Biology Reference
In-Depth Information
A
B
Loading pipette
CaSM
Loading pipette
CaSM
TP
PS
Fig. 1
Ca
2
þ
-selective microelectrode tip filling station. (A) Micropositioners on each side of an
upright microscope are used to position the tips of a loading pipette and a CaSM in the field of view.
The stage has been removed. The threaded plunger (TP) syringe and the plastic syringe (PS) are
connected to the loading pipette and CaSM via plastic tubing enabling application of pressure and
suction to control the length of liquid membrane loaded into the CaSM from the loading pipette.
(B) Higher magnification of A showing the close positioning of the glass loading pipette and glass
CaSM. The system is mounted on a large metal plate to reduce vibration during loading.
also the di
usion potential between the two ends of the column of liquid mem-
brane (
Bakker et al., 1997
). The di
V
usion potential is considered negligible as bulk
movement of Ca
2
þ
across the liquid membrane does not occur during common
usage with high-impedance electronics and no current flow. The inner phase
boundary potential is considered constant due to the rigorous clamping of Ca
2
þ
with bu
V
ers or with high concentrations of Ca
2
þ
in the backfilling electrolyte. The
external phase boundary potential, for an ideal ion-selective microelectrode, is
related to the extracellular ion activity by the Nernst equation,
V
E ¼ E
O
þ S
log
a
i
ð
1
Þ
where ''E
o
'' is the sumof constant potential contributions, 'S'is theNernstian slope
¼
(2.3RT)/(z
i
F)(R, T, and F hold their standardized meanings) and ''a
i
'' is the activity
of the primary ion. Constant potential contributions are comprised of the boundary
potentials and liquid junction potentials that exist across the circuit comprising the
reference and measuring electrodes. The valence ''z
i
'' of Ca
2
þ
produces a slope only
half as steep (
29 mV/order magnitude change in Ca
2
þ
) compared to monovalent
ions. The high selectivity of the two Ca
2
þ
liquidmembranes discussed here along with
the generally standard physiological media that are used enables us to perform flux
