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We have developed a computer-controlled,
scanning microelectrode measurement system for use in conductive
solutions. Commercially available or home-made microelectrodes will
work with the system hardware and software. |
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The
SVET (Scanning Vibrating Electrode Technique) |
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The
SVET (Scanning Vibrating Electrode Technique) can measure
voltage gradients down to nV at a
minimum speed of approximately 50 ms per scan point.
Voltage gradients are not disturbed by
the probe’s vibrations, hich are
typically 200 Hz to1 kHz. The 2D vibration is accomplished by use of
piezoelectric wafers driven by sine wave oscillators. Scanning is
done with a 3D stepper motor micromanipulator (CMC-4). The SVET
system is also capable of Electrochemical Impedance Spectroscopy
measurements. We call this SLEIS (Scanning Local Electrochemical
Impedance Spectroscopy). We are currently developing this technique
to improve system capabilities.
TYPICAL SVET/SLEIS SYSTEM
Note green wire from sample (WE) to provide
connection to a potentiostat. A counter
electrode and reference electrode can be easily introduced to the
sample with this configuration. The manual 3D micromanipulator
(KITE-R) is used to position a microelectrode for calibration.
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The
SLEIS (Scanning Localized Electrochemical Impedance Spectroscopy) |
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The
SLEIS (Localized Electrochemical Impedance Spectroscopy)
can measure below the 1.0 kHz range (typically 30-100 Hz).
Essentially, one leaves the microelectrode stationary
(non-vibrating) and then drives the sample with the oscillators in
the PSD-2 amplifier, either directly, or via a
potentiostat. Another mode is available as well to allow one
axis to measure as an SVET and the other to measure
as an LEIS simultaneously while scanning
the probe over a sample under potentiostat
control. These methods of measurement provide the user with high
sensitivity and a spatial resolution limited by the electrode tip,
typically 5-50 µm diameter. The
introduction of an FRA (Frequency Range Analyzer) is the usual
method if conducting SLEIS, though the PSDA-2 amplifier can provide
frequencies in the order of 100 to 1KHz.
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The
SIET (Scanning Ion-selective Electrode Technique) |
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The
SIET (Scanning Ion-selective Electrode Technique) can
measure ion concentrations down to picomolar
levels but they must be measured slowly at around 0.5 to 1 second
per point. This is mainly due to the mechanical disturbance of the
gradient by the electrode movement, although the time constant of
the LIX (Liquid Ion Exchanger) electrodes is also a factor. It takes
a fraction of a second to reestablish the gradient again. LIX
electrodes also have time constants in tenths of seconds (LIX
dependent, see LIX specs). The electrode is stepped from one
position to another in a defined sampling routine while being
scanned with the 3D micro-stepper motor manipulator (CMC-4). Setup
is the same as above except an ion headstage
or Polarographic head stage is
substituted for the vibrator assembly and signals fed to an IPA-2
amplifier instead of the PSDA-2 amplifier. Note that SIET/SPET scans
can be alternately done with SVET/SLEIS scans with two 3D stepper
motor manipulator and 2 CMC-4 systems.
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The
SPET (Scanning Polarographic Electrode
Technique) |
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The
SPET (Scanning Polarographic
Electrode Technique) can measure dissolved oxygen gradients in
aqueous media down to a fraction of a percent of concentration. The
electrode is polarized to create a reduction reaction on the
electrode tip. The system is programmed to do automatic polarization
plots of the electrode at different voltages to determine the best
operating voltage. This system is capable of detecting less than a
0.01% change in dissolved oxygen over a 10-micron excursion
using a computer adjustable repetitive positioning algorithm as with
the SIET. Different types of polarized electrodes can be utilized as
well. Currently, Clark and Whalen type polarized electrodes are
used. Nitric Oxide and Hydrogen peroxide electrodes can also be used
with the system. Basically, any kind of polarized electrode can be
used with the system.
