Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.
Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick’s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.
All animal cells are surrounded by a lipid bilayer membrane which separates the cytoplasm from the outside environment. The cell maintains an electrical membrane potential, negative inside, by active transport of ions1. The membrane potential is a stored energy source which the cell can utilize to operate various molecular devices in the membrane2. Neurons and other excitable cells have large membrane potentials. Rapid opening of sodium channels collapses the membrane potential (depolarization) and produces the action potential which is transported along the length of the neuron2. Aside from these rapid electrical changes, many tissues and organs generate and maintain significant long-term electrical potentials. For example, skin and corneal epithelia generate and maintain trans-epithelial potentials and extracellular electric currents by directional pumping of ions (mainly sodium and chloride)3.
While measurements of endogenous extracellular electric current using the vibrating probe4-6 and measurements of membrane or trans-epithelial potentials using the microelectrode system7-10 allow measurement of the electric parameters of cell membranes and epithelial cell layers, they give no indication of the ion species involved.
Microelectrodes with selective ionophore can measure specific ion concentration in solution. Ion gradients or flux could be measured with two or more electrodes at different positions. However, the intrinsic voltage drift of each probe would be different, causing inaccurate measurements or even detection of a gradient that was not present. A single electrode used in “self-referencing” mode whereby it moves at low frequency between two points solves this problem. Now the ion flux can be seen against the background of a relatively slow and stable signal drift (see Figure 3B).
The ion-sensitive measuring system uses ion-selective self-referencing microelectrodes to detect small extracellular fluxes of ions close to tissues or single cells. The system consists of an amplifier which processes the signal from the microelectrode and a micro stepper motor and driver to control the motion of the microelectrode. The ion-selective microelectrode and the reference electrode that close the circuit are connected to the amplifier via a headstage pre-amplifier (Figure 1A). Computer software determines the parameters of the microelectrode movement (frequency, distance) and also records the output of the amplifier. The stepper motor controls the microelectrode movement via a three-dimensional micropositioner. A low frequency vibrating ion-selective microelectrode was first developed in 1990 to measure specific calcium flux11. As well as calcium, commercially accessible ionophore cocktails are now available to make microelectrodes sensitive to sodium, chloride, potassium, hydrogen, magnesium, nitrate, ammonium, fluoride, lithium or mercury.
Basically, the self-referencing ion-selective microelectrode technique converts the activity of a specific ion dissolved in a solution into an electric potential, which can be measured by a voltmeter. The ionophore cocktail is an immiscible liquid (organic, lipophilic) phase with ion-exchange properties. The ionophore selectively complexes (binds) specific ions reversibly and transfers them between the aqueous solution contained in the microelectrode (electrolyte) and the aqueous solution in which the microelectrode is immersed (Figure 1D). This ion transfer leads to an electrochemical equilibrium and a variation of the electric potential between the microelectrode and the reference electrode is measured by the voltmeter. The voltage is proportional to the logarithm of the specific ion activity according to the Nernst equation allowing the calculation of the ion concentration (Figure 2A and B).
At present, several systems allow measurement of ion flux using a similar concept or principle. For example, the Scanning Ion-selective Electrode Technique (SIET)12,13 or the Microelectrode Ion Flux Estimation (MIFE) technique developed by Newman and Shabala14-16 are commercially available and widely used by the research community in order to determine specific ion fluxes occurring at cell membrane and tissue across a variety of animal, plant and single living cell models. Ion-selective microelectrodes have been used to measure hydrogen, potassium and calcium flux across plant roots17, chloride flux in rat cerebral arteries18 and in pollen tubes19, hydrogen flux in skate retinal cells20, calcium flux in mouse bone21, various ion fluxes in fungal hyphae22 and in rat cornea23, and finally calcium flux during single cell wound healing12,24. See also the following review for detailed information on ion-selective self-referencing microelectrodes25.
The following article describes in detail how to prepare and perform measurement of endogenous extracellular ion fluxes using the ion-selective self-referencing microelectrode technique at the single cell level.
1. Ion-selective Self-referencing Microelectrode Preparation
2. Ion-selective Self-referencing Microelectrode Calibration
3. Validation of the Ion-selective Microelectrode Technique
4. Preparation of Measuring Chamber
Note: Before experiments, consider the sample to be measured and how the sample is to be mounted and immobilized for microelectrode measurements.
