Biology Reference
In-Depth Information
R
¼
F
340
/F
380
and is shown in
Fig. 11
B. R
min
is the limiting value of R that is
reached during Ca
2
þ
deprivation, whereas R
max
is the limiting value of R reached
after treatment with ionophore at high [Ca
2
þ
].
24
The factor s
f,2
/s
b,2
is essentially
(F
0
f,380
BG
380
)/(F
0
b,380
BG
380
). Using these experimentally derived parameters
and a predetermined K
d
(224 nM;
Grynkiewicz et al.,1985
)in
Eq. (2)
, one can
convert the F
340
/F
380
ratio trace into a plot of [Ca
2
þ
]
i
as a function of time
(
Fig. 11
C).
This procedure has the advantage that all spectroscopically derived parameters,
namely R
min
, R
max
, and s
f,2
/s
b,2
, that are especially sensitive to environmental
changes are determined in situ with the indicator residing in the intracellular
environment. Only the equilibrium dissociation constant is determined in vitro.
R
min
determined by Ca
2
þ
deprivation is assumed to be the true value. In view of the
ine
ectiveness of currently available ionophores at low [Ca
2
þ
], one would be
justified in concluding that true R
min
would be di
V
cult to reach
25
and that R
min
is easy to overestimate. An overestimate of R
min
results in underestimation of
[Ca
2
þ
].
Finally, it is worthwhile to examine the e
Y
ects of errors in R
min
, R
max
, and
s
f,2
/s
b,2
on the derived value of [Ca
2
þ
]. For simplicity, one assumes that errors in
the three parameters are independent. Because s
f,2
/s
b,2
is related linearly to [Ca
2
þ
]
(see
Eq. (2)
), a percentage error in s
f,2
/s
b,2
translates into the same percentage error
in [Ca
2
þ
]. Inspection of
Eq. (2)
reveals that errors in R
min
should a
V
ect primarily
low values of [Ca
2
þ
] (corresponding to R values near R
min
). Error in R
max
, on the
other hand, a
V
ects the way in which all the R values are scaled and, therefore,
should influence all derived values of [Ca
2
þ
]. These expectations are borne out by
calculation.
26
V
24
From
Fig. 11
B, the ratio values near R
max
are seen to oscillate significantly because, at saturating
[Ca
2
þ
], the fluorescence of the indicator excited at 380 nm (F
b,380
¼
F
0
b,380
BG
380
) is very weak and
cannot be determined with high precision. In forming the ratio, because F
b,380
is a small number and
occurs in the denominator, noise fluctuations in F
b,380
become magnified into large-amplitude fluctua-
tions in R
max
. Therefore, one must average a large number of points to obtain a reliable estimate of
R
max
. Alternatively, the fluorescence intensity data (both F
340
and F
380
) can be smoothed first before a
ratio is formed.
25
Rather than estimating R
min
directly from the lowest values attained in the ratio trace, curve-
fitting the portion of the ratio trace that represents the slow descent towards R
min
is also a reasonable
approach. As expected, R
min
obtained by exponential curve-fitting is somewhat lower than that
estimated directly from the ratio trace.
26
When one uses parameters similar to those for Fura-2 inREF52 cells as determined on our instrument
(R
min
¼
0.5, R
max
¼
15, and s
f,2
/s
b,2
¼
12), a 10% overestimation of R
min
leads to
19% underestimation of
[Ca
2
þ
]at50nM,
10% at 100 nM, and
2% at 500 nM. A 10% overestimation of R
max
leads to
underestimation of [Ca
2
þ
]by
9.5% at 50 nM,
10.9% at 500 nM, and
12.5% at 1
m
M. A 10% under-
estimation of R
max
results in overestimation of [Ca
2
þ
]by
11.8% at 50 nM,
14% at 500 nM, and
16.5%
at 1
m
M.
