AAT Bioquest

What are the types of fluorescence quenching?

Posted January 13, 2023


The primary types of fluorescence quenching are: internal conversion and vibrational relaxation, Forster resonance energy transfer (FRET), intersystem crossing (ISC), Dexter electron transfer, and radiative energy transfer. When an electron reaches a higher vibration state through the absorption of a photon, the first relaxation process is known as vibrational relaxation. Fluorescence occurs when an atom relaxes through vibrational relaxation to the ground after being electrically excited. The exact frequencies of emission and excitation are dependent on the atom or molecule. Internal conversion occurs when there is enough overlap between vibrational modes of different electronic levels. However, there is a minimal chance to produce a full relaxation to an electron’s ground state due to the large energy gap between s0 and s1 levels.       

FRET is an energy transfer mechanism that occurs between light-sensitive donor and acceptor molecules. If the donor molecule becomes excited, it transfers non-radiative energy to the acceptor molecule as it relaxes and reaches its ground state; this process is known as resonance, which occurs due to dipole-dipole interactions. Resonance occurs at distances of approximately 10 nm but becomes more likely the closer dipoles are to one another; occurs at a rate of the inverse of the distance to the sixth power. The excited acceptor then relaxes radiatively to its excited state, and thus emitting a photon. There are three factors determining the efficiency of the FRET quantum yield. These factors are: the distance between the molecules, the approximate orientation of donor acceptor and emission absorption dipole moment, and the degree of overlap of the acceptor’s absorption spectrum and donor’s emission spectrum. It is important to note that donors do not emit photons during FRET.  

Intersystem crossing is a radiationless process during which an electron moves between two levels of the same energy but has a distinct multiplicity. For instance, intersystem crossing can occur between an excited triplet state (S=1, multiplicity=3) and an excited singlet state (S=0, multiplicity=1). In this specific situation, the electron flips its spin, and the excited singlet moves to an excited triplet state. 

In the Dexter mechanism, donor and acceptor molecules come close enough that their electron orbitals overlap. This allows for the excited electron of the donor molecule to be transported to an unoccupied orbital of the acceptor molecule; it then becomes its ground state. Simultaneously, an electron is transferred from the acceptor to the donor molecule, also in its ground state. This ensures that the total number of electrons in each molecule stays constant and this process can occur between singlets and triplets (multiplicity must remain constant). 

Radiative energy transfer is the process when an excited donor releases a photon when it relaxes to its ground state, and the photon is then reabsorbed by an acceptor molecule. Therefore, the absorption spectrum of the acceptor and emission spectrum of the donor must overlap. 

Additional resources

Fluorescence quenching: A tool for single-molecule protein-folding study

Förster Resonance Energy Transfer (FRET)

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