5. Ion Flux Measurement
6. Statistical Analysis and Data Presentation
We have previously shown that calcium influx appears after single cell wounding24. We therefore asked whether other ion fluxes occur upon single cell wounding. We used the X. laevis oocyte, a well-established model for single cell wound healing30-34 and electrophysiological recording24,35-39. Interestingly, potassium ions are more concentrated inside X. laevis oocytes (about 110 mM)40 than in the extracellular solution used (in MMR 1x: 1 mM) suggesting an efflux of potassium upon wounding. In order to confirm that hypothesis, we measured the potassium flux during the course of X. laevis oocyte cell membrane healing using the ion-selective self-referencing microelectrode.
To wound the oocyte, first pull a capillary electrode with a large tip size (~50 µm). Attach the electrode to a straight electrode holder and mount on a manual micro-positioner. Wound the oocyte by touching the membrane with the electrode tip24. Soon after wounding we detected a large efflux of potassium (up to 250 nmol cm-2 sec-1; Figure 3B–D). As the membrane wound healed, this flux diminished, returning to unwounded flux values seen in the intact membrane (~5 nmol cm-2 sec-1) when the wound healed (up to 16 min after wounding; Figure 3B–D). ANCOVA revealed a significant effect of time after wounding on potassium flux measurements (p <0.001). Post hoc analyses revealed significantly increased potassium efflux at 1-2 min (p <0.001) and at 5-6 min (p <0.05), but not at 15-16 min after wounding when compared to intact cell membrane condition (Figure 3D). We concluded that upon single cell wounding, an efflux of potassium appears at the level of the wound that decrease during the course of healing.
Ion | Ionophore cocktail | Electrolyte solution (100 mM) | Reference solution (3 M) | Artificial source solution (1 M) |
Ca2+ | Calcium Ionophore I Cocktail A (cat# 21048) | CaCl2 2H2O | KCl | CaCl2 2H2O |
Na+ | Sodium Ionophore II Cocktail A (cat# 71178) | NaCl | KCl | NaCl |
Cl– | Chloride Ionophore I Cocktail A (cat# 24902) | NaCl | CH2CO2K (Potassium Acetate) | NaCl |
K+ | Potassium Ionophore I Cocktail A (cat# 60031) | KCl | NaCl | KCl |
H+ | Hydrogen Ionophore I Cocktail A (cat#95291) | pH 7.0 | KCl | pH 4.0 |
Table 1: Examples of commonly used ionophore cocktails. Also shown are appropriate solutions to place in the microelectrodes, for the artificial source and to perform calibration. Catalog numbers are from Sigma-Aldrich.
Figure 1: Ion-selective microelectrodes. (A) Schematic representation of the ion-selective self-referencing microelectrode system. (B) Ion-selective microelectrode. (C) Reference electrode. (D) Ion exchange between the external solution and the microelectrode via the ionophore. (E) Scheme of the measuring chamber used for X. laevis oocyte.
Figure 2: Ion-selective microelectrodes calibration, artificial source and flux calculation. (A) Calibration curve. (B) Equation of the calibration curve and calculation of the ion concentration. (C) Calculation of the ion flux. (D) Ion flux measured at specific distances from the artificial source (1 M KCl).
Figure 3: Evolution of the potassium flux at X. laevis oocyte wound during healing. (A) Photograph and illustration of the excursion of the ion-selective microelectrode measuring ion concentration at X. laevis oocyte wound; the dashed line between ‘‘a’’ and ‘‘v’’ represents the animal–vegetal axis. (B) Illustration of the variation of potassium ion concentration at X. laevis oocyte wound during healing. (C) Scatter (xy) plot showing the mean and the standard error of potassium flux measurement at the level of the wound at different time during X. laevis oocyte wound healing. (D) Boxplot showing potassium flux measurement at the level of the wound at different time during X. laevis oocyte wound healing (n = 16; p values indicated as follow: *: p <0.05; ***: p <0.001).
The most critical steps for successful measurement of extracellular ion fluxes in vivo are: the reduction of the noise, the correct fabrication of the ion-selective microelectrodes and reference electrode, and the positioning of the sample and both electrodes.
