Principles of Fluorescence Spectroscopy

812 FLUORESCENCE CORRELATION SPECTROSCOPY
Figure 24.20. Confocal fluorescence images of giant unilamellar vesicles (GUVs) labeled with a cyanine (DiI-C 20 ) or a Bodipy (Bodipy-PC)-labeled
lipid. The lipid compositions are shown under the images. In the upper black and white images DiI-C 20 is on the left and Bodipy-PC on the right. The
bars indicate 10 µm. Revised from [67].
revealed the phenomenon of anomalous subdiffusion of
membrane-bound proteins where the G(τ) curve is spread
out over a larger range of τ values. 63–66
24.5.1. Biophysical Studies of Lateral Diffusion
in Membranes
The ability of FCS to measure widely different diffusion
coefficients has been useful in studies of lateral diffusion in
membranes. Giant unilamellar vesicles (GUVs) about 35
µM in diameter were used to allow observation of specific
regions of the membranes. Figure 24.20 shows confocal
fluorescence microscopy images of GUVs that were labeled
with two lipophilic dyes: DiI-C 20 and Bodipy-PC. 67 For
these images the GUVs were composed of two phospholipids:
dilauroyl phosphatidylcholine (DLPC) and dipalmitoyl
phosphatidylcholine (DPPC). At room temperature
DLPC bilayers are in the fluid state and DPPC bilayers in
the solid state. Because of the difference in length of the
acyl side chain, C12 for lauroyl and C16 for palmitoyl,
bilayers containing both lipids show lateral phase separation.
This does not mean that each phase is composed only
of DLPC or DPPC but rather that two phases exist: a fluid
phase rich in DLPC and a solid phase rich in DPPC.
The images in Figure 24.20 were taken through two
different emission filters to select for the shorter-wavelength
emission of Bodipy-PC (shown as green) or the
longer-wavelength emission of DiI-C 20 (shown as red). To
obtain the confocal images the GUVs were labeled with
both probes at relatively high concentrations. When the
GUV contains only DLPC both probes are distributed
homogeneously (left). When the GUVs contain a mixture of
DLPC and DPPC one finds liquid domains rich in Bodipy-
PC and solid domains rich in DiI-C 20 . As the mole fraction
of DPPC increases the extent of the solid phase containing
DiI-C 20 increases (middle and right). GUVs similar to those
shown in Figure 24.20 were studied by FCS. For FCS the
GUVs were labeled with only one probe DiI-C 20 at a lower
concentration to allow observation of only a few probe molecules.
For this system we expect the diffusion to be two
dimensional with different diffusion coefficients in each
phase. Assuming the probe brightness is the same in both
phases the autocorrelation function becomes
G(τ) C FD F(τ) C SD S(τ)
V eff(C F C S) 2
(24.33)
where F and S refer to the fluid and solid phases, respectively,
and the diffusion correlation function in each phase is
given by
D i(τ) ( 1 4D iτ
s 2 ) 1
(24.34)