Biomedical Engineering Reference
In-Depth Information
staining methods, and changes in the specifi c fl uorescent intensity of the dye are
measured with an imaging device such as a camera or photomultiplier mounted on
a microscope.
The primary determining factor for selection of an appropriate fl uorescent probe
for a particular experimental situation is its affi nity for Ca
2+
, which is indicated by
its dissociation constant (
K
d
). A wide variety of organic Ca
2+
probes are now avail-
able having sensitivities to [Ca
2+
]
i
which range from <50 nM to >50
M in
K
d
val-
ues. In a typical nerve cell, local [Ca
2+
]
i
can increase by a factor of between 10 and
100 in response to action potential generation. Therefore, if an indicator with very
high affi nity is used for imaging of neurons that fi re spontaneously at high fre-
quency, the indicator's fl uorescent signal could be saturated instantly and would not
monitor changes in the neural activity reliably. In such a case, an indicator with
lower affi nity would be chosen. On the other hand, if the experimental goal is to
image Ca
2+
infl ux from ligand-gated channels activated by synaptic inputs or Ca
2+
transients evoked by one or a few action potentials, then indicators with a relatively
high affi nity of about 200 nM in
K
d
value are suitable. Thus, even within a single
type of nerve cell, different indicators with different affi nities would be chosen to
image Ca
2+
signals associated with processes going on at different spatial and tem-
poral scales.
Another important consideration for the selection of an appropriate Ca
2+
indica-
tor is how the spectral characteristics of the fl uorescent dye are changed by Ca
2+
binding. The fl uorescent indicators are broadly classifi ed into singlemetric and
ratiometric dyes (Fig.
5.1
). The absorbance or fl uorescence intensity of a single-
metric dye is changed depending on [Ca
2+
]
i
without a shift in its emission spec-
trum. Typical singlemetric Ca
2+
indicators, including fl uo-3, fl uo-4, Calcium
Green, and Oregon Green 488 BAPTA, show a relative increase in the emission
fl uorescence with increase in [Ca
2+
]
i
. The behavior of fl uo-3, a popular singlemet-
ric indicator, is shown in Fig.
5.1a
. Each plot in this family of curves shows the
intensity of light emitted by the dye as a function of frequency, caused by illumina-
tion of the dye by light at 488 nm. Each different plot in the fi gure corresponds to
a different [Ca
2+
]
i
. By monitoring changes in the total amount of light emitted from
the sample within this range of 500-600 nm, the experimentor obtains a direct
measurement of changes in [Ca
2+
]
i
. Since most of the singlemetric fl uorescent dyes
are all excited by visible light, Ca
2+
imaging with these dyes requires a relatively
simple optical system and can be achieved with a standard confocal microscope.
Further, monitoring the fl uorescence at only a single wavelength enables high-
speed imaging of neural activity. However, singlemetric imaging in which [Ca
2+
]
i
change is expressed as a relative fl uorescence change with respect to the initial
value (
μ
F
/
F
0
) does not allow an effective cancellation of artifactual variation in the
fl uorescence signals resulting from photobleaching of the dye, fl uctuation of the
excitation light intensity, or movement of the sample. Therefore, in vivo Ca
2+
imaging in awake animals using singlemetric indicators is extremely problematic
and typically requires considerable ingenuity to eliminate the optical noise
(including, e.g., stabilization of the neural tissue during recording and off-line
post-processing using sophisticated software).
Δ
