PROTOCOL

Non-invasive measurement of bioelectric currents

with a vibrating probe
Brian Reid1, Richard Nuccitelli2 & Min Zhao1
1School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK. 2BioElectroMed Corp., 830 Southampton Ave., Suite 5100, Norfolk, Virginia
23510, USA. Correspondence should be addressed to M.Z. (m.zhao@abdn.ac.uk).

Published online 29 March 2007; doi:10.1038/nprot.2007.91

Small d.c. electrical signals have been detected in many biological systems and often serve important functions in cells and organs.
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

For example, we have recently found that they play a far more important role in directing cell migration in wound healing than
previously thought. Here, we describe the manufacture and use of a simplified ultrasensitive vibrating probe system for measuring
extracellular electrical currents. This vibrating probe is an insulated, sharpened metal wire with a small platinum-black tip
(10–30 lm), which can detect ionic currents in the lA cm
2 range in physiological saline. The probe is vibrated at about 300 Hz
by a piezoelectric bender. In the presence of an ionic current, the probe detects a voltage difference between the extremes of its
movement. The basic, low-cost system we describe is readily adaptable to most laboratories interested in measuring physiological
electric currents associated with wounds, developing embryos and other biological systems.

INTRODUCTION
Every cell in our body is surrounded by a plasma membrane that
pumps ions across itself to generate an ion concentration gradient.
These ion gradients work together with ion channels to generate a
voltage difference across the plasma membrane, typically on the
order of 70 mV, inside negative. Analogously, many tissues and
organs in our bodies are surrounded by an epithelium that also
generates a voltage difference across itself, typically 50 mV, inside
positive. Both of these voltage differences provide a built-in driving
force to move ion currents through cells and organs, and these
currents are integrally involved in all of our sensory systems.
Without them we would not be able to see, hear, taste or smell.
For excitable cells, such as neurons and muscle cells, the membrane
potential and its rapid changes (action potentials) are well known
and accepted as fundamental components of their physiology.
Patch-clamp techniques have been used to study single ion channel
activities and changes in whole cell membrane potentials. Elec-
trodes at the surface of the body have been used to detect the
electrical activity of organs such as the heart and brain, and these
signals are used routinely in both scientific research and medical
practice.
There is, however, some less well known electrical activity
associated with both cells and organs. This is the relatively steady
and long-lasting d.c. electric field, which is normally found in the
extracellular milieu. Typical examples of such fields are found at
wounds1 and within embryos during development2,3. The physio-
logical basis of such extracellular steady-state ionic currents is the
spatial separation of plasma membrane ion channels carrying
inward current from those carrying outward current, resulting in
a transcellular current flow. This has been observed in most
polarized cells4 such as the transcellular ‘‘dark’’ current in the
retinal rod5. In organs surrounded by epithelia, such currents are
typically found wherever the integrity of the epithelium has been
breached, as the transepithelial potential drives current out of the
newly formed low-resistance pathway. Although such steady-state
ionic currents are less well known than action potentials in excitable
cells, they are hardly new; they were detected flowing into the cut
end of neurons and out of skin wounds more than one and a half
centuries ago6–8. However, during the past 30 years, modern
techniques have been used to confirm and extend these earlier
findings. For example, these steady currents have been detected at
(i) amputated amphibian limbs and human finger tips9–12, (ii)
corneal and ocular lens epithelial wounds and skin wounds13–16,
(iii) damaged bone17 and (iv) transected site of spinal cord18.
Growth of microbes and wounds on plant roots are also accom-
panied by such small d.c. ionic currents19–22. Accumulating experi-
mental evidence suggests that these steady-state ionic currents may
have an important role in wound healing and regeneration14,16,23–33.
Similar steady-state ionic currents have also been measured during
development, and disruption of those currents alters normal
development34–40.
Although the physiological role and significance of this type of
electricity still needs much more detailed and well-controlled
investigation, recent experiments suggest that this signaling
mechanism may have far more significant roles than previously
appreciated. For example, we have found that electric signals of
physiological strength appear to have an overriding guidance effect
in directing cell migration in cornea and skin wound healing
models41. Detection of these physiological signals requires a very
sensitive instrument capable of measuring microamperes per cm2.
This instrument was invented 40 years ago and is called the
vibrating or self-referencing probe, because it vibrates a single
sensor between two points to measure the voltage difference
between them42. See Box 1 for a detailed description of probe
function. The original instrument used a coaxial electrode design,
in which the reference electrode was plated on the outer surface of a
gold-coated glass micropipette and the sensor electrode was plated
onto the inner conductor. The construction of this electrode was
time consuming and a substantial simplification was introduced by
Freeman et al.43 and Scheffey44 in the form of an insulated wire
probe and separate reference electrode in combination with
a differential amplifier that we use here. Helpful tutorials by
Scheffey45,46 and Robinson47 are available, and many other
improvements have appeared over the years. These include two-
dimensional versions that can detect the voltage vector in a

NATURE PROTOCOLS | VOL.2 NO.3 | 2007 | 661

 PROTOCOL

BOX 1 | HOW THE VIBRATING PROBE WORKS

The vibrating probe is used to measure the very small ionic currents near cells and tissues in physiological media. These currents generate a small
electric field as they flow through the medium. The probe’s signal-to-noise ratio is high enough to measure these small voltage differences.
The probe is an insulated, sharpened metal wire, typically platinum/iridium alloy or stainless steel, with a small platinum-black ball
electroplated to the exposed tip, and which has a relatively large surface area and capacitance. The probe is vibrated at about 300Hz between
two points in the electric field. As this capacitive probe moves between the two voltage values it will charge and discharge in a sinusoidal
pattern. This converts any potential difference between the two positions into a sinusoidal ac output whose amplitude is proportional to the
potential difference. This signal is detected by a frequency-amplifier (lock-in analyzer) ‘‘locked’’ to the vibration frequency so that signals at any
other frequency are effectively filtered out, which increases voltage sensitivity by about three orders of magnitude, as compared to measuring
the dc voltage difference of a stationary probe at two different locations. This sensitivity increase is due in part to the fact that the same probe
is used to sample the voltage at the two locations so that any noise in the probe itself is subtracted away in the final output.
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

plane48,49, as well as ion-selective self-referencing probes that can
detect transmembrane fluxes of different ion types and mole-
cules50–55. These ion-selective detectors were developed by and
are available from the BioCurrents Research Center (originally
called the National Vibrating Probe Facility) at the Marine Biolo-
gical Laboratory in Woods Hole, MA, USA. This center is also
developing a digitized vibrating probe system. Another extension of
this technique now available is based on the Kelvin probe and
vibrates the sensor in air to measure surface potentials and electric
fields in biological systems exposed to air rather than liquid,
such as human skin (see http://www.BioElectroMed.com and
http://www.kelvinprobe.com).
Here, we describe a low-cost, easy-to-use, simplified version of
the vibrating probe instrument for detecting electric currents that
can be readily built and used in the average biology laboratory, and
adapted to take measurements from a wide variety of samples. We
hope to stimulate interest in this method for exploring the electrical
nature of living things.

