Biology Reference
In-Depth Information
The basis for these limits is as follows. First, indicators are small molecules whose
extinction coe
cients almost never surpass 50,000. Second, with extremely rare
exceptions, cells do not exceed 50
m
m in diameter. Third, incubation with AM
esters usually achieves intracellular indicator concentration of several tens of
micromolar, and when indicators are introduced through a whole-cell patch elec-
trode, the concentration is usually kept below 100
m
M to ensure that excessive
Ca
2
þ
bu
Y
V
ering capacity is not introduced into the cell. Applying these limits gives
2
:
303
elc <
0
:
0575
1
1, e
x
Knowing that for x
1
x, we can recast
Eq. (A2.2)
as
I ¼ I
0
1
ð
2
:
303
elc
Þ ¼ I
0
2
:
303
elcI
0
ðA2:3Þ
which rearranges to a simple expression of the amount of light absorbed by the
sample:
I
0
I ¼ I
abs
¼
2
:
303
elcI
0
ðA2:4Þ
This approximate expression is accurate to within
1% for absorbances less than
0.06 (i.e.,
e
lc
<
0.06).
After absorbing a photon, a fluorescent molecule may reemit the absorbed
quantum of energy as fluorescence. The probability that after absorbing a photon,
a molecule will emit a photon of fluorescence is known as the quantum e
Y
ciency of
fluorescence. Quantum e
ciency is usually symbolized as Q or
f
(Greek phi). The
amount of fluorescence emission should be the amount of light absorbed multi-
plied by the quantum e
Y
Y
ciency:
F ¼
2
:
303
QelcI
0
ðA2:5Þ
In this expression, only Q and
e
are intrinsic molecular properties, and these can
di
er substantially between the Ca
2
þ
-free and Ca
2
þ
-bound forms of the indicator
(symbolized as In and CaIn, respectively). For typical nonratiometric indicators,
Q
CaIn
Q
In
, while
e
CaIn
e
In
. The total concentration of indicator, C
T
, is the sum
of the concentrations of the Ca
2
þ
-bound and Ca
2
þ
-free forms:
V
C
T
¼
½
CaIn
þ
½
In
A2:6Þ
Making use of the dissociation equilibrium constant:
In
Ca
2
þ
½
CaIn
CaIn
>
Ca
2
þ
þ
In
K
d
¼
½
we can deduce the fraction of indicator that is Ca
2
þ
-bound, f
CaIn
, and the fraction
that is Ca
2
þ
free, f
In
:
Ca
2
þ
K
d
Ca
2
þ
f
CaIn
¼
þ K
d
and
f
In
¼
þ K
d
ðA2:7Þ
Ca
2
þ
