Biomedical Engineering Reference
In-Depth Information
Figure 8.13
Principle of fluorescence confocal microscopy. No image is formed on a screen but the
fluorescence intensity is collected point by point by a sensor such as photomultiplier.
exposed the rest of the cone of illumination. To perform this task, an infrared (IR)
light is used for excitation. No fluorescence is directly excited by these IR photons.
However, if two of them combine, they can excite the dye to its excited state, so that
a photon is emitted when the molecule returns to its ground state. The probability
of such two-photon events is very small and needs a very high intensity to trigger a
detectable fluorescence. This condition on intensity is met only at the focal point of
the objective. Compared to classical confocal microscopy, there is no need for a pin-
hole and the corresponding optics. All the light coming from the excitation volume
is collected. Furthermore, as the excitation volume is confined at the focus, there is
no bleaching in the rest of the light cone and 3-D images can be reconstructed with
better accuracy even for very diffusing sample.
FRAP
As mentioned above, if a fluorophore is excited with a high intensity, it is irrevers-
ibly modified and loses its fluorescence. Therefore, if a high-energy light beam is
focused on a localized area, the so-called “bleached” area will appear as a dark spot
(note that this subsequent observation is performed with a lower intensity to avoid
affecting the fluorescent yield). Upon diffusion of the other fluorophores within
this dark area, it progressively recovers a higher level of fluorescence (Figure 8.14).
Fluorescence recovery after photobleaching (FRAP) quantifies this recovery of fluo-
rescence to extract dynamical characteristics of the system. Assuming a simple case
of a single diffusive species, the classical diffusion equation applies:
¶
c
2
D c
= Ñ
(8.5)
¶
t
Where
c
is the concentration and
D
the diffusion coefficient. This equation is then
solved to get the exact fluorescence profile in time and space. In practice, there
