The Light Spectrum and its Relationship with Fluorescence
Basic properties of light
Now that we’ve introduced the general process of fluorescence, let’s take a look at the basic properties of the light spectrum and its 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 longer wavelengths have lower frequency and lower energy.
The visible light spectrum.
An excited fluorophore molecule emits lower-energy light than the light it absorbs. 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.
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. So for each fluorescent dye, there is a specific wavelength—the excitation maximum—that most effectively induces fluorescence.
Typical representation of how fluorophore excitation range (bars) and fluorophore excitation maximum (stars) are displayed.
Let’s say that we have a tube that contains a particular fluorescent dye. If we shine 480 nanometer light at 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 we increase the excitation wavelength, say to 520 nanometers, more molecules are excited. However, this is still not the wavelength at which the proportion of excited molecules is maximal. For this particular dye, 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 energy and fluoresce, but again in smaller proportions. The range of excitation wavelengths can be represented in the form of a fluorescence excitation spectrum.
Explanation of fluorescent dye excitation range. Open circles represent fluorescent molecules in the excited state. Closed circles represent fluorescent molecules in the ground state. (A).Cross section of a fluorescent dye solution in ambient light. Dye solution excited by light at (B) 480 nm; (C) 520 nm; (D) 550 nm; (E) 595 nm; (F) Fluorescence excitation spectrum representing all of the excited dye molecules (open circles) at the different excitation wavelengths in B-E.
In summary, 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. So for each fluorescent dye, there is a specific wavelength—the excitation maximum—that most effectively induces fluorescence.
Now let’s look at the light that is emitted by the fluorophore molecules when they are excited at the optimal excitation wavelength. 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 of 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.
Example of fluorescent dye emission range. A) The dye solution is excited at 550 nm and the excited fluorophores are shown. B) Top view of the dye solution and the range of wavelengths being emitted by the excited fluorophores are indicated in the spectrum bar above.
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.
Fluorescence emission spectrum. A) Excited fluorophores in the dye solution emit light in a range of colors that comprise the emission spectra range. B) The majority of fluorophores emit light at the emission maximum of the dye.
The summary points of this introduction to fluorescence are:
- 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.
The complete tutorial is also available on this video.
Plot and compare spectra and check the spectral compatibility of multiple fluorophores.
For Research Use Only. Not for use in diagnostic procedures.