Biology Reference
In-Depth Information
Upper bound
∝
Q
CaIn
ε
CaIn
Δ
F
2
Δ
F
1
Lower bound
Q
In
ε
In
∝
0
0
[Ca
2+
]
Fig. 13
Indicator fluorescence intensity is a nonlinear function of [Ca
2
þ
]. At [Ca
2
þ
]
¼
0, all indica-
tor molecules in solution are in the Ca
2
þ
-free form, and indicator fluorescence is at the lower
bound. Whereas [Ca
2
þ
] can range from 0 to any arbitrarily large value, indicator fluorescence
cannot exceed an upper bound, which is reached when all indicator molecules in solution are in
the Ca
2
þ
-bound form. The relationship between fluorescence intensity and [Ca
2
þ
] is hyperbolic.
The consequence is that successive equal increments in [Ca
2
þ
] do not result in equal increments of
fluorescence intensity (compare the fluorescence increments
D
F
1
and
D
F
2
resulting from two equal
increments in [Ca
2
þ
]).
be calculated by using these parameters in
Eq. (4)
.
Figure 14
shows the relative
change in Fluo-4 fluorescence for di
erent increments in [Ca
2
þ
], up to a 10-fold
V
change ([Ca
2
þ
]/[Ca
2
þ
]
0
¼
10). Because resting [Ca
2
þ
]
i
is typically in the range 50-
100 nM, the starting [Ca
2
þ
] was assumed to be [Ca
2
þ
]
0
¼
75 nM for the calculation.
Figure 14
shows clearly that the relative change in fluorescence is never a good
measure of the true relative change in [Ca
2
þ
]. F/F
0
significantly underestimates
[Ca
2
þ
]/[Ca
2
þ
]
0
, and the error increases severely as the change in [Ca
2
þ
] becomes
larger.
VII. Measuring [Ca
2
þ
] in Mitochondria
As mentioned in
Section II
, when cells are incubated with the AM ester of
Rhod-2, the indicator preferentially loads into mitochondria. The structures of
two fluorescent dyes, TMRM and TMRE, which also accumulate preferentially
into mitochondria, and Rhod-2 AM are shown in
Fig. 15
A. The positively charged
structure in these molecules that enables preferential loading into mitochondria
is highlighted with thick lines in
Fig. 15
A.
Figure 15
B shows that, rather than
