WO2006017149A2

WO2006017149A2 - Systems and methods for detecting living organisms in sediments and rocks

Info

Publication number: WO2006017149A2
Application number: PCT/US2005/024190
Authority: WO
Inventors: Yue-Feng Sun
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2004-07-09
Filing date: 2005-07-11
Publication date: 2006-02-16
Also published as: WO2006017149A3

Abstract

The embodiments are directed to systems and methods for determining in-situ microorganisms in a solid matrix including sediments and rocks. The methods include collecting data such as, effective measured complex dielectric constant, propagation and attenuation time of an electromagnetic wave and porosity, using a multi-frequency impedance spectrometer, an electromagnetic propagation device and a density device. The method further includes calculating and comparing an effective complex dielectric constant with the measured complex dielectric constant and determining a cell concentration of the micro-organisms.

Description

SYSTEMS AND METHODS FOR DETECTING LIVING ORGANISMS IN SEDIMENTS AND ROCKS

Inventor: Yuefeng Sun CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 60/586,531 filed on July 9, 2004, which is incorporated in its entirety herein by reference.

BACKGROUND

Life in general and human beings in particular could merely be transient astrological and geological phenomena. Life in various forms develops its own way to emerge from and/or adapt to ever-changing natural environments. A dormant microorganism can remain in its form over a very long time span whereas higher life forms come and go. It seems to be a fact that a singular life in any corporeal form will eventually decay and decompose. Nevertheless, collective knowledge in astrobiology and biogeoscience in centuries to come, together with advances in astrophysics and biology, could ultimately provide a better understanding of life in general and a scientific guide for the future of life on Earth.

At our present knowledge, however, one of the fundamental questions in biology is still what is life, which remains a challenging pillar as basic as what is mass, a fundamental question in physics asked by Einstein. Astrobiology roadmaps suggest that fundamental concepts of life and habitable environments will assist us in recognizing biospheres that might be quite different from our own. Recent discoveries of various life forms in extreme environments on Earth also prompt investigation on what other life forms there are and to what extent and spatial distribution these enduring microorganisms exist. It is thus necessary to develop the science and technology that could quantitatively define, model, and measure the physical signatures of life in natural environments on Earth and in space.

The discoveries of microorganisms from depths ranging from the seafloor to 800 m below the seafloor beneath 5 km of water, along with many other discoveries of equal importance, prompt one to make renewed inquiries on the origin of life. The vast extent of extreme environments in which life can initiate, evolve, and sustain compels one to inquire the physical origin of life, including its primitive origination and differentiation from the non-life and related physical conditions at which it can form. Life may not necessarily be composed of the atomic or chemical elements of which currently known life forms are composed. Life may not even be composed of the atomic elements discovered on Earth. Life may not have the DNA structure as we know now. These seemingly unscientific and unsupported negations indeed expose us to the bottom of our ignorance and could cause despair and refutation among many. These are however necessary, to have a non-Earth- centric inquiry on the origin and distribution of life in the universe, required by the increasing evidence of microorganisms discovered in extreme environments and their implications. These negations are directly related to the fundamental question in astrobiology: how to define life.

SUMMARY OF THE INVENTION

Embodiments of the invention address the basic scientific questions in astrobiology and deep biosphere studies regarding the origin, distribution, and future of life. Two of the fundamental steps to help address these questions include defining various life forms in scientific and quantitative terms and mapping the extent of these life forms in their natural habitats. The embodiments of the present invention study the geophysical signatures of living microorganisms in sediments by performing dielectric measurements of life-bearing sediment samples. The embodiments provide evidence of the feasibility that downhole bio- logging tools can provide for quantitative detection of in-situ living microorganisms in natural sediments and rocks on Earth and other planets.

More particularly, the embodiments include methods to determine signatures of living microorganisms in sediments and rocks and to obtain high resolution and accurate estimates of bacterial density and viral density in sediments and rocks, using multi-sensor dielectric probes and logging devices. The dielectric measurements are obtained in both frequency and time domains with varying waveform bandwidths up to, for example, 10 GHz. The systems of the present invention include electromagnetic propagation devices that directly result in quantitative indicators of subsurface life and its volume concentration or bio-logging.

In accordance with an embodiment, a method for determining in-situ micro¬ organisms in a solid matrix including sediments and rocks, includes the steps of collecting data indicative of an effective measured complex dielectric constant ( έ measured) using a multi-frequency impedance spectrometer; collecting data indicative of propagation time and attenuation of a sinusoidal electromagnetic wave using an electromagnetic propagation device; collecting data indicative of the porosity and effective dielectric constant of the solid matrix (ε_s ^* ); calculating a value indicative of an effective complex dielectric constant (ε*) using the effective dielectric constant of the solid matrix (£^*) and an effective dielectric constant of a fluid mix comprising micro-organisms {ε_f ^* ) determined from a theoretical model and the porosity; comparing the values of the effective complex dielectric constant (ε*) measured and calculated ε*; and determining a cell concentration of the micro¬ organisms from the value of έ_f when ε* is equal to ε* measured.

