Fluorescence-based techniques are valuable tools for studying cellular structure and function as well as the interactions of molecules in biological systems.  Fluorescence is also important in the detection and quantitation of nucleic acids and proteins in gel electrophoresis, microarrays, and fluorescence spectroscopy.

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 blog post 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.  The content summarized here can also be found as a video series of tutorials on the Life Technologies website.

Fluorescent Dyes
Examples of widely used fluorescent dyes are ethidium bromide, Alexa Fluor dyes, Cy dyes, and fluorescein.

States of Molecules
How and why do these dyes and stains emit different colors of light?  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 and results in the emission of light energy.  This process is called fluorescence.  To “fluoresce” means to emit light via this process.

A. Ground State
A fluorophore is a molecule that is capable of fluorescing.  In its ground state the fluorophore molecule is in a relatively low-energy stable configuration and it does not fluoresce.

B. Absorption
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 will become excited.

C. Excitation
The molecule reaches a higher-energy state called an excited state.  This process is known as “excitation”.  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 10-19 seconds.

Fluorescence Emission
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.

The cyclical fluorescence process can be summarized as:
1. Excitation of a fluorophore through the absorption of light energy.
2. A transient excited lifetime with some loss of energy.
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.

Learn more about products for fluorescence imaging.