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Applicable Electronics, Inc. equipment
is software independent and easily
modifiable. |
Science Wares' ASET software was programmed to
utilize these components. The software is mostly hardware
independent. Together, this system is capable of automated voltage
and current measurements in aqueous media, as well as intracellular
measurements of voltage potentials and ion concentrations. |
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The microelectrode is
repeatedly moved in a programmable move-wait-measure routine (1,2 or
3D) and also grid or vector scanned or positioned with a 3D
motorized, micro-stepping
micro-manipulator. (CMC-4 system) |
Essentially, the electrode-sampling scheme is
either controlled by hardware, as in the case of the SVET or by
software as in the case of the SIET (Ion) and SPET (Polarographic)
measurements. The electrode measures over a small space (5-50 µm) in
a repeatable manner. This electrode-sampling scheme is then
positioned at different points by a scan routine from the computer.
Programmable grid and vector scans are available as well as single
point observations. For the SIET and SPET micro-gradients and
absolute concentration measurements are recorded simultaneously. The
system is also capable of multiple, independent measurements of
different phenomena either simultaneously, if permissible, or
alternately. |
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The system also works as
an ion concentration measurement system. |
For example, the 3D stepper motor manipulator
can be programmed to step in discrete, vertical increments, at
different locations in mud sediments or bio-films. More than one
microelectrode can be mounted for simultaneous measurements of
different phenomena. The IPA-2 amplifier (Ion/Polarographic-2
Channel) can also measure intra-cellular voltages or concentration
with one head while the other head stage simultaneously makes an
extra-cellular measurement. Auxiliary equipment can also be
integrated into the experimental setup, such as epithelial clamps,
patch clamps, potentiostats,
thermometers, etc. |
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The major limitation
when measuring extra-cellular micro-gradients is the time domain of
data collection. |
The SIET/SPET technique works from DC to 1.0
Hz. With computer control there is no need for continuous human
interaction unless your specimen will move or grow. Automated scan
sequences provide the investigator with an auto-scanning system. A
polarized electrode has an effect on local concentration at the
electrode tip as a function of the reduction reaction at the
electrode tip/media interface. This may be of concern with some
biological investigators. |
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The ASET software is the
heart of the system. |
This software is the culmination of over 15
years of program development. ASET is a hardware independent
platform, though it is focused on the hardware designs of Applicable
Electronics, Inc. The programming makes the system very diverse and
easily configurable. It is written for the WINDOWS-XP environment.
Data is convertible to text making it transportable to commercially
available spreadsheet and plotting programs. Video frames are also
stored and stored on disk in a bitmap format (.BMP) for
transportability to common photo programs. |
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A system for canceling the
Nernst potential or background current
on a microelectrode is done with a simple hardware/software design
to maximize the dynamic range of the A/D board. |
Utilizing the properties of differential
amplification, a hardware gateway has been designed for a D/A
computer output to provide operating electrode potential
cancellation, periodically and when needed. With this feature, the
electronic amplifiers used for SIET and SPET can be DC-coupled
without saturation of the hardware in situations where electrode
offsets, such as Nernst potentials, are
beyond the dynamic range of the system due to amplifier gain. |
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A micro-stepping motor
positioning system has also been designed. |
Advances in stepper motor technology have
allowed us to utilize low cost, commercially available units to
precisely position microelectrodes. Stepper motors have 2 major
disadvantages over linear motor positioning systems. One is that
current is applied to the motor when at rest, which produces an
electromagnetic field around the stepper motors. The other problem
is the stepping (staccato) action of the motor. Software minimizes
these effects to a point where they are not a real concern since
these are move-wait-measure techniques. Shielding prevents inductive
noise pickup from the stepper motors. The motion control system is
very versatile and can stand-alone for other micro-positioning
needs. It is factory set for 16 micro-steps with dipswitch settings.
This gives the system a mechanical movement of approximately 50
nanometers per micro-step providing sub-micron accuracy and
repeatability. |
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Video images with vector
overlays or surface plots over video images provide great
visualization of data. |
Video is extremely valuable for visualizing
the experiment. It also provides a wealth of information synchronous
with data collection. Since these techniques are non-invasive the
distance between the microelectrode and the surface of what is being
measured is critical to quantifying data. A programmable video
capture routine also provides a time lapse video recording system. |