MATERIALS
REAGENTS
. Silver-loaded epoxy adhesive (Farnell ionacure SL65; available from many
suppliers, including RS Components) ! CAUTION Irritant; avoid contact
with eyes and skin; in case of contact, rinse immediately with water
. Acetone (Sigma) ! CAUTION Highly flammable, irritant; in case of contact
with eyes, rinse immediately with water
. Perchloric acid (Sigma) ! CAUTION Causes severe burns, heating may cause
explosion, contact with combustible material may cause fire; wear suitable
protective clothing; in case of contact with eyes, rinse immediately with
water
. Potassium dicyanoaurate (0.2% w/v KAu(CN)2 in dH2O) (Aldrich Chemical
Company) ! CAUTION Toxic by inhalation, skin contact and if swallowed;
irritant; do not breathe dust, wear gloves; in case of contact with eyes, rinse
immediately with water
. Platinic chloride (10% w/v H2PtCl6 in dH2O) (Sigma) ! CAUTION Toxic if
swallowed, causes burns, may cause sesitization by inhalation and skin
contact; do not breathe dust, wear gloves; in case of contact with eyes, rinse
immediately with water
. Lead acetate (1% w/v (C2H3O2)2Pb.3H2O in dH2O) (Sigma) ! CAUTION
Toxic by inhalation and if swallowed; do not breathe dust, wear gloves
EQUIPMENT
. Elgiloy/parylene-coated stainless-steel microelectrode, 3 inch, 7 MO (item
no. SSM33A70; World Precision Instruments; also available from Micro
Probe Inc.; http://www.microprobes.com/index.html)
. R30 Gold connectors (http://www.vectorelect.com/Catpdf/Page%2070.pdf)
. Optics: virtually any inverted, compound, upright or video zoom
microscope can be used that has at least 25 magnification, for example,
Motic microscopes; http://www.motic.com
. Variable d.c. power supply unit (ours was built in-house by Dr D.I. Gray. It
is also commercially available from many companies, such as Telonic
Instruments Limited; http://www.telonic.co.uk/htdocs/products/categories/
dcps/dcinfo.asp)
. Two-phase lock-in analyzer (EG&G model 5208; Princeton Applied
Research; http://www.princetonappliedresearch.com); virtually any
commercially available lock-in amplifier can be used, for example, model
PSDA-2, Applicable Electronics; http://www.applicableelectronics.com or
Ametek Princeton Applied Research; http://www.princetonappliedresearch.com)
. Thurlby intelligent digital multimeter (model 1905a; obtained from RS
Components)
. Calibration chamber (manufactured in-house; see below) For more accuracy in
calibration, a 3M KCl-filled glass microelectrode with a sub-micron tip (10–
20 M tip resistance) can be used as a point source of current for calibration
. Piezoelectric drive (20–1,500 Hz; model 2DPI or 2DITL (Applicable
Electronics) or model PZT-5H (Vernitron Corp.))
. Three-dimensional micro-manipulator (L.S.Starrett Co. Athol). Dissecting
microscope and manipulator are on a vibration-isolation air table
(Wentworth Laboratories Ltd). Personal computer (with digital I/O card;
PC+ card; National Instruments; http://www.ni.com)
. Pre-amplifier (model 100; Vibrating Probe Co.)
. Probe vibrator power supply (model N-802) and a constant-current
calibrator (model N-600) from the Vibrating Probe Co. m CRITICAL As the
Vibrating Probe Co. no longer exists, complete turnkey vibrating probe
systems may be obtained from Applicable Electronics Inc.
(http://www.applicableelectronics.com), called SVET (Scanning Vibrating
Electrode Technique)
EQUIPMENT SETUP
Parts and complete systems See Figure 1 for a schematic of our vibrating probe
setup. Parts and complete systems are available from the following companies:
http://www.applicableelectronics.com—computer-controlled, non-invasive,
multi-dimensional, 3D scanning microelectrode systems with fast scan speed and
video imaging. Available, turnkey systems include SVET (scanning vibrating
electrode technique), a two-dimensional vibrating probe system, SIET (scanning
ion-selective electrode technique, SPET (scanning polarographic
electrode technique), both SIET and SPET are 3D measurement, and 3D scanning
systems. Also, the newly invented DVIT (differential video imaging technique)
can image ion movement in solution in real time over an entire digitized image
area. Although recently invented, DVIT has proven to be more sensitive than
SVET when applied to metal surface corrosion analysis. All systems have spatial
resolution in the 1–10 mm range and can be used simultaneously with many other
methods of measurement, such as patch clamp, intracellular techniques, among
others. ASET-XP software http://sciencewares.com controls all Applicable
Electronics Inc. scanning systems and techniques.
http://youngerusa.com—This is the exclusive agent for marketing and service
and support for Applicable Electronics Inc. technologies in China and the
Far East.
http://www.bioelectromed.com and http://www.kptechnology.ltd.uk/—Scanning
Kelvin probes for non-contact surface potential measurements. See also
http://www.kelvinprobe.com/.

662 | VOL.2 NO.3 | 2007 | NATURE PROTOCOLS

 PROTOCOL

Calibration chamber Our calibration chamber was manufactured in-house.
Combined with the constant current calibrator, it generates a constant current
of 1.5 mA cm
2 between the two electrodes. Care is taken to insure that the probe
is in the center of the chamber and that the calibration solution has a flat
meniscus to insure a uniform voltage gradient across the chamber with no
field distortion. The chamber consists of a 9 cm plastic Petri dish lid into
which is glued a 55 mm plastic Petri dish lid (see Fig. 2a,b). Plastic strips were
glued into this (using Dow Corning 3140 RTV silicone rubber coating) to
form a watertight chamber 35 mm long by 25 mm wide. Down the long sides
on the inside of this rectangular chamber are glued stainless-steel electrodes.
A BNC (British Naval Connector) coaxial cable was soldered to the
electrodes, making one electrode a current source (+/
mA) and the other
a ground (sync); the other end of the cable was connected to the constant
current calibrator.

