The basics of fluorescence

Dyes and stains have long been used to detect and visualize structures and processes in biological samples. Today, many of the favored dyes and stains have a fluorescent component because fluorescent molecules can be detected with extraordinary sensitivity and selectivity.  This module will give you a basic introduction to the fluorescence process and definitions of some key terms that you will encounter as you learn more about fluorescence.

Topics in this section:
Defining fluorescence

Some molecules are capable of being excited, via absorption of light energy, to a higher energy state, also called an excited state. The energy of the excited state—which cannot be sustained for long— “decays” or decreases, resulting in the emission of light energy. This process is called fluorescence. To “fluoresce” means to emit light via this process. A fluorophore is a molecule that is capable of fluorescing. Some examples of fluorescent molecules include Invitrogen™ Alexa Fluor™ dyes, structural stains like DAPI or Invitrogen™ MitoTracker™ Red, and green fluorescent protein.

Ground state

In its ground state, the fluorophore molecule is in a relatively low-energy, stable configuration, and it does not fluoresce. When light from an external source hits a fluorophore molecule, the molecule can absorb the light energy. If the energy absorbed is sufficient the molecule reaches a higher-energy state, called an excited state. This process is known as “excitation”.

Excitation state in fluorescence.  (A) Light emitted from external source is absorbed by the molecule which reaches an excited state (B).

Excited state

There are multiple excited states or energy levels that the fluorophore can attain, depending on the wavelength and energy of the external light source. Since the fluorophore is unstable at high-energy configurations, it eventually adopts the lowest-energy excited state, which is semi-stable. The length of time that the fluorophore is in excited states is called the excited lifetime, and it lasts for a very short time, ranging from 10-15 to 10-9 seconds.

Schematic of the excited lifetime of a fluorophore.

Emission state

Next, the fluorophore rearranges from the semi-stable excited state back to the ground state, and the excess energy is released and emitted as light. The emitted light is of lower energy, and thus longer wavelength, than the absorbed light. This means that the color of the light that is emitted is different from the color of the light that has been absorbed. Emission of light returns the fluorophore to its ground state. The fluorophore can absorb light energy again and go through the entire process repeatedly. 

Fluorescence emission (A) and return to ground state (B).

Summary of fluorescence process

The cyclical fluorescence process can be summarized as:

Step 1. Excitation of a fluorophore through the absorption of light energy.

Step 2. A transient excited lifetime with some loss of energy.

Step 3. Return of the fluorophore to its ground state, accompanied by the emission of light.

The light energy emitted is always of a longer wavelength than the light energy absorbed, due to the energy lost during the transient excited lifetime, as shown in Step 2. 

Summary of the fluorescence process. (A) Jablonski diagram demonstrating the electron transition states of the fluorophore during the fluorescence process. (B) Three step process of fluorescence.