Biomedical Engineering Reference
In-Depth Information
Figure 8.7
Excitation (black) and emission (gray) fluorescence spectra of fluorescein.
8.2.1.2 Fluorescent Molecules and Particles
Because of its versatility, fluorescence has been used in very different situations.
Fluorescent particles can be chemically synthetized (exogenous) or directly synthe-
tized by the cell or the bacterium (endogenous) by inserting the right gene in the
sequence of the protein. In this last case, it is attached at a precise position on a
given protein.
Exogenous Fluorophores: Organic Molecules
It first has to be realized that the situation is different if one wants to deal with in
vitro situations (isolated and/or purified molecules or fixed cells) or in vivo situa-
tions (live cells). Fluorophores are characterized by their excitation and emission
spectra; their quantum yield, defined as the number of emitted photon per absorbed
photon, quantifies their efficiencies. The fluorescence properties of these molecules
depend on their immediate environments. For instance, they can become sensitive
and local sensors for pH or viscosity. They can also detect ions in solutions: Cal-
cium or other divalent ions for instance are readily and quantitatively detected by
the FuraRed molecule even within live cells. Most of these organic fluorophores can
be coupled to proteins and a routinely used technique is to attach them to a given
antibody in order to specifically detect, localize, or measure the concentration of the
corresponding protein in vitro or in fixed cells. However, they are often toxic and
their use is consequently restricted mostly to fixed cells.
Unfortunately, these molecules cannot switch indefinitely between their excited
state and their ground state. After a certain number of these transitions (this num-
ber is extremely variable from one molecule to the other), they permanently lose
their fluorescence properties, a phenomenon called photobleaching that is enhanced
by the presence of dissolved oxygen in the solution. By carefully degassing solutions
and keeping the excitation to a minimum, this effect can be minimized at the cost
of a much decreased fluorescence intensity.
