Biology Reference
In-Depth Information
[Ca
2
+
]
free
Free
[Ca
2+
]
3.0
Buffer
curve
(
M)
m
F2
1.5
F1
[Ca
2
+
]
total
(
m
M)
300
150
0
0.5
1.0
Time (s)
1.0
0.5
T1
T2
0
Total [Ca
2
+
]
Fig. 9
Illustration of the conversion of increments in total cellular Ca
2
þ
to free Ca
2
þ
signal using the
cellular buffer power. Increase of the total cellular Ca
2
þ
by equivalent amounts (T1 & T2) causes a rise
of free [Ca
2
þ
] of F1 and F2 due to differences in the background [Ca
2
þ
]. Cellular Ca
2
þ
buffer is
illustrated by the relationship between total cellular Ca
2
þ
and free Ca
2
þ
. Note that while the amplitude
of the transient increase in free [Ca
2
þ
] depends on the increase in total Ca
2
þ
and the cellular buffer
power, the time course of the decrease will depend on the extent of activation of cellular Ca
2
þ
pumps
and exchangers. Generally, the rate of these processes depends on the free [Ca
2
þ
], therefore the decay of
the Ca
2
þ
transients of different amplitudes may differ substantially.
XVI. Calibration of Single Wavelength Dyes
Calibration of single wavelength dyes is based on two main assumptions: firstly,
that the dye is at equilibrium with intracellular Ca
2
þ
, that is, the kinetics of the
change of intracellular Ca
2
þ
are slow compared to the rate constants for associa-
tion and dissociation. For the commonly used Fluo-type dyes at an intracellular
concentration of
<
100
m
M, the half time for association and dissociation are of the
order of
1 ms and therefore for most circumstances the equilibration assumption
holds. The second assumption is that all the dye molecules sense the same amount
of light, that is, there is no filtering intrinsic to the biological preparation or by the
dye itself. Typically, the absorption coe
cient of Ca
2
þ
indicators is of the order of
50,000 M
1
cm
1
, therefore significant absorbance (
Y
>
0.05) would occur in a cell
10
m
m thick containing
100
m
M dye. This consideration is important for single-
photon excitation and is one of the constraints that limit single-photon imaging to
thin (
>
<
50
m
m) specimens.
