Biology Reference
In-Depth Information
Fluo-3, Fluo-4, and Oregon Green all show dramatic increase in fluorescence
upon binding to Ca 2 þ , while Fura-Red fluorescence decreases upon Ca 2 þ binding.
The optical arrangement required for these dyes is the simplest, that is, single
excitation and emission wavelengths.
Spectral shift:Inthiscase,Ca 2 þ binding causes a shift in either the excitation
or emission wavelengths. Fura-2 and related dyes show a shift in the excitation
spectrum of the dye with minimal changes in the emission ( Fig. 7 B). In contrast,
Indo-1 and related dyes show large shifts in the emission spectrum of the dye
with minimal changes in the excitation spectrum. This spectral shift is used to
allow ratiometric measurements. In the case of Fura-based dyes, while exciting
at
340 nm, a rise of Ca 2 þ will cause an increase in fluorescence, whereas while
exciting at 380 nm causes a decrease. The ratio of fluorescence at these two
wavelengths is a unique function of the [Ca 2 þ ] and is independent of the
concentration of the dye. This is particularly useful when measuring cells that
move and thereby alter the amount of dye within the light path, or comparing
cell compartments with di
erent dye concentrations (e.g., nucleus versus the
cytosol). However, despite all these advantages, the Fura- and Indo-based dyes
are rarely used in confocal microscopy because excitation wavelengths are
short (
V
400 nm) and therefore not convenient for commonly available lasers.
The shortest wavelength routinely available in commercial systems is 405 nm,
which can be used to excite Fura-based dyes close to the 380 nm excitation
maximal. In this mode, Fura acts as an inverse indicator, a property that has
some value in single-photon confocal and 2P imaging ( Ogden et al.,1995;
Wokosin et al., 2004 ).
Fluorescence lifetime: In this case, the decay of fluorescence at the end of a pulse
of excitation light will take a finite time (ns) to decay. The time course of decay is
a
<
ected by the binding of Ca 2 þ ( Fig. 7 C). In the case of Fluo-3, the decay of
fluorescence or lifetime is shorter for the Ca 2 þ bound form ( Sanders et al., 1995 ).
Since the lifetime is independent of the concentration of the dye, the relative
population of bound and free dye can easily be calculated from this technique.
However, the nontrivial analysis of emission decay required to obtain lifetime data
has minimized the use of this technique.
FRET e Y ciency: In this case, a donor molecule absorbs photons and can transfer
the associated energy to a close by acceptor molecule via a nonradiative process that
operates at distances less than the wavelength of light (up to 10 nm). The closely
adjacent molecule accepts the energy transfer (thus excites) and then emits light at a
distinct wavelength ( Fig. 7 D). The most popular pairs of donor and acceptor
molecules are cyan fluorescent proteins (donor) and yellow fluorescent proteins
(acceptor). The e
V
ciency of the energy transfer depends on the proximity of the
two molecules; within 10 nm, the e
Y
ciency is high but this drops dramatically as
the distance between the two molecules increases (proportional to 1/(distance) 6 ).
This mechanism can be used to detect Ca 2 þ binding to either construct since the
change in tertiary structure will alter FRET e
Y
Y
ciency and therefore altered
FRET signal.
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