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Fluorescence

  • Fluorescence explained

Definition

Fluorescence is the emission of electromagnetic radiation, especially of visible light, stimulated in a substance by the absorption of incident radiation and persisting only as long as the stimulating radiation is continued.

Cycling of Fluorescence

Some molecules are capable of being excited via absorption of light energy to a higher energy state, an excited state. The energy of the excited state decays resulting in the emission of light energy. This process is called Fluorescence.

A fluorophore is a molecule capable of fluorescencing. In its ground state the fluorophore molecule is in a relatively low energy configuration, and it does not fluoresce.

  • Excitation

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 an exited state. This process is known as excitation.

S_{0}+hv_{ex}\rightarrow S_{1}

There are multiple excited states or energy levels that the flourophore can attain depending on the wavelength and energy of the external light source.

  • Transient excited lifetime

Since the fluorophore is unstable at high energy configurations, it eventually adopts the lowest energy excited state, which is semi-stable. In this process there is a loss of energy. The length of time that the fluorophore is in an excited state is called the excited lifetime. It last for a very short time, ranging from 10-15 to 10-9 seconds.

  • Emisssion

Next, the flourophore rearranges form the semi-stable excited state back to the ground state and the excess energy is released and emitted as light.

S_{1}\rightarrow hv_{em}+heat+S_{0}

The emitted light is of lower energy and has a 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.

Photobleaching

An important characteristic of fluorophores is that they can go through the cycle of fluorescence repeatedly, in theory, indefinitely. However, in reality the fluorophore instability during the excited lifetime makes it susceptible to degradation. High intensity illumination can cause the fluorophore to change its structure and no longer fluoresce. This phenomenon is known as photobleaching.

Fluorescence Spectra

  • Fluorescence excitation spectrum

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 others. This range of wavelengths reflects the range of possible excited states that the fluorophore can achieve. Therefore, for each fluorescent dye there is a specific wavelength, the excitation maximum, which most effectively induces fluorescence.

The range of excitation wavelengths can be represented in the forms of the fluorescence excitation spectrum.

  • Fluorescence emission spectrum

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.

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 fluorophores all emit the same intensity of light, the wavelengths are not homogeneous. Collectively however, the population fluoresceses most intensely at the maximum indicated by the distribution, represented by the fluorescence emission spectrum.

Stokes shift

The basic fluorescent properties of a fluorophore; excitation and emission, are often presented in the form of line graphs. These curves describe the likelihood that excitation and emission will occur as a function of wavelength. They also provide important information about the expected behavior of the irradiated fluorophore.

The emission maximum for the fluorophore is always at a longer wavelength than the excitation maximum. The difference between the excitation and the emission maximum is called the Stoke shift. The magnitude of the Stoke shift is determined by the electronic structure of the fluorophore and it is one of its characteristics.

The Stoke shift is due to fact that some of the energy of the excited fluorophore is lost through molecular vibrations that occur during the brief lifetime of the molecule’s excited state. This energy is dissipated as heat to surrounding solvent molecules as they collide with the excited fluorophore.



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