Biomedical Engineering Reference
In-Depth Information
10.2 Microspectroscopy in Quantitative Biology:
Where and How
Static localization of the macromolecules and proteins in cells and tissues is
not su
cient by itself to elucidate the key mechanisms that regulate funda-
mental processes of the cell. The activity of a biological molecule is not just
a function of its structure but also of its
dynamic
behavior in the cell i.e.,
localization and interactions and their modifications in time. Thus, great im-
portance has to be given to the spatio-temporal dynamics of the molecular
interactions within cells in situ and, in particular, in vivo. In the following
sections, we describe two spectro-microscopy approaches that, combined with
the FPs chimera technology, are of growing importance in modern biology.
10.2.1 Fluorescence Correlation Spectroscopy
Fluorescence correlation spectroscopy (FCS) was introduced by Elson, Magde,
and Webb in 1972 [11]. In 1990, Denk and Webb demonstrated a new type of
microscope on the basis of two-photon excitation of molecules [12]. In 1995,
Berland, So, and Gratton put together the two technologies, two-photon exci-
tation microscopy and FCS, and demonstrated the potential of this method-
ology in intracellular measurements [13]. FCS is now a widely used technique.
The reader is invited to refer to the many good articles published on the
physics of the two-photon excitation process [14-16] and on the FCS tech-
nique [17-20]. Here, we discuss in specific the use of FCS in cell biology as
it provides information about mobility (diffusion coe
cients), concentration
(number of particles), association (molecular brightness), and localization (im-
age) of the target molecules. All this information helps understanding the
complex molecular interaction networks, which are at the basis of cellular
processes.
Chemical reactions and diffusion of molecules in solutions can be followed
by a variety of methods but when we need to study them in living cells, most
methods fail abruptly. The appeal of FCS is that, especially using two-photon
excitation, living cells can be explored anywhere by a subfemtoliter volume
without disrupting the cell integrity. The most stringent requirement for FCS
to work is the possibility to observe the fluorescence signal in a small volume
and at very high sensitivity and dynamic range (Fig. 10.6).
Only if the volume is so small at any instant of time, it might contain just
one or few molecules. Because of these requirements, FCS is also known as
a
single molecule
spectroscopy. If the number of fluorescent molecules in the
volume does not change with time and if the quantum yield of the fluorophore
is constant, then the average number of the emitted photon is constant. How-
ever, the instantaneous number of detected photon is not constant, because
of the nature of the emission/detection process, which follows the Poisson
statistics (
2
). This added shot-noise is independent of time.
2
The Poisson distribution describes events that occur rarely in a short period
