3.3 FRET Sensors
The phenomenon of Förster or Fluorescence Resonance Energy Transfer (FRET)
was first described by Theodor Förster in 1946 [126]. In principle FRET is a physical
phenomenon in which a donor fluorophore in its excited state non-radiatively transfers its excitation energy to a neighboring acceptor fluorophore which is located in
close proximity, thereby causing the acceptor to emit its characteristic fluorescence.
As FRET involves non-radiative transfer of energy, it is highly sensitive to the
distance between donor and acceptor dipoles within the 1–10 nm range [127]. Thus,
FRET has found extensive application as a spectroscopic ruler in monitoring molecular interactions, because the distances that can be measured are much shorter than
the diffraction limit of conventional microscopy and even super resolution microscopy. In case of fluorescent protein-based FRET sensors, the donor and the acceptor
are fluorescent proteins with distinct spectral properties. The two proteins upon
interaction undergo conformational change and alter the proximity of chromophores
and its orientation, which promotes the occurrence of FRET [128]. The efficiency of
FRET is inversely proportional to the sixth power of distance within the short range
of 10 nm [129]. The inverse sixth power law relation leads to detectable change in
energy transfer even for change in orientation/proximity at the molecular scale
between the interacting chromophores. Such small-scale molecular interactions
include protein-protein interactions, conformational changes, intracellular ion concentrations, and enzyme activities (Fig. 4).
The efficiency of FRET (i.e., the quantum yield of the energy transfer) between
any two FPs is determined by the overlap of the emission spectrum of the donor
and the excitation spectrum of acceptor, quantum yield of donor fluorescence, and
extinction coefficient of the acceptor. A 30% or higher overlap in the emission
spectra of donor and excitation spectra of acceptor is a prerequisite for achieving
sufficient FRET signal with a reliable detection limit. Apart from this, the proximity
of the donor and acceptor governs the FRET efficiency by inverse power law. The
quantum yield and extinction coefficient of fluorophores determine the sensitivity
and yield of FRET signal. For any given pair of chromophores involved in FRET, an
integral factor representing the abovesaid parameters is denoted by the Förster
distance (R 0 ), which is the distance at which the FRET efficiency is 50% of its
maximal value.
With the advent of a wide range of GFP variants with distinct excitation and
emission spectra, the possibility of donor/acceptor combination has increased dramatically. Initial FRET-based biosensors were predominantly based on BFP as
energy donor, which was hampered by its instability and lower brightness. Recently,
cyan fluorescent protein (CFP) and donor yellow fluorescent protein (YFP) have
become the most useful FRET pairs for many in vitro studies. Following this trend,
two novel FRET pairs (mAmetrine/tdTomato and mCitrine/mTFP1) were developed
for simultaneous imaging of two different enzymes in a single-cell level.
Applications of Fluorescent Protein-Based Sensors in Bioimaging
165
The phenomenon of Förster or Fluorescence Resonance Energy Transfer (FRET)
was first described by Theodor Förster in 1946 [126]. In principle FRET is a physical
phenomenon in which a donor fluorophore in its excited state non-radiatively transfers its excitation energy to a neighboring acceptor fluorophore which is located in
close proximity, thereby causing the acceptor to emit its characteristic fluorescence.
As FRET involves non-radiative transfer of energy, it is highly sensitive to the
distance between donor and acceptor dipoles within the 1–10 nm range [127]. Thus,
FRET has found extensive application as a spectroscopic ruler in monitoring molecular interactions, because the distances that can be measured are much shorter than
the diffraction limit of conventional microscopy and even super resolution microscopy. In case of fluorescent protein-based FRET sensors, the donor and the acceptor
are fluorescent proteins with distinct spectral properties. The two proteins upon
interaction undergo conformational change and alter the proximity of chromophores
and its orientation, which promotes the occurrence of FRET [128]. The efficiency of
FRET is inversely proportional to the sixth power of distance within the short range
of 10 nm [129]. The inverse sixth power law relation leads to detectable change in
energy transfer even for change in orientation/proximity at the molecular scale
between the interacting chromophores. Such small-scale molecular interactions
include protein-protein interactions, conformational changes, intracellular ion concentrations, and enzyme activities (Fig. 4).
The efficiency of FRET (i.e., the quantum yield of the energy transfer) between
any two FPs is determined by the overlap of the emission spectrum of the donor
and the excitation spectrum of acceptor, quantum yield of donor fluorescence, and
extinction coefficient of the acceptor. A 30% or higher overlap in the emission
spectra of donor and excitation spectra of acceptor is a prerequisite for achieving
sufficient FRET signal with a reliable detection limit. Apart from this, the proximity
of the donor and acceptor governs the FRET efficiency by inverse power law. The
quantum yield and extinction coefficient of fluorophores determine the sensitivity
and yield of FRET signal. For any given pair of chromophores involved in FRET, an
integral factor representing the abovesaid parameters is denoted by the Förster
distance (R 0 ), which is the distance at which the FRET efficiency is 50% of its
maximal value.
With the advent of a wide range of GFP variants with distinct excitation and
emission spectra, the possibility of donor/acceptor combination has increased dramatically. Initial FRET-based biosensors were predominantly based on BFP as
energy donor, which was hampered by its instability and lower brightness. Recently,
cyan fluorescent protein (CFP) and donor yellow fluorescent protein (YFP) have
become the most useful FRET pairs for many in vitro studies. Following this trend,
two novel FRET pairs (mAmetrine/tdTomato and mCitrine/mTFP1) were developed
for simultaneous imaging of two different enzymes in a single-cell level.
Applications of Fluorescent Protein-Based Sensors in Bioimaging
165