Perrin-Jablonski diagram

The Perrin-Jablonski diagram (figure 1) is convenient for visualizing the different

processes involved in the interaction of a molecule with light: photon absorption, internal
conversion, fluorescence, intersystem crossing and phosphorescence.

When a molecule is excited from the ground state to an upper excited singlet state (S1) by
absorption of a photon of light {1}, it usually ends up in one of the upper vibrational
levels of that state. A molecule in a high vibrational level will quickly fall to the lowest
vibrational level of this state by a process called “vibrational relaxation”, which does not
involve any light emission {4}.
When the molecule has attained the lowest vibrational level in the excited state, one of
three things can happen:
• return to the singlet ground state by the emission of a photon of light (fluorescence)
without changing the spin state {2}. The lifetime of this process is very short
(nanoseconds);
• decay back to the ground state through Vibrational collisions (radiationless decay) with
other molecules {6};
• the spin of the excited electron reverses, leaving the molecule in an excited triplet state
(T1); this mechanism is called “InterSystem Crossing” (ISC) {5}. The T1 state is of a
lower electronic energy than the excited singlet state. The T1 state can decay to the
ground state by photon emission in a process called “phosphorescence”, but the transition
is forbidden and this results in very long excited state lifetimes (up to 10’s of seconds).
In the case of petroleum fluorescence, only the first two processes are significant
Förster resonance energy transfer
Förster resonance energy transfer (FRET), also known as fluorescence resonance
energy transfer, resonance energy transfer (RET) or electronic energy transfer
(EET), is a mechanism describing energy transfer between two chromophores.
A donor chromophore, initially in its electronic excited state, may transfer energy to an
acceptor chromophore (in proximity, typically less than 10 nm) through nonradiative
dipole–dipole coupling. This mechanism is termed "Förster resonance energy transfer"
and is named after the German scientist Theodor Förster. When both chromophores are
fluorescent, the term "fluorescence resonance energy transfer" is often used instead,
although the energy is not actually transferred by fluorescence.

Primary Conditions for FRET

1. Donor and acceptor molecules must be in close proximity (typically 10–100 Å).
2. The absorption spectrum of the acceptor must overlap the fluorescence emission
spectrum of the donor .
3. Donor and acceptor transition dipole orientations must be approximately parallel.

Theoretical basis
1. The transfer rate constant from donor to acceptor, , is given by

where and are the emission rate constant and the lifetime of the excited donor in
the absence of transfer, respectively, is the distance between the donor and the acceptor
and is the critical quenching radius or Förster radius, i.e., the distance at which
transfer and spontaneous decay of the excited donor are equally probable ( ).
is given by

The orientation factor is given by

where is the angle between the donor and acceptor moments, and and are the
angles between these, respectively, and the separation vector; is the angle between the
projections of the transition moments on a plane perpendicular to the line through the
centres. can in principle take values from 0 (perpendicular transition moments) to 4
(collinear transition moments). When the transition moments are parallel and
perpendicular to the separation vector, . When they are in line (i.e., their moments
are strictly along the separation vector), . For randomly oriented transition
(dipole) moments, e.g., in fluid solutions, .

2. The FRET efficiency (E) is the quantum yield of the energy transfer transition, i.e. the
fraction of energy transfer event occurring per donor excitation event:

where kET is the rate of energy transfer, kf the radiative decay rate and the ki are the rate
constants of any other de-excitation pathway.

The FRET efficiency depends on many parameters that can be grouped as follows:

 The distance between the donor and the acceptor

 The spectral overlap of the donor emission spectrum and the acceptor absorption
spectrum.
 The relative orientation of the donor emission dipole moment and the acceptor
absorption dipole moment.

E depends on the donor-to-acceptor separation distance r with an inverse 6th power law
due to the dipole-dipole coupling mechanism:

The FRET efficiency relates to the quantum yield and the fluorescence lifetime of the
donor molecule as follows:

where τ'D and τD are the donor fluorescence lifetimes in the presence and absence of an
acceptor, respectively, or as

where F'D and FD are the donor fluorescence intensities with and without an acceptor,
respectively.
Experimental Confirmation of the Fӧrster resonance energy
transfer theory
The inverse sixth power distance dependence of Fӧrster resonance energy transfer was
experimentally confirmed by Stryer and Haugland using a donor and an acceptor
separated on an oligoproline helix. Haugland, Yguerabide and Stryer also experimentally
demonstrated the theoretical dependence of Fӧrster resonance energy transfer on the
overlap integral by using a fused indolosteroid as a donor and a ketone as an acceptor.

Selected Applications of FRET

Structure and conformation of proteins
Spatial distribution and assembly of protein complexes
Receptor/ligand interactions
Immunoassays
Probing interactions of single molecules
Structure and conformation of nucleic acids
Real-time PCR assays and SNP detectio
Detection of nucleic acid hybridization
Primer-extension assays for detecting mutations
Automated DNA sequencing
Distribution and transport of lipids
Membrane fusion assays
Membrane potential sensing
Fluorogenic protease substrates
Indicators for cyclic AMP and calcium