In order to minimize the noise, the recording system should be in an earthed (grounded) Faraday cage preferably with a metal-topped (vibration isolation) table which is also earthed. In addition, the microscope chassis should also be earthed. Sources of electric noise include the light source. A fiber-optic 'fan-less' light source causes minimal electric noise. Finally, keeping the silver wire and pellet in the microelectrode holders chlorided also minimizes noise (dip in sodium hypochlorite bleach and rinse in dH2O).The presence of air bubbles in the ion-selective microelectrode or in the reference electrode will result in measurement failure as the conductivity of the microelectrode will be nil or compromised. Thus, it is crucial to verify the electrodes under the microscope before mounting them on the holders. See the protocol for detailed procedure to remove air bubbles. The correct positioning of both the sample and the microelectrodes are required in order to ensure reliable and reproducible results. The measurement of ion flux is dependent on the excursion of the microelectrode and its position relative to the sample. It is important to identify precisely the area of interest that will be measured on the sample and position the microelectrode to have a perpendicular movement from the sample. Any excursion of the microelectrodes in a way that is not perpendicular to the sample will result in altered ion fluxes measurements and increased variability among samples.
Ionophore cocktails dedicated to measure specific ions, for example potassium, may also sense the presence of other ions, such as sodium. In the case that the measuring solution contains a high amount of a competing ion for the ionophore cocktail, it is important to determine the selectivity of the ionophore cocktail by using the artificial source experiment. Here, the solution used to culture X. laevis oocytes (MMR) contains a high concentration of sodium. Thus, it is important to assess whether the potassium ionophore cocktail used also senses sodium. By using the potassium ionophore cocktail filled microelectrode, we can attempt to measure a sodium flux using an artificial source that contains high sodium concentration (1 M NaCl; see Table 1) keeping the same measuring solution. The chemical gradient favors the efflux of sodium, but ideally no sodium flux should be detected by the potassium-specific ionophore cocktail. If a significant flux is measured, the experimental condition should be optimized. For example, the concentration of the competing ion could be lowered down to the point the microelectrode does not sense it anymore, while this may affect the sodium flux across the plasma membrane leading to potential interference during potassium flux measurements. Ideally, a correcting factor calculated from the artificial source experiment can be applied to the data, or another ionophore cocktail can be tested. Ion flux measurements using the ion-selective self-referencing microelectrode allow the measurement of ion fluxes occurring at cells and tissue in aqueous solution. Measurements of ion fluxes in cells or tissues that are normally in contact with an air environment requires the presence of a solution that is not naturally present in their environment and that can alter the ion flux and exchange that occurs under normal conditions. Specific attention has to be made to define the content of such solution and minimize the deviation from the original, physiological environment. The spectrum of ions that can be measured by the ion-selective self-referencing microelectrode technique depends on the availability and existence of specific ionophore cocktails selective to the ion of interest.
Ion flux measurements performed with the ion-selective self-referencing microelectrode are done in solution, usually near the surface of cells or tissues, allowing the non-invasive measurement of extracellular ion fluxes. This method does not allow the measurement of ion fluxes inside tissues, between cell and intercellular space. The ion-selective self-referencing microelectrode is not the only method that allows measurement of ion fluxes in vivo. An alternative new method uses fluorescent bioelectricity reporters41 which enables the measurement of ion fluxes that are not possible using microelectrodes. These dyes permit measurements of ions fluxes inside tissues and cells and can achieve subcellular localization. This technique can acquire spatial information of the ion flux inside tissues and cells but not ion exchange between the tissue and the extracellular space. Furthermore, the fluorescent bioelectricity reporters usually generate semi-quantitative data. The use of microelectrode-based technology to measure ion fluxes is still valid and necessary and brings additional information to the use of fluorescent bioelectricity reporters, making them complementary rather than competing techniques. In addition, interesting recent developments include amperometric self-referencing detectors of oxygen, nitric oxide and neurotransmitters dopamine and glutamate42,43. Amperometric sensing is based on a chemical reaction at the sensor tip. New fiber-optic microelectrodes (“optrodes”) have been developed to measure non-invasively metabolic oxygen flux34,35 and pH44 with high selectivity and sensitivity45,46. There is now also an enzyme-based nanoparticle-coated probe sensitive to glucose47.
We have seen that the ion-selective self-referencing microelectrode enables measurements of extracellular ion fluxes in vivo. Ions are not only exchanged between the cells/tissues and extracellular space but also between cells and tissues within living organisms. It is important to combine this technique with others such as fluorescent bioelectricity reporters in order to appreciate the spatial resolution of the ion fluxes inside tissues in addition to the actual measurements of the ion fluxes near its surface. In addition, ion fluxes represent an important part of the bioelectric state that defines cells and tissues together with cell membrane potential, trans-epithelia potential or extracellular electric currents. It is important, in addition to the measurement of ion fluxes, to measure, in combination, cell membrane and trans-epithelia potential as well as extracellular electric currents24.
The authors have nothing to disclose.