PROCEDURE
Probe manufacture: constructing probes
1| Cut the microelectrode 25 mm behind the tip. Remove 5 mm of parylene insulation from the cut end by scraping with a
scalpel blade. This ensures a good electrical contact. Care should be taken to avoid any damage to the fine tip, or the insulation
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

between the tip and the cut end. Hold the electrode by clamping it in a pair of small artery clamps, with their jaws sheathed in
silicon tubing to protect the insulation on the electrode.

2| Use silver-loaded epoxy resin to fill the cavity of a gold R-30 male connector pin. Insert the cut end of the electrode into
the epoxy-filled connector as far as it can go, making sure the epoxy surrounds the electrode up to the top of the connector,
with no gaps. Align the electrode as close as possible to be parallel to the long axis of the connector. Leave the probe vertical
(e.g., stuck in a lump of plasticine) for 24 h to allow the epoxy to harden. Note that MPI now supply mounted, ready-to-plate
electrodes that are 1.5 or 2.0 cm long (tip of the electrode to end of the connector).
’ PAUSE POINT Probes can be stored indefinitely in a sealed container at room temperature (exact temperature is not critical)
before electroplating.
Probe manufacture: electroplating probes
3| Before electroplating with gold, clean the tip of the probe by immersing in acetone. The probe should be connected to
the piezoelectric drive. The circuit we use to electroplate probes is shown in Figure 1a. While viewing under the inverted
microscope (60), place the probe tip into the gold plating solution (0.2% potassium dicyanoaurate). Turn on the variable d.c.
power supply unit (see below) and switch to ‘‘low’’. Apply a current of 2 nA (monitor current on the meter) for 5 min, then
increase to 20 nA until the probe tip diameter is about half
the final desired diameter (approximately 10–15 mm). Monitor a Piezoelectric
bender
tip size with an eyepiece reticule with scale lines on it (1 mm
with 100 divisions). The variable d.c. power supply unit we Test
Probe Off
use was manufactured in-house. It has a series of resistors to Plate
Ground
step-down the current to the nanoampere range required for wire
electroplating probes. Pre-amplifier

m CRITICAL STEP Gold plating is necessary only if you use Petri dish bath
stainless-steel electrodes. If platinum/iridium electrodes are
used, this step can be eliminated. 056.4 RD 7 8 9
Off Low
4 5 6
+/-

4| Rinse the probe tip in distilled water and place in plati- ** st H R 0 1 2 3 On High

nizing solution (1% platinic chloride, with 0.1% lead acetate). Digital multimeter Variable D.C.
Switch the variable d.c. power supply unit to ‘‘high’’ and apply power supply

a current of 200 nA for 5 min. Then increase the current to

500 nA until the tip diameter is approximately 80% of the b Ref.
5208 input Ch.1 Amplitude
–0.23 ref. Freq.
312
final desired diameter. Increase current to 1 mA and apply
in 0.5 s bursts until the final tip diameter is obtained Ch.1
Ch.1
Channel output Frequency HZ
(about 30–35 mm). 1 2
Vibrating probe power
Figure 1 | Probe electroplating and measuring circuits. (a) Circuitry for Two phase lock-in Signal supply
electroplating vibrating probes. Under a dissecting or inverted microscope, analyzer input A

the probe tip can be monitored visually at 60, preferably with an eyepiece Signal
reticule to measure tip size. The ‘‘bath’’ dish is mounted under the microscope Calibrator Test
output Ref. Off Pre-amplifier
and contains the appropriate electroplating solution, or dH2O to wash the
Ground Plate
probe. The pre-amplifier is switched to ‘‘plate’’ and the current (monitored on
wires
the multi-meter) adjusted with the variable d.c. power supply. (b) Circuitry Output
for calibrating and measurement with probes. The calibration chamber Lock-in + 15
output Calibration
applies a current of exactly 1.5 mA cm
2 and the polarity is switched using – 1.5 chamber
the constant current calibrator. Probe vibration frequency is adjusted with
the probe power supply, and measurement settings such as time constant Constant current To
calibrator computer
and sensitivity are set with the lock-in analyzer.

NATURE PROTOCOLS | VOL.2 NO.3 | 2007 | 663

 PROTOCOL

Figure 2 | Probe calibration. (a) Photograph of the calibration chamber with

probe and earth wires in position for calibrating. The stainless-steel electrodes
a Ground wires
Probe
c Positive Negative

are glued to two opposing inner sides of the calibration chamber. The probe
is aligned parallel to the electrodes so that the direction of vibration is
parallel to the current flowing between the electrodes. The chamber holds 643
–2
approximately 3 ml of fluid. (b) Diagram of calibration chamber showing mV 1.5 µA cm
probe mounted on piezoelectric drive and with probe tip in solution in the Calibration Electrodes
chamber baseline (zero level)
chamber. A BNC wire carries the current to the electrodes from the constant
current calibrator. The electrodes are highlighted red (see the EQUIPMENT b 10 s 100 mV 674
SETUP section for detailed description of calibration chamber). Inset: probe BNC mV
wire
showing ‘‘double image’’ of tip seen during vibration. Scale bar, 100 mm. d
(c) Calibration traces. Above: diagram showing the direction of the current
To constant Probe vibration
(yellow arrows) when positive and negative calibrations are applied in the current calibrator
calibration chamber. Probe vibration is shown diagrammatically—the probe is
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

vibrated approximately 1 tip-width, so that a ‘‘double image’’ of the probe tip

is seen, with the two images just touching. Below: typical calibration trace.
Current of 1.5 mA cm
2 in the positive direction gives an upward deflection. Probe trace returns to zero level (baseline) when current is switched off.
The reversed current direction gives a downward deflection. In this example, the so-called plus and minus calibrations, when analyzed using the computer, gave
readings of 643 and 674 mV, respectively. The size of these millivolt readings is proportional to the size of the current being measured, although their actual
value is arbitrary and depends on the setting of the input voltage range in the software and the sensitivity setting on the amplifier; for example, they could be
64.3 and 67.4 mV. Saline used for calibration had a resistivity of 800O-cm. (d) Traces showing (above) slight noise (wavy sinusoidal trace line) but fairly level
baseline and good response, and (below) unstable baseline. The probe making the top trace is usable but not the bottom one. The noisy probe may be
‘‘stripped’’, that is, the platinum ball removed and the probe replated.

5| Rinse in distilled water and store at room temperature in a sealed container (e.g., a micropipette storage jar).
’ PAUSE POINT Probes can be stored indefinitely in a sealed container at room temperature after electroplating. They can also
be left in air overnight between experimental sessions, without adverse effects, provided dust or debris do not accumulate on
the probe tip. Cleaning and re-plating of the same tip can also be done.

