Biology Reference
In-Depth Information
With these two criteria satisfied, the Ca
2
þ
concentration can be calculated from
the fluorescence from single wavelength dyes using the following equation:
¼ K
d
F F
min
Ca
2
þ
ð
Þ=F
max
F
ð
Þ
where F is the fluorescence signal from the cell/tissue; K
d
the dissociation constant
of Ca
2
þ
for the indicator (units M); F
min
the minimum fluorescence achieved when
the dye is essentially Ca
2
þ
free, which practically can be approximated by exposing
the dye to a [Ca
2
þ
], that is, 0.01K
d
of the dye; and F
max
is the fluorescence achieved
when the dye is completely Ca
2
þ
bound, which practically can be achieved with
[Ca
2
þ
] of 100K
d
of the dye. Measurements of these constants with some degree of
precision inside a cell are, however, di
cult. The dissociation constant can be
measured outside the cell in solutions approximating the intracellular mileau, but it
has been a frequent observation that the value of the K
d
is altered by the intracel-
lular environment in a way that is di
Y
cult to mimic by solution chemistry, for
example, mimicking intracellular viscosity and the range of negatively charged
intracellular proteins (
Poenie, 1990
). Therefore, the best practice is to measure the
dissociation constant within the cell type of interest. The easiest way to achieve this
is by using a glass microelectrode to gain access to the intracellular space. The use
of a series of solutions with a high concentration of Ca
2
þ
bu
Y
er (EGTA or
BAPTA) with specific [Ca
2
þ
] can be used to make a series of single cell measure-
ments to allow estimation of K
d
. However, it is important to note that this
technique cannot be applied to multicellular preparations where multiple cells in
a tissue are di
V
V
erentially loaded with the dye.
XVII. Estimation of
F
max
Values
This should be measured on a cell-to-cell basis even within multicellular prepara-
tions and involves exposing the inside of the cell to
50
m
MorhigherCa
2
þ
, depend-
nity of the dye for Ca
2
þ
. These levels are generally toxic to cells, but if
tolerated for a short time (1-2 s), this may be su
ing on the a
Y
cient to estimate F
max
.These
intracellular [Ca
2
þ
] levels can be achieved rapidly within single cells by perfusion
with a Ca
2
þ
ionophore and raised extracellular Ca
2
þ
(
Loughrey et al.,2003
).
A second ingenious method used in single voltage clamp experiments is to use an
amphotericin-containing patch pipette that facilitates monovalent cation exchange
across the membrane within the patch and therefore allows low resistance access to
the cell. At the end of the experiment, themembrane is ruptured under the patch using
a rapid pressure step and the resultant influx of Ca
2
þ
from the patch pipette generates
a rapid rise of intracellular [Ca
2
þ
] that can be used to assess F
max
(
Diaz et al.,2001
).
A simpler but less reliable method is to simply use the microelectrode to penetrate the
cell and allow extracellular Ca
2
þ
influx in order to record F
max
, but generally Ca
2
þ
influx occurs in parallel with a rapid loss of intracellular dye so the signals would have
to be interpreted with caution.
Y
