Biomedical Engineering Reference
In-Depth Information
As shown in fi gure panels 1B and 1C, the absorption spectra “shift” when [Ca
2+
]
i
changes. These more complicated response characteristics enable an internally cali-
brated measurement of [Ca
2+
]
i
that circumvents the problems listed in the preceding
paragraph for singlemetric dyes. To achieve this independence from artifact, the
experimentor must record a shift in the absorption spectrum of the indicator and
thus must record two independent measurements of the indicator's fl uorescence for
each determination of [Ca
2+
]
i
.
To illustrate, consider Fura-2, which is one of the most popular ratiometric Ca
2+
indicators. Figure
5.1b
shows the excitation spectra of Fura-2 in the presence of dif-
ferent internal calcium concentrations. Note that these plots are very different from
those shown in Fig.
5.1a
: in panel a, each plot shows the intensity of light emitted
by the dye as a function of frequency, caused by illumination of the dye by light at
488 nm. In panel b, each curve shows the excitation spectrum, not the emission
spectrum: each curve plots the amplitude of fl uorescence emitted by the indicator at
a wavelength of 510 nm for a range of different excitation wavelengths between 250
and 450 nm. Each blue curve is the excitation spectrum at a different [Ca
2+
]
i
. During
an experiment using Fura-2, the experimentor monitors the dye's fl uorescence emit-
ted at 510 nm in response to sequential illumination with light at two different wave-
lengths. The amplitude of the 510 nm fl uorescence excited by 340 nm light increases
with increasing [Ca
2+
]
i
, while the amplitude of the 510 nm fl uorescence excited by
380 nm light decreases with increasing [Ca
2+
]
i
(Fig.
5.1b
). When two excitation
beams with 340 and 380 nm wavelengths are delivered alternatively, the ratio of
fl uorescence intensity emitted at 510 nm by each excitation beam (
F
340
/
F
380
) can
thus be used to calculate the change in [Ca
2+
]
i
.
Since the fl uorescent images of Fura-2 excited at 340 and 380 nm are not acquired
simultaneously, however, it becomes problematic to monitor neural activity at high
temporal resolution using that indicator. The data acquisition rate is limited by the
necessary exposure time at each of the two wavelengths. If a higher temporal resolu-
tion is required than can be achieved with Fura-2, another type of indicator may
solve the problem. An example of such a one-excitation/two-emission type of ratio-
metric dye, Indo-1, is illustrated in Fig.
5.1c
. This panel shows a family of emission
spectra, as in panel A: each plot shows the intensity of light emitted by Indo-1 as a
function of frequency, caused by illumination of the indicator by light at 338 nm.
Each different plot in the fi gure corresponds to a different [Ca
2+
]
i
. Note that the peak
of the fl uorescence spectrum to excitation by 338 nm light shifts from 400 to 480 nm
with increased Ca
2+
binding (Fig.
5.1c
). Thus, the ratio of the fl uorescence intensity
at 480 nm to that at 400 nm (
F
480
/
F
400
) indicates [Ca
2+
]
i
. For simultaneous measure-
ment of the fl uorescence intensities at 400 and 480 nm, the emission light is spec-
trally dispersed by a dichroic mirror, and two fl uorescent images are divided with a
special optical system (e.g., W-View System by Hamamatsu Photonics, DualView
System by PHOTOMETRICS). Two simultaneous images are acquired with two
cameras or onto the right and left halves of the imaging area of a single camera. The
elimination of time lag in the acquisition of two images enables high-speed ratio-
metric imaging. FRET-based genetic probes such as “Cameleon” are another type
of one-excitation/two-emission ratiometric indicator.