Probe setup: selecting the optimal vibration frequency

6| First, determine the optimum operating frequency of probe vibration by observing the probe tip in the dissecting
microscope (40 magnification) while the vibration frequency is slowly adjusted to determine a resonant frequency (maximum
excursion of probe with minimum amplitude of oscillator signal). We select an optimal frequency of 310 Hz in order to be at
least 5 Hz away from any multiple of the a.c. power line frequency (50/60 Hz).

Probe setup: selecting the optimal phase angle

7| Place the probe tip in saline solution in the calibration chamber (see EQUIPMENT SETUP and Fig. 2a,b) and vibrate at an
amplitude approximately equal to the tip diameter (a ‘‘double image’’ of the probe tip should be seen, with the two ball images
just touching; see Figs. 2b, c). Apply a d.c. current of +1.5 mA cm
2 and adjust the phase angle on the lock-in analyzer. Turn
off the current and adjust baseline, then apply current again to adjust phase angle for no response. Repeat until there is no
response with current on or off to find the zero or no response angle. Then add a 901 phase shift to this angle to give maximum
response. Figure 2c shows a typical calibration trace, showing calibration responses in both directions (+/
1.5 mA cm
2)
from the horizontal base line. Other important settings on the lock-in analyzer are as follows: sensitivity, which is typically
set to 20 mV, and time constant (response time, typically 10 s). It takes five time constants for a stable reading.

Data recording setup

8| Establish a method for data recording. The output from the lock-in analyzer (d.c. volts) can be fed into a personal
computer via a National Instruments Lab PC+ digital/analog I/O card. The data can be recorded using Strathclyde
Electrophysiology Software’s Whole Cell Electrophysiology Program (WCP Ver. 1.7b; available for download from http://
spider.science.strath.ac.uk/PhysPharm/showPage.php?pageName¼software_ses. This is a basic electrophysiology recording
software package, which is easy to use and free to download. Further information about WCP can be found in Box 2.

BOX 2 | IMPORTANT SETTINGS IN WCP (VER. 1.7B)

First press F10 to select the lab interface you are using. Then go to the Record to Disc sub-menu (F1), then the Set-up menu (F9). Set record size
to 2048. Input voltage range (IVR) depends on the size of the currents you are measuring and the conductivity of the bathing solution.
Examples of different IVR settings are shown in Table 1. Digital sampling interval (DSI) determines how quickly the WCP program scans the
incoming data. For example, a DSI of 0.5 will give one full-screen scan per second. This is appropriate for some electrophysiological methods
(e.g., recording miniature endplate potentials from muscle fibers) but for vibrating probe studies a much slower scan speed is required, so we use
a DSI of 200. With this setting, one full-screen scan takes approximately 6 min 50 s.

664 | VOL.2 NO.3 | 2007 | NATURE PROTOCOLS

 PROTOCOL

TABLE 1 | Input voltage range (IVR) software settings for different samples and bathing solutions.
Sample Bathing solution Conductance (mS) IVR Reference
Cornea Artificial tear solution 13.5 0.25–0.5 Reid et al.14
Skin Phosphate-buffered saline 11 0.5–1 Zhao et al.41
Dictyostelium Developing buffer 1.6 2.5 NA
Tadpole Marc’s modified Ringer’s 1.2 2.5 NA
Plant roots Artificial pond water 0.2 2.5–5 van West et al.22
Ocular lens Artificial tear solution 13.5 0.25–0.5 Wang et al.15
Hippocampus HBSS 13 0.25–0.5 NA
Aorta DMEM 13.5 0.25 NA
Solution conductance was measured with a Kent EIL 5003 conductivity meter (Kent Industrial Measurements Ltd, Chertsey, UK). HBSS ¼ Hanks’ balanced salt solution; DMEM ¼ Dulbecco’s modified Eagle’s medium.
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

Other electrophysiology software packages can be used, for example, Spike2 by Cambridge Electronic Design Limited, Cambridge,
UK (http://www.ced.co.uk).
Measuring with the probe: calibration
9| Vibrate the probe in saline solution or dH2O for 10–15 min to hydrate the probe before calibrating and making
measurements.
m CRITICAL STEP Hydration of the probe reduces noise and improves signal response.
10| The probe should be calibrated in the same solution in which the specimen is to be measured, and at the same temperature,
for example, artificial tear solution for cornea, Marc’s modified Ringer for tadpoles, etc. (see Table 1). See Box 3 for the theory
of probe calibration. Place the solution into the calibration chamber with the probe parallel to the long axis of the chamber
(Fig. 2) and vibrate the probe tip in the solution. As before, vibrate at an amplitude approximately equal to the tip diameter
so that a double image of the probe tip is seen (shown diagrammatically in Fig. 2c). Begin recording when a stable and level
baseline is obtained (see Fig. 2c,d), then apply ‘‘positive’’ current via the constant current calibrator. This sends a current of
+1.5 mA cm
2 through the calibration chamber, in the direction of ‘‘south to north’’ (Fig. 2c). When the trace has leveled-off,
switch off the current. The trace will fall back to baseline. Apply a reverse current of
1.5 mA cm
2, to give a downward
deflection. The voltage output from the amplifier (Fig. 2c) is in millivolts and is proportional to the size of the measured
current. The actual size in millivolts is arbitrary and depends on the sensitivity setting of the amplifier and the input voltage
range setting in the WCP software.
m CRITICAL STEP For ion substitution experiments, the probe must also be calibrated in ion-free solution. A correction factor for
data acquired in a solution different from that in which it was calibrated can be calculated if you know the resistivity of the solution.

BOX 3 | THEORY OF PROBE CALIBRATION

Generating a known current density near the probe can be done in several ways. Passing a known current between two parallel wires or plates is
one way illustrated in Fig. 2. The total current flowing is divided by the total surface area of the cross-section through which the current flows.
Another option is to pass current through a micropipette acting as a point source. As this current will flow away in all directions from this point,
the cross-sectional area is given by the surface area of a sphere with a radius, r, determined by the distance between the pipette tip and the center
of vibration of the probe. The expected current density measured by the probe when a current, I, is passed through the pipette is given by I/4pr2.
The current density in the solution is related to the solution’s resistivity and electric field present by the following equation:
J ¼ E/r ¼ V/dr where
J ¼ current density in A cm
2
E ¼ electric field in V cm
1
r ¼ resistivity of the medium in O-cm
V ¼ peak-to-peak voltage difference measured by the probe in volts
d ¼ distance between extremes of vibration of the probe in cm
Lock-in amplifier outputs usually provide the root mean square (RMS) value of the voltage measured and this must be multiplied by 2.83 to
obtain the peak-to-peak (ptp) value.
Therefore, given a calibration signal of 1.5 mA cm
2, a 30 mm vibration amplitude and a resistivity of 800 O-cm, the expected ptp voltage
difference that the probe will measure is
V ¼ Jdr ¼ 1.5 mA cm
2  0.003 cm  800 O-cm ¼ 3.6 mV.
The lock-in will report the RMS value of 3.6 mV/2.83 ¼ 1.27 mV.
The lock-in amplifies this signal by a gain that depends on the sensitivity setting selected on the front panel. The full-scale output of the lock-in
is ±10 V RMS and this corresponds to ±10 mV on the most sensitive scale (gain of 106) and ±20 mV on the scale that we most commonly used here
(gain of 500). On this 20 mV sensitivity setting, a 1.3 mV signal will read 0.65 mV. The 643 mV signal in Fig. 2 means that the pre-amplifier and
software gain combined for an additional 1,000-fold increase in signal.

