Biomedical Engineering Reference
In-Depth Information
Time
Time
Fig. 13.1.
Optical response of molecules bombarded by highly focused laser beam.
Rapidly diffusing small molecule (
left
); slow, large molecule, with its large well-
defined bursts of optical energy (
right
)
Fig. 13.2.
Autocorrelation of fluorescence response. High time resolution is espe-
cially useful for sub-nanosecond response dynamics
Dual-color cross-correlation FCS measures the cross-correlation of the flu-
orescence intensities of two distinct dyes [18]. Thus, it becomes possible to
detect different molecules without reference to their diffusion characteristics.
More recently, a combination of the two methods has been reported to re-
duce the need for a distinct, often rather large mass ratio between the two
molecule types [19]. A typical FCS setup is shown in Fig. 13.3. A highly fo-
cused laser beam is directed towards the detection volume. The reflected light
is applied to the high sensitivity optical sensor through a prism. A digital
correlator matches the autocorrelation function with a database of known re-
sponses. Generally, in FCS time resolutions of a few tens of picoseconds and
sensitivities equivalent to a few hundred photons are needed.
