Biology Reference
In-Depth Information
Table II
CaSMs based on ionophore ETH1001 possess short response times in
physiological saline over a range of [Ca
2
þ
]
Response times (t
95%
ms)
0.1-1 mM
1-10 mM
10-1 mM
1-0.1 mM
48
7
53
10
58
9
81
10
Physiological saline consists of (in micromolar) 120 NaCl, 5 KCl, 2 MgCl
2
,
10 HEPES with the CaCl
2
concentration listed above. CaSMs remained stationary
during the experiment, while three adjacent streams of media (1 mL/min) were rapidly
positioned (
<
8 ms) in front of the measuring electrode. These measurements describe
the response time of the entire measuring system for 4 CaSMs.
10
6
G
O
are typically used to accommodate ion-selective microelectrodes that
have high resistances, 1-20 G
O
(
Ammann, 1986
). Even with the best electronics,
the time constant (RC, resistance times capacitance) of the CaSM itself imposes a
low pass filter. Resistance is primarily dependent on the tip diameter and length of
the column of liquid membrane and capacitance is primarily dependent on the
thickness and dielectric constant of the wall of the glass micropipette. To reduce
the resistance of the CaSMs, they are fabricated with relatively large tips of 2-3
m
m
inner diameter and with short columns of liquid membrane
30
m
m. The short
columns are achieved by tip loading the liquid membrane as discussed above.
Capacitance can be lowered by using thicker walled borosilicate glass (1.5 mm O.
D. 0.84 mm I.D. cat# 1B150-6, WPI Sarasota, FL). The construction design listed
above has produced CaSMs with response times shorter than 100 ms,
Table II
.
In practice, a slight deviation from the expected length does not change the
response time very much, at least when considering the use of these electrodes in
the self-referencing application.
IV. Self-referencing of CaSMs
V
A. Di
erential Concentration Measurement
Self-referencing of CaSMs was developed to measure extracellular Ca
2
þ
gradi-
ents/currents that may have existed near previously characterized extracellular
voltage gradients (
K¨htreiber and Ja
V
e, 1990
). For example, relatively steady
ux of Ca
2
þ
across the plasma membrane gives rise to a gradient of Ca
2
þ
with
a higher concentration near the cell. Self-referencing of CaSMs is implemented by
measuring the [Ca
2
þ
] at two points in that Ca
2
þ
gradient. The electrical variation
of a single CaSM due to thermal noise,
e
Z
100-200
m
V, of the high-impedance
sensors and chemical drift is too large to enable measurement of such small
di
erences in extracellular Ca
2
þ
. As a result, a frequency sensitive method of
V