NATURE PROTOCOLS | VOL.2 NO.3 | 2007 | 665

 PROTOCOL

a Measurement at corneal wounds c e Current at bovine

cornea wounds

Current (µA cm )
–2
1.5 µA cm

–2
10 s 8
6 Normal
Wound
4
N = 12
2
a b c d e f g h i j k 0
0 1 2 3 4
Distance from wound center (mm)
b Mouse cornea wound d 12
Mouse cornea wound current profile
a b c d e f g h i j k

% wound current
120

–2
Probe 8

µA cm
80
4
40
0
a b c d e f g h i j k 0
0 100 200 300
Wound edge time course (min)
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

Figure 3 | Measurement of endogenous wound electric currents in cornea. (a) Chamber for mounting and measuring eye. The eye is held in wire loops and the
probe has free access to the front of the eye. Scale bar, 2 cm. (b) Photograph (top view) of corneal wound. Red dots indicate measuring positions ‘‘a’’ to ‘‘k’’.
Probe is in measuring position ‘‘f’’. White arrows indicate wound edges. Scale bar, 500 mm. (c) Probe measurement traces (taken from WCP electrophysiology
software) from various positions across the wounded cornea in b. All were at an input voltage range of 0.5 except ‘‘a’’ and ‘‘k’’, which were 0.25. (d) Graph
showing electric current profile across wound. (e) Bovine cornea wound currents. Wounds were 2 mm in diameter, thus position 0 ¼ wound center, 1 ¼ wound
edge, and 2, 3, 4 ¼ outside wound. Time-lapse graph shows wound edge current change over 5 h following wounding. Bovine eyes were mounted in a chamber
similar to that for mouse or rat eyes but scaled-up in size.

Also, to compensate for any changes in the solution’s resistivity due to evaporation, the probe calibration should be checked at the
end of the experiment in the solution used during the experiment.
Experimental measurements
11| Mount the immobilized biological specimen to be studied in an appropriate chamber. The sample must be immobilized in
such a way that it is held firm and immobile without causing damage and to allow free access to the probe. Figures 3–6 show
examples of various samples mounted for measurement. To access different regions of the sample, for example, head and tail of
a tadpole, we have found that it is easier to rotate the whole sample chamber rather than move the sample within the chamber
or alter the probe angle.
12| With vibration turned off, use the microscope (magnification depends on sample; usually 25 or 40 magnification) to
align the probe vertically in the same plane of focus as the point on the specimen to be measured. Start vibration and move the
probe to a reference position as far away as possible from the sample. Start recording a baseline or zero level (see Fig. 2c,d).
When a stable baseline has been established, move the probe to the measuring position (see Figs. 3b and 4b,e). Note that the
probe measures current flowing in the direction of vibration only; so to measure the current entering or exiting a flat structure,
the probe is aligned parallel to the surface so that the vibration is perpendicular to it (see Figs. 3b and 4b,e). The distance
from the sample surface is usually about 50 mm. Observe the trace until it levels-off at a level above or below baseline (outward
or inward current, respectively). Move the probe back to the reference position. The trace should fall back to baseline. Normally
we save one file for each measurement position, for example, for the cornea wound profile in Figure 3, files were named
‘‘eye2a.wcp’’ through to ‘‘eye2k.wcp’’. The probe can be moved in the x, y and z directions, but cannot be quickly or easily
rotated. Therefore, it is easier to rotate the sample chamber to access different areas of the sample, for example, while
measuring across the curved surface of a cornea, the eye (or
the whole chamber) can be readily rotated horizontally about a Mouse skin wounds d Human skin wounds
its center to access the whole corneal surface. For time-lapse
experiments, where measurements are made maybe every
20 min for several hours, the probe can be left vibrating in the
bath throughout the length of the experiment, with calibra-
tions at start and end. Calibration at the end of an experiment b e
in the saline used throughout the experiment compensates for
changes due to evaporation.

Figure 4 | Measurement of endogenous wound electric currents in mouse and

human skin. (a,d) Wounds were made with a number 11 scalpel blade and c Wound current (µA cm
–2
) f Wound current (µA cm
–2
)
6 10
measured with the probe at fixed time intervals after wounding. (b,e) Probe in
4 5
measuring position near wound. Scale bars, 500 mm. (c,f) Graphs showing 2
change of wound current over time. Wound current in human skin had larger 0 0
amplitude but similar time course to mouse skin wound. Note: ethical –2 –5
0 100 200 300 400 0 100 200
permission for experiments on humans was obtained from Grampian Health
Board, UK. Time after wounding (min) Time after wounding (min)

666 | VOL.2 NO.3 | 2007 | NATURE PROTOCOLS

 PROTOCOL

Data analysis a Tadpole tail measurement

13| We use the Microsoft Windows version of WCP (WinWCP Ver.
3.6.9; also available from the website above) to analyze the
probe data. The calibration and measurement values are
compiled in a Microsoft Excel spreadsheet. The formula for
calculating the current values uses the appropriate calibration
value, for example, the value of the current is calculated using b Amputated tail d Zebrafish embryo
g f
the formula B5(1.5/32.96), where cell B5 is the size of the peak a
b e
measured in WCP and 32.96 is the size of the positive calibration Dorsal Ventral c d

trace produced when +1.5 mA cm
2 was applied in the cali- fin fin
0.05
bration chamber. Note that this current was outward, hence the a bcde

Current (µA cm )
–2
0.03
positive value; therefore, positive calibration is used (32.96). Blood
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

0.01
Spinal vessel
If there are inward currents, negative calibration is used. cord –0.01
Muscle

 TIMING –0.03

–0.05
Steps 1 and 2: manufacture—30–40 min to prepare four c Regenerative
Non-regenerative
a b c d e f g

probes, plus 24 h for epoxy to harden 0.4

Stump current (µA cm )

e

–2
Dictyostelium

Outward
current
“Mound” stage
Steps 3 and 4: electroplating—approximately 30 min per 0.2 0.4
probe 0
0.3

