II) CHROMOPHORES AND THE PRINCIPLE OF THE F.R.E.T
1) Definition of the chromophors
Employed like adjective, this term indicates a whole of atoms at the origin of the color of a molecular entity and, by extension and more generally, at the origin of a given electronic transition. This optical property results from a capacity to absorb the energy of photons in a range of the visible spectrum while the other wavelengths are transmitted or diffused. For example, the carotene is the chromophoric one which gives their color to many fruits (like carrots), this molecule indeed absorbs the wavelengths in the blue range of the visible spectrum but reflects the lower wavelengths (orange and red). There are two types of chromophoric:
• systems with connections pi combined: The presence of a sufficiently long sequence of double connections combined in an organic molecule creates a delocalized electronic cloud being able to enter in resonance with the incidental radiation.
• metal complexes around a metal of transition: Orbital D of the metal atom are distributed between this one and the ligand. The absorption of an incidental photon results in a jump of an electron into orbital the higher. One finds this type of chromophors as well in biological molecules then in inorganic compounds.
In our experiment we not used one but two chromophoric known as coupled. Between these two chromophors can occur a transfer of energy by resonance, called F.R.E.T for Fluorescent Resonance Energy Transfer, whose mechanism is developed below.
2) Interest and definition of FRET
The precise localization and the nature of the interactions between the specific species of molecules of the alive cells are a subject of important interest in biological research but these last are often limited by the resolution of the optical instruments used. The conventional fluorescence microscopes can roughly detect two molecules within the limits of the criterion of Rayleigh i.e. 200 nanometers. However, to include/understand the physical interactions between proteins partners intervening in typical biomolecular processes, the relative proximity of the molecules must be given more precisely than allow it the traditional methods of imagery optical which are limited by diffraction. It is there that the technique of FRET intervenes which used with an optical microscope only allows the determination of the approach of two distant molecules of some nanometers. The transfer of energy between fluorescent molecules or transfer of energy by resonance of the Förster type is a quantum phenomenon bringing into play two fluorescent molecules (for us here the two chromophors ones), namely a donor fluorescent molecule in an electronic state excited and being able to transfer its energy from excitation to an acceptor. This energy transfer by resonance is a quantum mechanical process nonradiative which does not require a collision between the chromophoric ones. Nevertheless so that this transfer takes place, it is necessary that the emission spectrum of the donor overlaps the absorption spectrum of the acceptor and that the distance separating them is lower than ten nanometers. Here thus diagrams recapitulating the conditions necessary to FRET.
Zone of stepping of of the donor and the absorption emission spectra of the acceptor (spectral overlap area)
Caption diagram: • Absorption will spectra: absorption spectrum • Emission will spectra: emission spectrum • CFP: fluorescent protein of color cyan (the donor) • DsRFP: red fluorescent protein (the acceptor)