Biology Reference
In-Depth Information
Therefore, Eq.
(20.3)
can be rewritten as follows:
t
1
app
¼ t
1
b1
þ t
1
b2
þ t
1
off
(20.8)
Because
t
app
is the lifetime directly determined from the histogram in single-molecule
colocalization experiments, and
t
b2
can be measured using the histograms of
the photobleaching times for individual non-colocalized spots (therefore, this measure-
ment is reliable because the photobleaching lifetimes are measured simultaneously
with the apparent colocalization durations),
t
b1
and
t
off
can be obtained as follows:
h
i
1
t
off
¼ t
1
t
1
b1
t
1
b2
(20.9)
app
In single-color experiments,
h
i
1
t
off
¼ t
1
t
1
b1
app
2
(20.10)
For example, the colocalization lifetime of Cy3-Fab-labelled CD59 was estimated to
be 151 ms from the single exponential decay fitting (see
Fig. 20.6
C). Because the
photobleaching lifetime of the Cy3 probe on Fab is 5.4 s, the true colocalization life-
time was estimated using Eq.
(20.10)
to be 160 ms.
Acknowledgments
This work was supported in part by Grants-in-Aid for Specific Research (B) (No. 24370055)
and on Innovative Areas (No. 2311002, Deciphering sugar chain-based signals regulating in-
tegrative neuronal functions) from the Ministry of Education, Culture, Sports, Science and
Technology of Japan.
References