–2
Step 5: probe storage—months 0.2

µA cm
current
Inward
–0.2 0.1
Step 6: setting frequency—5 min
0.0
Step 7: setting phase angle—20 min –0.4
a b c d e –0.1
Step 8: software setup—5 min Center Edge

Step 9: hydration of the probe—10–15 min

Step 10: calibration—10 min
Steps 11 and 12: sample measurement—time to measure
samples depends on sample type, number of measurements,
etc.; for example, to profile a cornea with wound (six positions
of measurement), it takes about 30 min
Step 13: analysis—again, time for analysis depends on the
number of files to be analyzed; for example, the data in
Figure 3a–d took about 20 min to analyze
Figure 5 | Endogenous current measured at tadpole tail stump, zebrafish
embryo and Dictyostelium. (a) The tadpole chamber is made from a 55-mm
tissue culture dish. An insulated stainless-steel wire (thick black line) is glued
in place to hold the sample. Scale bar, 2 cm. (b) Cut end of tadpole tail
showing internal structures and positions of probe measurement (a–e). Scale
bar, 1 mm. (c) Currents measured in regenerative (stage 40) are different
from those in non-regenerative (stage 45) tails. (d) Mapping current around
zebrafish embryo. Current is outward at head, inward at tail. (e) Mound stage
of aggregating Dictyostelium discoideum amoeba has outward current at center
and inward current around edge.

? TROUBLESHOOTING
Probes
The desired properties of a probe are a combination of low noise, level baseline and good response (large signal-to-noise ratio).
The calibration trace in Figure 2c shows the response of a good probe with all these qualities. Often probes made at the same
time and appearing otherwise identical will have totally different properties. A probe with slight noise is usable, but a variable
baseline makes it difficult to measure the size of the peaks as it is essential to have a level baseline against which to measure.
For example, Figure 2d shows a trace with slight noise but a fairly level baseline (top trace), whereas the bottom trace has
a variable baseline. The probe making the top trace is usable but not the bottom one. If a probe becomes dirty, the signal
response can be reduced. Routine washing in distilled water following measurement can reduce build-up of dirt. Acetone can
also be used to remove dirt, and will not affect the probe. To thoroughly clean a probe that has accumulated significant debris,
the probe can be vibrated in 60% perchloric acid for 5 min. The probe may need re-plating after this.

Equipment
As in all electrophysiology, it is essential to electrically isolate and/or ground equipment, to minimize electronic noise in your
measurements. Although it is not necessary to use a Faraday cage because electrode access resistance is about 10–50 kO,
giving it a low Johnson noise, it is important to avoid any ground loops, which create noise. Everything should be grounded
via only one path to a single grounding point such as the equipment main power ground or local plumbing (cold water pipe).
In our system, we have an uninterruptable power supply (Smart UPS 1400; APC Ltd, W. Kingston, RI, USA) between the
main electrical supply and the three major components of equipment (lock-in analyzer, vibrating probe power supply and
pre-amplifier; see Fig. 1). The vibration-isolation table is grounded, as is the dissecting microscope and the micro-manipulator.
The shielding of the BNC wire, which carries the signal from the probe to the pre-amplifier (red wire in Fig. 1b), is grounded,
and the signal cable for the reference electrode used for the differential input should run physically close to the signal cable
from the probe. In any system like this, the grounding process is determined by the location the system is in. It is best to
check each piece of equipment to insure that they are grounded properly and do not add noise. Each unit can be powered off

NATURE PROTOCOLS | VOL.2 NO.3 | 2007 | 667

 PROTOCOL

Figure 6 | Endogenous current measured in rat hippocampus and rat ocular

lens. (a) Rat hippocampus was wounded with fine forceps. Unwounded had
a Hippocampus wound current
2.5
an outward current (red column) and wounding induced a significant inward
Rat hippocampus 1.5
current. (b) Cutting the rat lens capsule (as in cataract surgery) induces a

–2
0.5

µA cm
large inward current, which may contribute to the formation of secondary
cataract due to increased cell proliferation. (c–e) Control measurements using –0.5

inert material and fixed tissue. Vibrating probes can be prone to ‘‘reflection –1.5
artifacts’’ due to proximity of a solid surface during measurements. Here, we –2.5
0 5 10 30 60 90 120 180 210
show control data to test for such artifacts. (c) Plastic dialysis tubing cut to
Time after wounding (min)
size is used as an artificial tadpole tail. (d) Graph shows mean wound current b Rat ocular lens
and control current. (e) Dictyostelium slug and mound stage control values a b c d
4.0

–2
0.0
were obtained by fixing the tissue in methanol.

µA cm
Normal –4.0 a b c d
Eye –8.0
–12.0
to find noise sources. Too many grounds can be worse than
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

c 4.0
too few and can cause ground loops. The constant current a b d

–2
0.0

µA cm
calibrator is powered by a 9 V battery (PP9) to eliminate noise Lens
Cut –4.0 a c d
–8.0 b
from main power supply. –12.0

Control measurements c d 0.3 µA cm –2

The vibrating probe can be prone to artifacts, which can occur Control measurements 0.2
0.1
owing to stirring of the solution near a solid surface, and Tadpole tail Control: plastic 0
which can result in small currents apparently being measured wound dialysis tubing
–0.1
Wound Control

where no current is actually flowing. It is possible to test

for these small ‘‘reflection’’ anomalies by making control e Dictyostelium
Slug Slug-fixed
measurements using inert materials or fixed dead tissue. You
can also measure the same place with the lock-in phase set Mound Mound-fixed
to zero to see if it is an artifact. In a real electrical field, you
should see zero if you switch the lock-in by 901. For example,
we tested for reflection artifacts using plastic dialysis tubing as a false tadpole tail (Fig. 6c,d) and also made measurements
from fixed, dead Dictyostelium culminating mound and slug (Fig. 6e). If the control values are zero or close to zero, then we
can conclude that these artifacts do not contribute significantly to the measurements from living samples.

ANTICIPATED RESULTS
The basic vibrating probe system described here is extremely sensitive if care is taken to prepare good probes and the system is
configured to minimize electrical noise. With a properly tuned and calibrated system, one can detect currents as low as 0.1 mA
cm
2 in physiological saline. Using this basic vibrating probe system, we have successfully measured endogenous extracellular
currents in a wide variety of samples, including rat and mouse cornea and skin14, rat ocular lens15, human skin41 Xenopus
tadpole, zebrafish embryo, rat hippocampus, rat aorta, Dictyostelium slugs and plant roots22. Examples of some of these data are
shown in Figures 3–6. A step-by-step example of measurement from a cornea wound is shown in Figure 3a–d. We have also
used the probe to monitor current in applied electric field experiments.
A number of published papers containing useful techniques, hints, troubleshooting guides, etc. have been included in the
reference list43–47,56–58.

