Biology Reference
In-Depth Information
10
3
times before digitizing. Typically, this mode employs a high-pass filter with a
time constant of 10 s. RC subtract allows amplification for systems with large drift
but involves a correction factor to o
set the high-pass filter. The correction factor
will be dependent on the time constant of the high-pass filter and the period of data
acquisition. For standard conditions, a period of 3.3 s (0.3 Hz translation frequen-
cy), 40
m
m/s translation speed, 10 s time constant of the high-pass filter along with
data selection of the last 70% of the half cycle, we calculate that the signal is 7%
smaller than a square wave with similar rise time.
Automated, repetitive positioning of the CaSMs is controlled by three stepper
motors arranged in an X, Y, Z configuration with the Z plane parallel to the plane
of the stage of the microscope. Smooth linear motion is obtained by coupling each
of the stepper motors to a lead screw controlling the position of three small
translational plates connected together to form a three-dimensional positioner
(BioCurrents Research Center, Woods Hole, MA). Low voltage control of the
stepper motors prevents electrical feedback to the high-impedance headstage of the
CaSM. Positioning can be achieved over a working distance of 3-4 cm with
submicron resolution and repeatability (
Danuser, 1999
). A computer interface
enables repetitive motion and positional control with the Faraday cage closed.
V
B. Di
V
erential Concentration Determination
The relationship between the measured di
erential
ion concentration between the two poles of excursion for an ideal CaSM can be
determined using the Nernst equation.
V
erential voltage and the di
V
E
1
E
2
¼ E
O
þ S
log
C
i
ð
Þ
1
E
O
þ S
log
C
i
ð
Þ
2
DE ¼ S
log
C
i
ð
Þ
1
S
log
C
i
ð
Þ
2
DE ¼
log
C
S
1
i
ðÞ
log
C
S
2
!
i
ðÞ
C
S
1
i1
ð
2
Þ
ðÞ
C
S
2
i
ðÞ
DE ¼
log
''E
1
'', ''C
i(1)
'', and ''S
1
'' are the measured voltage, [Ca
2
þ
] and slope of the voltage-
log(C
i
) graph for the near pole of excursion. The subscript 2 labels the same
parameters for the far pole of excursion. The slow changing constant potential
contributions ''E
o
'' are reduced if not eliminated by calculating the di
V
erence
between potentials over short periods of time.
Equation (2)
enables a clear picture of the relationship of the sensitivity of
detection to the background ion concentration during measurements. For a
given [Ca
2
þ
] change due to cellular flux, the concentration in the position next to
the cell, C
i(1)
, is the sum of the background ion concentration and the concentra-
tion change generated by the source while C
i(2)
, in most cases, is close to the
background ion concentration. It is easier to generate a larger
D
E when the
background concentration of the measured ion is lower as the ratio of C
i(1)
/C
i(2)
