Biology Reference
In-Depth Information
ciency. Thus, the presence of Ca
2
þ
creates FRET by the
cameleon, which may be readily detected. Moreover, because Ca
2
þ
a
with much greater e
Y
nity can be
tuned by incorporating mutations into the calmodulin protein, cameleons may
detect free Ca
2
þ
concentrations in the range 10 nM-10 mM, and this has been
done to visualize local Ca
2
þ
signals in the nucleus, sarco/endoplasmic reticulum,
and the cytosol, by transfecting chimeras of the cameleon that also have the
appropriate localization signals encoded in the complementary DNA (
Miyawaki
et al., 1997
). A typical example of a cameleon used for FRET imaging of Ca
2
þ
has
an excitation spectrum peak at 442 nm for the donor (blue mutant of GFP), with
the associated emission peaking at 486 nm. This wavelength FRET-excites the
GFP on the acceptor side, which then emits longer wavelength fluorescence with a
peak at 530 nm (
Fig. 4
).
Several protocols for detecting and measuring FRET e
Y
ciency have been
developed, of which some are more applicable to imaging of Ca
2
þ
signals than
others. These include measuring donor quenching, that is, measuring the decrease
in the emission from the donor fluorophore, which appears because some of the
energy emitted by the donor fluorophore is used to excite the acceptor chromo-
phore/fluorophore. This is done by taking the ratio between donor and acceptor
fluorescence during FRET as the numerator, and the same ratio in the absence of
FRET (i.e., by removing either of the donor or acceptor fluorophores) as the
denominator. Like all ratiometric quantifications, this has the advantage that the
measure becomes independent of local variations in fluorescence. The disadvan-
tage is that both the donor and acceptor fluorophores may be quenched by other
factors that would misrepresent the results. Another method for measuring FRET
e
Y
ciency is by measuring donor quenching and acceptor photobleaching, that is,
the intensity of the donor emission in the presence of an acceptor relative to the
intensity of the donor emission in the absence of an acceptor. In practice, the
latter is done by first photobleaching the acceptor by illuminating it with light at
the peak excitation spectrum of the acceptor fluorophore before measuring the
donor emission. This protocol relies on the fact that fluorescing itself causes the
fluorophores to lose their ability to fluoresce, a process called photobleaching.
However, because photobleaching may take many minutes to achieve (up to
Y
20 min), this may become less available in live specimens. Finally, FRET
combined with fluorescence lifetime imaging (FLIM) has introduced a robust
option for Ca
2
þ
measurements, because it largely is una
ected by experimental
conditions such as fluorophore concentrations and excitation intensities. In this
combined FRET-FLIM approach, the change in donor lifetime is measured in
the presence and absence of an acceptor. The principle behind this is that the
period of time the donor will fluoresce (i.e., the lifetime of the donor) depends on
the presence or absence of an acceptor (
Levitt et al., 2009
). As described above,
the measurement of donor emission in the absence of an acceptor may be done by
first photobleaching the acceptor. However, once the photobleached control
images have been captured, this protocol allows for detailed imaging of Ca
2
þ
signals over a short time period.
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