CONCLUSION
In conclusion, we describe here a basic, low-cost but highly sensitive vibrating probe system capable of non-invasively
detecting the very small bioelectric currents generated by living cells, tissues and organs.

ACKNOWLEDGMENTS We thank Neil Gow for allowing us to re-assemble
his vibrating probe system, Ann Rajnicek for technical advice and help with
Figure 6c,d and Guy Bewick for photographic assistance. Bing Song contributed to
Figures 4a–c and 5e, Christine Pullar to Figure 3e, Yao Li to Figure 6a and Noemi
Lois to Figure 6b. We thank the Wellcome Trust for continuous support (058551,
068012). We are grateful to Ken Robinson, Alan Shipley and Peter Smith for
many helpful comments on the manuscript. RN is a member of the BioCurrents
Research Center Physiology of Development and Polarity Module (NIH:NCRR
P41RR01395) and is also supported by NIH R44 GM069194. We thank expert
referees and editors for comments and suggestions that greatly improved the
manuscript.
COMPETING INTERESTS STATEMENT The authors declare that they have no
competing financial interests.
Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions
1. Barker, A.T., Jaffe, L.F. & Vanable, J.W., Jr. The glabrous epidermis of cavies
contains a powerful battery. Am. J. Physiol. 242, R358–R366 (1982).
2. Hotary, K.B. & Robinson, K.R. Endogenous electrical currents and the resultant
voltage gradients in the chick embryo. Dev. Biol. 140, 149–160 (1990).
3. Metcalf, M.E.M., Shi, R.Y. & Borgens, R.B. Endogenous ionic currents and voltages
in amphibian embryos. J. Exp. Zool. 268, 307–322 (1994).
4. Nuccitelli, R. Transcellular ion currents: signals and effectors of cell polarity. Mod.
Cell Biol. 2, 451–481 (1983).
5. Hagins, W.A., Penn, R.D. & Yoshikami, S. Dark currents and photocurrent in retinal
rods. Biophys. J. 10, 380–412 (1970).