This work was supported by National Science Foundation grant MCB-0951199, and in part by the NIH grant EY01910, California Institute of Regenerative Medicine grants RB1-01417 and by the Fundação para a Ciência e Tecnologia (FCT) grant SFRH/BD/87256/2012.
IonAmp | BioCurrents Research Center, Woods Hole, MA, USA | none | amplifier created by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from “XBL function matters” (http://www.xuyue.org/) or from “YoungerUSA” (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/) |
IonAmp32 | BioCurrents Research Center, Woods Hole, MA, USA | none | software created by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from “XBL function matters” (http://www.xuyue.org/) or from “YoungerUSA” (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/) |
Headstage pre-amplifier | BioCurrents Research Center, Woods Hole, MA, USA | INA116 | BSR Voltage Follower INA116, designed by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from “XBL function matters” (http://www.xuyue.org/) or from “YoungerUSA” (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/) |
MicroStep Driver | BioCurrents Research Center, Woods Hole, MA, USA | none | three MicroStep drivers are required for X, Y and Z-positioning; created by the BioCurrents Research Center, Woods Hole, MA, USA; Similar system can be purchased from “XBL function matters” (http://www.xuyue.org/) or from “YoungerUSA” (http://www.youngerusa.com/) or from Applicable Electronics(http://www.applicableelectronics.com/) |
Manual micropositioner | World Precision Instruments | Model KITE-R | Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/) |
Magnetic stand | World Precision Instruments | Model M10 | Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/) |
Vibration isolation table | Newport Inc. | Model VW-3036-OPT-023040 | Similar system can be purchased from Applicable Electronics(http://www.applicableelectronics.com/) |
Part of three dimentional micropositioner: angle bracket, 90°, slotted faces | Newport Inc. | Model 360-90 | Assemblage of the three dimantionnal micropositionner requires also Three electric rotary motors for X, Y, Z control, MPH-1 mounting arm with MCA-2 adjustable-angle post and Various Newport connectors and screws to bolt onto vibration table |
Part of three dimentional micropositioner: Peg-Joining Dovetail Stage 0.5 inch X Travel | Newport Inc. | 460PD-X | none |
Part of three dimentional micropositioner: Quick-Mount Linear Stage, 0.5 inch XY Travel | Newport Inc. | 460A-XY | none |
Kwik-Fil thin walled borosilicate glass capillaries without filament | World Precision Instruments | TW150-4 | none |
Electrode puller | Narishige | PC-10 | none |
Metal rack | Made in-house | none | Metal electrode holder made in-house by drilling 2 mm wide holes half centimeter spaced in a 10cm by 15cm rectangular base of steel |
Oven | QL | Model 10 Lab Oven | none |
Silanization solution I | Sigma-Aldrich | 85126 | Hazardous, handle as recommended by provider |
Glass Petri dish; Pyrex | Fisher Scientific | 316060 | none |
Electrode/micropipette storage jar | World Precision Instruments | E215 | none |
Glass dessicator | Fisher Scientific | 08-595E | Contains Drierite dessicant (W.A. Hammond Drierite Co. Ltd, Xenia, OH, USA). Place petroleum jelly on the seal to make it airtight. |
Plastic Pasteur pipette | Fisher Scientific | 11597722 | none |
Bunsen burner | Fisher Scientific | S97329 | none |
Microscope slide | Sigma-Aldrich | S8902 | none |
Straight microelectrode holder | Warner Instruments | QSW-A15P | with a gold 1 mm male connector and Ag/AgCl wire |
Straight microelectrode holder | World Precision Instruments | MEH3S | with a AgCl(Ag+)pellet inside and a gold 2 mm male connector |
6 cm Petri dish | VWR | 60872-306 | none |
Nitex mesh | Dynamic Aqua-Supply Ltd. | NTX750 | none |
Glue; Loctite epoxy | VWR | 500043-451 | Mix glue and hardener in equal parts in a plastic weighing boat and mix thoroughly. Sets quickly but leave at RT for 24 h for full curing |
Deionized water | Sigma-Aldrich | 99053 | none |
Sodium Chloride | Sigma-Aldrich | S7653 | none |
Potassium Chloride | Sigma-Aldrich | P9333 | none |
Calcium Chloride | Sigma-Aldrich | C1016 | none |
Magnesium Chloride | Sigma-Aldrich | M8266 | none |
Hepes | Sigma-Aldrich | H3375 | none |
Sodium Hydroxyde | Sigma-Aldrich | S8045 | none |
Potassium Acetate | Sigma-Aldrich | P1190 | none |
Agarose | Sigma-Aldrich | A9539 | none |