Biomedical Engineering Reference
In-Depth Information
Figure 8.14
Principle of a FRAP experiment. After being bleached by a high-intensity laser beam,
the fluorescence progressively increases by diffusion and/or active transport and this recovery is
monitored. This intensity often does not come back to its initial value because of the immobile frac-
tion of molecules of interest.
might be a fraction of the fluorescence that is never recovered (corresponding to im-
mobile molecules). For the other molecules, the diffusion coefficient
D
is given by:
∼
2
D w /
τ
(8.6)
where
w
is the width of the bleached spot, and
τ
the characteristic time of the fluo-
rescence recovery. The proportionality coefficient depends on the geometry of the
experiment and on the boundary and initial conditions; it is calibrated with known
molecules.
FRET
Fluorescence resonance energy transfer (FRET) consists of using two dyes in such a
way that the emission spectrum of the first dye significantly overlaps the excitation
spectrum of the second one. This way, when the two molecules are close enough,
exciting the first dye results in a decrease of its emission and, conversely, to an in-
crease of emission for the second one. Typically, the molecules cannot be further
apart by more than a distance called the Forster radius that is of the order of 5 nm
[27]. FRET is thus well adapted to intramolecular distance measurements or to
other situations where the interacting molecules are very close. It is often described
as a “molecular ruler.”
TIRF
Total internal reflection fluorescence microscopy (TIRF) is very useful to image
phenomena close to an interface. When light propagating in glass is totally re-
