Why fluorescence occurs

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Copy Link. Powered by Social Snap. Copy link. Copy Copied. Various radiative and non-radiative processes can de-populate the excited state so the total decay rate is the sum over all rates:. If the rate of spontaneous emission or any of the other rates are fast, the lifetime is short. The average lifetime of fluorescent compounds that emit photons with energies from the UV to near infrared are within the range of 0.

This relationship shows that fluorescence intensity is proportional to concentration. Fluorescence rarely results from absorption of UV-radiation of wavelengths shorter than nm because this type of radiation is sufficiently energetic to cause deactivation of the excited state by predissociation or dissociation. Most organic molecules have at least some bonds that can be ruptured by energies of this strength. Fluorescence commonly occurs from a transition from the lowest vibrational level of the first excited electronic state to the one of the vibrational levels of the electronic ground state.

The average lifetime is 10 -7 to 10 -9 seconds for? Figure 2 is a schematic of a typical filter fluorimeter that uses a source beam for fluorescence excitation and a pair of photomultiplier tubes as transducers. The source beam is split near the source into a reference beam and a sample beam. The reference beam is attenuated by the aperture disk so that its intensity is roughly the same as the fluorescence intensity. Both beams pass through the primary filter, with the reference beam being reflected to the reference photomultiplier tube.

The sample beam is focused on the sample by a pair of lenses and causes fluorescence emission. The emitted radiation passes through a second filter and then is focused on the sample photomultiplier tube. The electrical outputs from the two transducers are then processed by an analog to digital converter to compute the ratio of the sample to reference intensities, which can then be used for qualitative and quantitative analysis.

To obtain an emission spectrum, the excitation monochromator is fixed and the emission monochromator varies. To obtain an excitation spectrum, the excitation monochromator varies while the emission monochromator is fixed.

For this purpose proteins of interest can be tagged with genetically encoded fluorescing molecules like GFP green fluorescing protein. Molecules of interest e. GFP -derivates. Luminescence describes the occurrence of luminous effects that are caused by the change of an electron from an excited state to a state with lower energy.

Electrons can exist in different energy states. The ground state is a very stable state for an electron and has the lowest energy level.

If electrons absorb energy, they can be elevated to a higher energy level, an excited state. As the excited state is of higher energy than the ground state, energy has to be released when an electron returns to its ground state. This energy can be released in the form of emitted photons. There are several forms of luminescence differing in the way the system is excited, e.

Photoluminescence can further be divided into two sub-groups, fluorescence and phosphorescence. The main difference between fluorescence and phosphorescence is the duration of their luminescence. Fluorescence immediately ends when illumination is stopped. In contrast, phosphorescence can last for hours after the excitation has ended. Fluorochromes will only fluoresce if they are illuminated with light of the corresponding wavelength.

The wavelength depends on the absorption spectrum of the fluorophore and it has to be ensured that an appropriate quantity of energy is delivered to elevate the electrons to the excited state. After the electrons are excited they can dwell in this high energy state for a very short time only. When the electrons relax to their ground state or another state with a lower energy level, energy is released as a photon. As some of the energy is lost during this process, light with an increased wavelength and lower energy is emitted by the fluorochrome compared to the absorbed light.

As phosphorescing molecules can luminesce for a much longer time than fluorochromes, there must be a difference in the way they store the excitation energy. The basis for this discrepancy is found in the two forms of excitation levels, the singlet excited state and the triplet excited state, which are based on different spin alignments. Spins are an attribute of electrons. In simplified terms, the spin describes the angular momentum of the electron caused by its rotation.