In practice, FCS is combined with confocal microscopy to yield a good
signal-to-noise ratio for examining individual molecules. In order to ensure that all
of the processes that are being analyzed actually are statisticalfluctuations around
an equilibrium condition, the samples under investigation are maintained at thermal
equilibrium. Depending on what category offluorescent markers are used, the
excitation light sources can be argon or argon-krypton multiline lasers, single-line
He–Ne lasers, or laser diodes.
Theflexibility of the FCS method can be increased by using twofluorescent
dyes and employing separate photodetectors for the two emission spectra. This
method is calledcross-correlation. Two other common methods based on using
fluorescencefluctuations to probe molecular interactions arephoton counting his-
tograms(PCH) andfluorescence intensity distribution analysis(FIDA). In addition,
confocal fluorescence coincidence analysis is a highly sensitive and ultrafast
technique for probing rare events in the femtomolar range [ 22 – 24 ].
9.4 Elastic Scattering Spectroscopy
Elastic scattering spectroscopy(ESS), which also is known by the namesdiffuse
reflectance spectroscopy(DRS) andlight scattering spectroscopy(LSS), has found
important applications for the in vivo diagnoses of diseases in a wide range of
organs, such as brain, breast, colon, esophagus, oral cavity, pancreas, and skin. ESS
is based on the analysis of elastic scattering in tissue of broadband light that can
range anywhere from the UV through the visible to the near-infrared regions [ 25 –
31 ]. In an elastic scattering process, photons that impinge on tissue components are
scattered without experiencing a change in their energy. This means that the pho-
tons will change their travel direction but not their wavelength.
An example of an ESS opticalfiber probe is shown in Fig.9.12. The light is
injected into a tissue sample (which is a turbid medium) through a dual-fiber optical
fiber probe. Typical core diameters can be 400 and 200μm for the illumination and
collection opticalfibers, respectively, with a center-to-center separation of 350μm.
The spectrum of the injected broadband light, which ranges from 330 to 760 nm,
undergoes several scattering events in a typical depth of between 200 and 600μm
from the surface of the tissue.
The collected photons come from the characteristic banana-shaped region
between the illumination and the collectionfibers. This region has a volume of
approximately 0.06 mm^3 for the probe configuration shown in Fig.9.12. The
information contained in the collected light depends on the anisotropy factor (the
angular scattering probability distribution; see Sect.6.3) and the distance between
the illumination and detectionfibers. The optical probe generally is placed in direct
contact with tissue. The diagnosis of the backscattered signal can be done within
milliseconds of the tissue illumination. Note that only light that has experienced
multiple scatterings can be collected by the detectionfiber.
9.3 Fluorescence Correlation Spectroscopy 273