, because of the energy that is lost during the
transient excited state. Both excitation and emission have a range of wavelengths at which they absorb or emit light. Each of these ranges have a maximum, which is the wavelength or color of light that produces the maximum absorbance or emission, known as the excitation maximum and the emission maximum, respectively. The difference between the excitation and emission maximum is known as the Stokes Shift. This difference in wavelength is due to the energy that is lost during the excited lifetime. The value of the Stokes Shift is dependent on the specific fluorophore that is used.
This process of fluorescence can be repeated multiple times by the same fluorophore.
However, after repeated exposures to light, the fluorophore may become denatured and no longer be able to fluoresce, a term known as photobleaching.
For fluorescence microscopy, there are many different dyes or fluorophores that can be used, as well as different light or excitation sources. One of the most common excitation sources that is used are broadband sources. A broadband source produces white light that contains the entire spectrum of visible light. Because of this, it will give many different peaks of fluorescence. This method is often used with filters that will be used to block out or inhibit undesirable wavelengths or colors, which will give better selectivity. Another common external light source are lasers. Some lasers have an output of just one wavelength, while others might produce multiple wavelengths, in which filters may still be needed to eliminate the undesired light. Two different filters can be used. If only a single dye is used, a long pass emission filter is useful, as it filters out excitation light to reduce background noise but transmits everything else.
If multiple dyes are used, a bandpass emission filter is useful, as it isolates the emission of each