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many other receptors exhibit this frequent colocalization, codiffusion, and separation
(
Hern et al., 2010; Kasai et al., 2011; Suzuki et al., 2012
).
For determining the colocalization duration, the histogram of the durations of indi-
vidual colocalization events is fitted with a single exponential decay curve, using non-
linear least-squares fitting by the Levenberg-Marquardt algorithm, provided in the
MicroCal Origin 7.5 package (
Fig. 20.6
C). The colocalized durations are then corrected
for the photobleaching lifetimes of the probes, based on the equations described in
20.3.3, thus giving
t
CCD
. The log-rank test is a useful statistical survival analysis for
examining whether distributions of colocalization lifetimes are distinguishable.
20.3.3
Colocalization lifetimes of receptor dimers in dual-color
experiments
In two-color simultaneous single-fluorescent-molecule tracking experiments that use
two fluorophores—such as pairs of Alexa488 and Alexa594, rhodamine 110 and tet-
ramethylrhodamine, or ATTO488 and ATTO594—the two full images synchro-
nously obtained in different colors are spatially corrected and overlaid (
Koyama-
Honda et al., 2005
). A predefined mask is simultaneously imaged through each path
to determine the individual path properties. This mask has a lattice of 1
m
m diameter
optical holes spaced 5
m
m apart. Using this grid, we can map a point imaged through
one of the imaging paths to the same point imaged through the other path. In this
manner, a third-order spline fit can be used to correct for translations and rotations
between the two observation arms and the optical distortions and inhomogeneities
inherent in both the optical paths and each camera. The details of this procedure have
been published previously (
Koyama-Honda et al., 2005
).
In two-color simultaneous single-fluorescent-molecule tracking experiments, the
distance between the two molecules can be measured directly from the coordinates
(
x
and
y
positions) of each molecule (as in photoactivated localization and stochastic
optical reconstruction microscopies), which can be determined independently in
each image in different colors (
Koyama-Honda et al., 2005; Suzuki, Fujiwara,
Edidin, et al., 2007; Suzuki et al., 2012
). Even when pairs of different colored mol-
ecules are known to be truly associated, the probability of scoring the two molecules
as associated is limited by the localization accuracies of each molecule and the ac-
curacies of superimposing the two images. Based on a method developed previously
(
Koyama-Honda et al., 2005; Suzuki, Fujiwara, Edidin, et al., 2007
) and the accu-
racies determined herein, we found that for truly associated molecules, the probabil-
ity of scoring the two molecules as associated increases to 99% using the criterion
that the molecules lie within 240 nm of each other. Therefore, we used this criterion
as the definition of colocalization in simultaneous two-color single-molecule obser-
vations. The distance of 240 nm coincided with the definition of colocalization in
single-color experiments. Given this coincidence, we defined the colocalization of
two fluorescent molecules as the event in which the two fluorescent spots represent-
ing these molecules become localized within 240 nm of each other.
