Fluorescence - The Light Spectrum, Fluorphore Excitation and Emission
I. Introduction
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 article 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 in this article can also be found as a fluorescence webinar on the Life Technologies website.
II. Cycling of Fluorescence
A fluorophore can repeatedly undergo the fluorescence process-in theory, indefinitely. This is extremely useful because it means that one fluorophore molecule can generate a signal multiple times. This property makes fluorescence a very sensitive technique for visualizing microscopic samples – even a small amount of the stain can be detected.
III. Photobleaching
The fluorophore's structural instability during the excited lifetime makes it susceptible to degradation. High-intensity illumination can cause the fluorophore to change its structure so that it can no longer fluoresce. This is called photobleaching. When a fluorescent sample such as a slide with mounted tissue is photobleached the fluorophores are no longer promoted to an excited state even when the required light energy is supplied.
IV. Properties of the light spectrum
Now that we've introduced the general process of fluorescence, let's take a look at the basic properties of the light spectrum and it's importance in fluorescence. The visible spectrum is composed of light with wavelengths ranging from approximately 400 nanometers to 700 nanometers.
Light waves with shorter wavelengths have higher frequency and higher energy. Light waves with linger wavelengths have lower energy light than the light it absorbed. Therefore, there is always a shift along the spectrum between the color of the light absorbed by the fluorophore during excitation and the color emitted.
Example – Let's say that we have a tube that contains a particular fluorescent dye. If 480 nanometer light is shined on the dye solution, some of the fluorophore molecules will become excited. However, the majority of the molecules are not excited by this wavelength of light. As the excitation wavelength is increased to say 520 nanometers more molecules are excited. However, this is still not the wavelength at which the proportion of excited molecules is maximal. For the particular dye being used in this example 550 nanometers is the wavelength that excites more fluorophores than any other wavelength of light. At wavelengths longer than 550 nanometers the fluorophore molecules still absorb and fluoresce but again in smaller proportions. The range of excitation wavelengths can be represented in the form of a fluorescence excitation sprectrum.
V. Excitation Range and Maximum
A. Range
A fluorescent dye absorbs light over a range of wavelengths- and every dye has a characteristic excitation range. However, some wavelengths within that range are more effective for excitation than other wavelengths. This range of wavelengths reflects the range of possible excited states that the fluorophore can achieve.
B. Maximum
For each fluorescent dye there is a specific wavelength- the excitation maximum- that most effectively induces fluorescence.
VI. Emission
Just as fluorophore molecules absorb a range of wavelengths, they also emit a range of wavelengths. There is a spectrum of energy changes associated with these emission events. When we excite the previously described dye solution at its excitation maximum, 550 nanometers, light is emitted over a range of wavelengths. A molecule may emit at a different wavelength with each excitation event because of changes that can occur during the excited lifetime, but each emission will be within the range. Although the fluorophore molecules all emit the same intensity of light, the wavelengths, and therefore the colors of the emitted light, are not homogeneous. Collectively, however, the population fluoresces most intensely at 570 nanometers. Based on this distribution of emission wavelengths, we say that the emission maximum of this fluorophore is 570 nanometers. The range of wavelengths is represented by the Fluorescence Emission Spectrum.
VII. Summary
Fluorophores are molecules that upon absorbing light energy can reach an excited state then emit light energy. The three-stage process of excitation, excited lifetime, and emission is called fluorescence. Fluorophores absorb a range of wavelengths of light energy and also emit a range of wavelengths. Within these ranges are the excitation maximum and the emission maximum. Because the excitation and emission wavelengths are different, the absorbed and emitted light are detectable as different colors or areas on the visible spectrum.
Watch the most recent fluorescence webinar on the Life Technologies website.
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 article 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 in this article can also be found as a fluorescence webinar on the Life Technologies website.
II. Cycling of Fluorescence
A fluorophore can repeatedly undergo the fluorescence process-in theory, indefinitely. This is extremely useful because it means that one fluorophore molecule can generate a signal multiple times. This property makes fluorescence a very sensitive technique for visualizing microscopic samples – even a small amount of the stain can be detected.
III. Photobleaching
The fluorophore's structural instability during the excited lifetime makes it susceptible to degradation. High-intensity illumination can cause the fluorophore to change its structure so that it can no longer fluoresce. This is called photobleaching. When a fluorescent sample such as a slide with mounted tissue is photobleached the fluorophores are no longer promoted to an excited state even when the required light energy is supplied.
IV. Properties of the light spectrum
Now that we've introduced the general process of fluorescence, let's take a look at the basic properties of the light spectrum and it's importance in fluorescence. The visible spectrum is composed of light with wavelengths ranging from approximately 400 nanometers to 700 nanometers.
Light waves with shorter wavelengths have higher frequency and higher energy. Light waves with linger wavelengths have lower energy light than the light it absorbed. Therefore, there is always a shift along the spectrum between the color of the light absorbed by the fluorophore during excitation and the color emitted.
Example – Let's say that we have a tube that contains a particular fluorescent dye. If 480 nanometer light is shined on the dye solution, some of the fluorophore molecules will become excited. However, the majority of the molecules are not excited by this wavelength of light. As the excitation wavelength is increased to say 520 nanometers more molecules are excited. However, this is still not the wavelength at which the proportion of excited molecules is maximal. For the particular dye being used in this example 550 nanometers is the wavelength that excites more fluorophores than any other wavelength of light. At wavelengths longer than 550 nanometers the fluorophore molecules still absorb and fluoresce but again in smaller proportions. The range of excitation wavelengths can be represented in the form of a fluorescence excitation sprectrum.
V. Excitation Range and Maximum
A. Range
A fluorescent dye absorbs light over a range of wavelengths- and every dye has a characteristic excitation range. However, some wavelengths within that range are more effective for excitation than other wavelengths. This range of wavelengths reflects the range of possible excited states that the fluorophore can achieve.
B. Maximum
For each fluorescent dye there is a specific wavelength- the excitation maximum- that most effectively induces fluorescence.
VI. Emission
Just as fluorophore molecules absorb a range of wavelengths, they also emit a range of wavelengths. There is a spectrum of energy changes associated with these emission events. When we excite the previously described dye solution at its excitation maximum, 550 nanometers, light is emitted over a range of wavelengths. A molecule may emit at a different wavelength with each excitation event because of changes that can occur during the excited lifetime, but each emission will be within the range. Although the fluorophore molecules all emit the same intensity of light, the wavelengths, and therefore the colors of the emitted light, are not homogeneous. Collectively, however, the population fluoresces most intensely at 570 nanometers. Based on this distribution of emission wavelengths, we say that the emission maximum of this fluorophore is 570 nanometers. The range of wavelengths is represented by the Fluorescence Emission Spectrum.
VII. Summary
Fluorophores are molecules that upon absorbing light energy can reach an excited state then emit light energy. The three-stage process of excitation, excited lifetime, and emission is called fluorescence. Fluorophores absorb a range of wavelengths of light energy and also emit a range of wavelengths. Within these ranges are the excitation maximum and the emission maximum. Because the excitation and emission wavelengths are different, the absorbed and emitted light are detectable as different colors or areas on the visible spectrum.
Watch the most recent fluorescence webinar on the Life Technologies website.
