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during single-photon excitation were also paralleled by similar spectra at
800-950 nm during 2P excitation, but longer wavelengths failed to produce the
increased fluorescence anticipated from the single-photon spectrum. The behavior
of the Rhod-dyes (related structurally to Fluo-dyes) has yet to be examined in
detail mainly because accessing wavelengths
>
Y
cult.
The practical application of 2P excitation to excite Ca
2
þ
-sensitive dyes is limited;
the majority of applications used
1100 nm is technically di
800 nm to excite either Fluo-3 or Rhod-2.
In doing so, these studies are using the higher powers available from lasers at
these wavelengths, rather than attempting to access the higher quantum yields that
may be present at longer wavelengths. Further work is clearly required to deter-
mine the optimal 2P conditions to excite these readily available dyes at 1000-
1100 nm, or alternatively design new dyes with fluorophore structures that are
more easily excited by the 2P approach (
Kim et al., 2008
).
XV. Is It Worth Converting the Intracellular Fluorescence
Signal to [Ca
2
þ
]?
Ca
2
þ
-sensitive dyes are frequently used in conjunction with confocal microscopy
to simply indicate the timing or frequency of transient Ca
2
þ
events. Under these
circumstances, it is not considered necessary to convert the fluorescence signal into
estimates of intracellular [Ca
2
þ
]. This is avoided partly for experimental ease, since
conversion requires knowledge of the concentration of the dye and its a
nity for
Ca
2
þ
, and partly because absolute Ca
2
þ
concentrations are not considered vital to
the interpretation of the experiments. In a number of cases this may be justified,
but whenever amplitude or time course of a Ca
2
þ
transient is considered an impor-
tant variable, then calibration becomes essential for the following several reasons:
1. It is important to distinguish changes in background Ca
2
þ
from one experi-
mental scenario to the next since this determines the subsequent intracellular Ca
2
þ
bu
Y
er power. Any event that generates a Ca
2
þ
transient, for example, Ca
2
þ
influx
via plasmalemmal Ca
2
þ
channels or Ca
2
þ
release from an internal store, will
increase total intracellular Ca
2
þ
by a specific amount, but the extent to which
this increases the free cytosolic Ca
2
þ
concentration will depend on the cellular
bu
V
er power for Ca
2
þ
. This is illustrated in
Fig. 9
, where total cellular Ca
2
þ
is
increased by a standard amount; the subsequent free Ca
2
þ
transient amplitude can
be
V
10%) changes in resting [Ca
2
þ
]. There-
fore, if the basal Ca
2
þ
concentration changes, the interpretation of changes in
transient amplitude has to be made with caution.
2. Intracellular [Ca
2
þ
] levels that almost saturate the indicator cannot be used to
examine moderate changes in the amplitude of the Ca
2
þ
transient. In the absence
of information concerning the maximal Ca
2
þ
signal, it is di
40% larger simply because of small (
cult to know how close
the dye is to saturation and therefore how sensitive the signal is to changes in peak
Ca
2
þ
level.
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