668 | VOL.2 NO.3 | 2007 | NATURE PROTOCOLS


6. DuBois-Reymond, E. Vorläufiger Abriss einer Untersuchung uber den sogenannten
Froschstrom und die electomotorischen Fische. Ann. Phy. U. Chem. 58, 1–30
(1843).
7. Piccolino, M. Luigi Galvani and animal electricity: two centuries after the
foundation of electrophysiology. Brain Res. Bull. 46, 381–407 (1997).
8. Piccolino, M. The bicentennial of the Voltaic battery (1800–2000): the artificial
electric organ. Trends Neurosci. 23, 147–151 (2000).
9. Borgens, R.B., Vanable, J.W., Jr. & Jaffe, L.F. Bioelectricity and regeneration. I.
Initiation of frog limb regeneration by minute currents. J. Exp. Zool. 200,
403–416 (1977).
10. Borgens, R.B., Vanable, J.W., Jr. & Jaffe, L.F. Bioelectricity and regeneration: large
currents leave the stumps of regenerating newt limbs. Proc. Natl. Acad. Sci. USA
74, 4528–4532 (1977).
11. Borgens, R.B., Vanable, J.W., Jr. & Jaffe, L.F. Role of subdermal current shunts in
the failure of frogs to regenerate. J. Exp. Zool. 209, 49–56 (1979).
12. Illingworth, C.M. & Barker, A.T. Measurement of electrical currents emerging
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols
during the regeneration of amputated fingertips in children. Clin. Phys. Physiol.
Meas. 1, 87–89 (1980).
13. Chiang, M., Robinson, K.R. & Vanable, J.W., Jr. Electrical fields in the vicinity
of epithelial wounds in the isolated bovine eye. Exp. Eye Res. 54, 999–1003
(1992).
14. Reid, B., Song, B., McCaig, C.D. & Zhao, M. Wound healing in rat cornea: the role
of electric currents. FASEB J. 19, 379–386 (2005).
15. Wang, E., Reid, B., Lois, N., Forrester, J.V., McCaig, C.D. & Zhao, M. Electrical
inhibition of lens epithelial cell proliferation: an additional factor in secondary
cataract? FASEB J. 19, 842–844 (2005).
16. Robinson, K.R. & Messerli, M.A. Left/right, up/down: the role of endogenous
electrical fields as directional signals in development, repair and invasion.
BioEssays 25, 759–766 (2003).
17. Borgens, R.B. Endogenous ionic currents traverse intact and damaged bone.
Science 225, 478–482 (1984).
18. Borgens, R.B., Jaffe, L.F. & Cohen, M.J. Large and persistent electrical
currents enter the transected lamprey spinal cord. Proc. Natl. Acad. Sci. USA 77,
1209–1213 (1980).
19. Gow, N.A. & McGillivray, A.M. Ion currents, electrical fields and the polarized
growth of fungal hyphae. in Ionic Currents in Development (ed. Nuccitelli, R.)
81–88 (Alan R. Liss, New York, 1986).
20. Gow, N.A. Circulating ionic currents in micro-organisms. Adv. Microb. Physiol. 30,
89–123 (1989).
21. Weisenseel, M.H. & Meyer, A.J. Bioelectricity, gravity and plants. Planta 203
(suppl. 1): S98–S106 (1997).
22. van West, P. et al. Oomycete plant pathogens use electric fields to target roots.
Mol. Plant Microbe Interact. 15, 790–798 (2002).
23. Borgens, R.B., Roederer, E. & Cohen, M.J. Enhanced spinal cord regeneration in
lamprey by applied electric fields. Science 213, 611–617 (1981).
24. Borgens, R.B., Blight, A.R. & McGinnis, M.E. Behavioral recovery induced by
applied electric fields after spinal cord hemisection in guinea pig. Science 238,
366–369 (1987).
25. Borgens, R.B. Are limb development and limb regeneration both initiated
by an integumentary wounding? A hypothesis. Differentiation 28, 87–93
(1984).
26. Jaffe, L.F. & Vanable, J.W., Jr. Electric fields and wound healing. Clin. Dermatol. 2,
34–44 (1984).
27. Lee, R.C., Canaday, D.J. & Doong, H. A review of the biophysical basis for the
clinical application of electric fields in soft-tissue repair. J. Burn Care Rehabil. 14,
319–335 (1993).
28. McCaig, C.D. & Zhao, M. Physiological electrical fields modify cell behaviour.
BioEssays 19, 819–826 (1997).
29. Altizer, A.M., Moriarty, L.J., Bell, S.M., Schreiner, C.M., Scott, W.J. & Borgens, R.B.
Endogenous electric current is associated with normal development of the
vertebrate limb. Dev. Dyn. 221, 391–401 (2001).
30. Ojingwa, J.C. & Isseroff, R.R. Electrical stimulation of wound healing. J. Invest.
Dermatol. 121, 1–12 (2003).
31. Nuccitelli, R. Endogenous electric fields in embryos during development,
regeneration and wound healing. Radiat. Prot. Dosimetry 106, 375–383
(2003).
32. Nuccitelli, R. A role for endogenous electric fields in wound healing. Curr. Top. Dev.
Biol. 58, 1–26 (2003).
33. McCaig, C.D., Rajnicek, A.M., Song, B. & Zhao, M. Controlling cell behaviour
electrically: current views and future potential. Physiol. Rev. 85, 943–978
(2005).
PROTOCOL
34. Shi, R. & Borgens, R.B. Embryonic neuroepithelial sodium transport, the resulting
physiological potential, and cranial development. Dev. Biol. 165, 105–116
(1994).
35. Shi, R. & Borgens, R.B. Three-dimensional gradients of voltage during
development of the nervous system as invisible coordinates for the establishment
of embryonic pattern. Dev. Dyn. 202, 101–114 (1995).
36. Hotary, K.B. & Robinson, K.R. Endogenous electrical currents and the resultant
voltage gradients in the chick embryo. Dev. Biol. 140, 149–160 (1990).
37. Hotary, K.B. & Robinson, K.R. Endogenous electrical currents and voltage
gradients in Xenopus embryos and the consequences of their disruption. Dev. Biol.
166, 789–800 (1994).
38. Levin, M. Bioelectromagnetics in morphogenesis. Bioelectromagnetics 24,
295–315 (2003).
39. Levin, M. The embryonic origins of left–right asymmetry. Crit. Rev. Oral Biol. Med.
15, 197–206 (2004).
40. Jaffe, L.F. & Stern, C.D. Strong electrical currents leave the primitive streak of
chick embryos. Science 206, 569–571 (1979).
41. Zhao, M. et al. Electrical signals control wound healing through
phosphatidylinositol-3-OH kinase-g and PTEN. Nature 442, 457–460 (2006).
42. Jaffe, L.F. & Nuccitelli, R. An ultrasensitive vibrating probe for measuring steady
extracellular currents. J. Cell Biol. 63 (2 Part 1): 614–628 (1974).
43. Freeman, J.A. et al. Steady growth cone currents revealed by a novel circularly
vibrating probe: a possible mechanism underlying neurite growth. J. Neurosci.
Res. 13, 257–284 (1985).
44. Scheffey, C. Two approaches to construction of vibrating probes for electrical
current measurement in solution. Rev. Sci. Instr. 59, 787–792 (1988).
45. Scheffey, C. Pitfalls of the vibrating probe technique, and what to do about them.
in Ionic Currents in Development (ed. Nuccitelli, R.) 3–12 (Alan R. Liss, New York,
1986.
46. Scheffey, C. Electric fields and the vibrating probe, for the uninitiated. in Ionic
Currents in Development (ed. Nuccitelli, R.) xxv–xxxvii (Alan R. Liss, New York,
1986).
47. Robinson, K.R. Endogenous and applied electrical currents: their measurement
and application. in Electric Fields in Vertebrate Repair (eds. Borgens, R.B.,
Robinson, K.R., Vanable, J.W., McGinnis, M.E., McCaig, C.D.) 1–25 (Alan R. Liss
Inc., New York, 1989).
48. Freeman, J.A., Manis, P.B., Samson, P.C. & Wikswo, J.P., Jr. Microprocessor
controlled two- and three-dimensional vibrating probes with video graphics:
Biological and electro-chemical applications. in Ionic Currents in Development
(ed. Nuccitelli, R.) 21–36 (Alan R. Liss, New York, 1986).
49. Hotary, K.B., Nuccitelli, R. & Robinson, K.R. A computerized 2-dimensional
vibrating probe for mapping extracellular current patterns. J. Neurosci. Methods
43, 55–67 (1992).
50. Smith, P.J.S.S., Sanger,, R.H. & Jaffe, L.F. The vibrating Ca2+ electrode: a new
technique for detecting plasma membrane regions of Ca2+ influx and efflux.
Methods Cell Biol. 40, 115–134 (1994).
51. Smith, P.J.S.S., Hammar, K., Porterfield, D.M., Sanger, R.H. & Trimarchi, J.R. Self-
referencing, non-invasive, ion selective electrode for single cell detection of
trans-plasma membrane calcium flux. Microsc. Res. Tech. 46, 398–417 (1999).
52. Marenzana, M., Shipley, A.M., Squitiero, P., Kunkel, J.B. & Ribinacci, A. Bone as an
ion exchange organ: evidence for instantaneous cell-dependent calcium efflux
from bone not due to resorption. Bone 37, 545–554 (2005).
53. Kunkel, J.G., Cordeiro, S., Xu, Y., Shipley, A.M. & Feijo, J.A. The use of non-
invasive ion-selective microelectrode techniques for the study of plant
development. in Plant Electrophysiology—Theory and Methods (ed. Volkov, A.G.)
109–137 (Springer-Verlag, Berlin/Heidelberg, 2006).
54. Messerli, M.A., Robinson, K.R. & Smith, P.J.S.S. Electrochemical sensor
applications to the study of molecular physiology and analyte flux in plants. in
Plant Electrophysiology—Theory and Methods (ed. Volkov, A.G.) Ch. 4, 73–107
(Springer-Verlag, Berlin/Heidelberg, 2006).
55. Smith, P.J.S.S., Sanger,, R.S. & Messerli, M.A. Principles, development and appli-
cations of self-referencing electrochemical microelectrodes to the determination
of fluxes at cell membranes. in Methods and New Frontiers in Neuroscience
(ed. Michael, A.C.) Ch. 18, 373–405 (CRC Press, Boca Raton, Florida, USA 2007).
56. Nuccitelli, R. Vibrating probe technique for studies of ion transport. in
Noninvasive Techniques in Cell Biology Ch. 11, 273–310 (Wiley-Liss Inc., Boca
Raton, Florida, USA 1990).
57. Nawata, T. A simple method for making a vibrating probe system. Plant Cell
Physiol. 25, 1089–1094 (1984).
58. Dorn, A. & Weisenseel, M.H. Advances in vibrating probe techniques. Protoplasma
113, 89–96 (1982).
NATURE PROTOCOLS | VOL.2 NO.3 | 2007 | 669