Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 825
Figure 24.38. Optical filters for FCCS using RhG and Cy5. In the bottom
panel the solid line is the dichroic mirror and the dashed line is
the emission filter. Revised from [125].
Figure 24.37. Instrumentation for dual-color FCS. Revised from [125].
understood as each autocorrelation function being the
weighted sum for each species (G and R) as seen through
one of the channels. For instance, the G fluorophore is
detected in the G channel if it is present on the G or GR particle.
It is important to notice that the amplitude of the
cross-correlation function is proportional to the concentration
of the doubly labeled species CGR .
Because of the different excitation wavelengths the
dimension of the observed volume will be different for each
color, and different for the cross-correlation function. For
each channel
V G eff π 3/2 s 2 Gu G, τ G D s2 G
4D
V R eff π 3/2 s 2 Ru R, τ R D s2 R
4D
For the cross-correlation measurement
V GR
eff π3/2
2 3/2(s2 G s 2 R)(u 2 G u 2 R) 1/2
τ GR s2 G s 2 R
8D
(24.53)
(24.54)
(24.55)
(24.56)