Biology Reference
In-Depth Information
custom computer program, which takes the cross correlation of the observed image
using a reference two-dimensional Gaussian function with a full width of 200 nm
(
Fujiwara et al., 2002
). In addition to identifying fluorescent spots, this method gen-
erates the local peaks in the correlation image (
Fig. 20.4
B) and thus determines
whether an observed spot represents one unresolvable spot or two resolvable spots.
In this manner, the number density of distinguishable spots can be determined.
Because the number of dyes attached to individual anti-Fab fragments exhibits
Poisson distributions, the absolute number of molecules located within a single colo-
calized spot cannot be determined using fluorescent Fab fragments. Because ACP- or
Halo7-tagged receptor molecules can be labeled with dyes at a 1:1 D/P ratio and high
efficiency, the absolute number of molecules in a single colocalized spot can be de-
termined by measuring the fluorescence intensities of individual spots of ACP- or
Halo7-tagged receptors.
After each individual spot in the image is identified, the fluorescence intensities
of the identified spots are determined, yielding the histograms.
Figure 20.5
A shows
the distribution of fluorescence intensities of individual spots of ACP-CD59 labeled
with Dy547 in CHO-K1 cells. The histograms are fitted by the sum of three log-
normal functions (
Mutch et al., 2007
) and provide the spot fractions of monomers,
dimers, and tetramers via comparison with the histogram for the fluorescence inten-
sities of individual spots of monomer reference molecules. To determine the fluores-
cence intensity distribution of monomers, we often measure the individual spot
intensities of ACP- or Halo7-tagged proteins linked to the TM domains of nonraft
molecule low-density lipoprotein receptors (ACP-TM or Halo-TM), which are
FIGURE 20.5
Fractions of monomers, homodimers, and homo-oligomers of acyl carrier protein (ACP)-
CD59. (A) Distributions of the signal intensities of individual spots of Dy547-labeled ACP-
CD59 expressed at densities of 0.90 (
788) copies/mm
2
and fitted with three log-normal
functions that represent the distributions for monomers, homodimers, and apparent
homotetramers of ACP-CD59. This result was obtainable because more than 95% of ACP was
fluorescently labeled. (B) Distribution of the signal intensities of individual spots of Dy547-
labeled ACP transmembrane (ACP-TM; nonraft monomer reference protein; 0.16 spots/mm
2
,
n¼
n¼
868 spots) fitted with a single log-normal function.