The step of collecting data indicative of the effective measured complex dielectric constant (ε* measured) includes collecting data from a plurality of electrodes at a plurality of frequencies, such as, for example, 100Hz, 500Hz, IKHz, 5KHz, 10KHz, 100KHz, IMHz, 5MHz, 10MHz and 32MHz. The method further includes collecting data indicative of a density measurement, the density measurement determining the porosity and effective dielectric constant of the solid matrix ε_s ^* . The method includes the use of lookup tables from a plurality of theoretical models for a plurality of micro-organisms to determine the cell concentration.

In accordance with another aspect of the invention, a system for determining in-situ micro-organisms in a solid matrix including sediments and rocks, includes a multi-frequency impedance spectrometer to provide data indicative of an effective measured complex dielectric constant; an electromagnetic propagation device; and a processor for processing data provided by at least the multi-frequency impedance spectrometer and the electromagnetic propagation device to generate values indicative of cell concentration of the micro-organisms. The system also includes a display device for displaying at least one of intermediary measurements, final measurements and processed values. The system also includes a memory device to store data and processing instructions. A density measurement device can also be included in the system. The multi-frequency impedance spectrometer device includes at least four electrodes, wherein at least two electrodes are current electrodes and two electrodes are voltage electrodes.

The foregoing and other features and advantages of the invention will be apparent from the following more particular description of embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of dielectric constant versus frequency for E. coli with a volume concentration of 15% in 0.25% NaCL water, seawater, and quartz mineral in accordance with an embodiment of the present invention.

FIGS. 2A-2C are graphical representations of high-resolution cm-scale estimation of gas hydrate saturation (concentration) (FIG. 2B) in natural sediments using a 1.1 GHz dielectric logging device, FIG. 2A is a plot of porosity (%) versus depth (m); and FIG. 2C is a plot of cm-scale formation microimage (FMI) image structures.

FIGS. 3A-3C illustrate a living cell, a cell with no applied electric field and a cell under applied electric field, respectively.

FIGS. 4A-4D illustrate microbes and their transport in sediments and rocks. FIG. 5 graphically illustrates a comparison of the results of theoretical modeling with experimental data on E. coli.

FIGS. 6A and 6B illustrate graphically strong effects of bacterium concentration on dielectric measurements in accordance with embodiments of the present invention.

FIGS. 7A-7D illustrate graphically the strong effect of porosity on dielectric-constant and conductivity in accordance with embodiments of the present invention.

FIGS. 8A-8C graphically illustrate model results for signature of living bacterium in sediments and porous rocks in accordance with an embodiment of the present invention.

FIG. 9 illustrates schematically a four-electrode multifrequency impedance spectrometer in accordance with an embodiment of the present invention.

FIG. 10 illustrates a flow chart of a method to collect and analyze data to determine in-situ life in sediments and rocks in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION

The embodiments of the present invention have a practical emphasis on providing a basis for downhole logging sensors or devices for remote sensing of living microorganisms under in-situ conditions in natural sediments and rocks in the deep Earth Biosphere and on other planets. The bio-logging tools/devices can be tested at, without limitation, the well- documented and well-characterized sites in extreme environments on Earth including the test sites that have been used in NASA-related research such as geothermal sites in Yellowstone National Park, high saline ponds, impact craters (for example, Haughton crater on Devon Island), low pH mine drainage locations, Death Valley, and others, as well as space mission related tests. The embodiments of the present invention are predicated on the realization that the dielectric signatures of living microorganisms in sediments can be measured and distinguished from their host environmental materials.

The existence of subsurface life in extreme environments seems to be a near certainty. Bacteria have been found within marine sediments, sedimentary rocks, glassrims of pillow basalts, and in great depths within ocean basaltic rocks. Microorganisms have been discovered from depths ranging from the seafloor to 800 m below the seafloor beneath 5 km of water. Both bacterial density and viral density are as high as 109 cells/ml near the seafloor and normally they decrease with depth. At a depth of 1 km below seafloor and 6 km below sea level, the bacterial density can still be as high as 106 cells/ml at least. Thermophilic bacteria, methanogens, and other microbes in extreme environments can have higher or less concentration, depending on the energy source available for the microbial community. Thermophilic Fe(III)-reducing and magnetic iron oxide-producing bacteria at temperatures up to 85°C and at a depth of more than 2 km below continental surface have been reported. Magnetite and Fe-sulfϊde-bearing carbonate globules formed at ~700°C along fractures and in pore spaces in Martian meteorite to infer the possible past Martian biota have also been reported. Most recently, the first-time evidence for eukaryotic, fungal fossil in carbonate- filled vesicles of massive lava flow in deep ocean crust near the Pacific Farallon Ridge axis have been reported.

From the viewpoint of philosophy and metaphysics, it seems that there is no distinction of life from non-life in the same way as there is no distinction of mass from energy. As we understand now, it is easy to turn life into non-life and to turn mass into energy. However, we have little knowledge on how to define and form life from non-life just as we do not know how to define and make mass from energy. Nevertheless as the definition of life is concerned, electromagnetic force is a common basic cause acting on all life forms that we know so far from virus and prokaryote to plant and man. And all life forms and ordinary non-life matters have definite electromagnetic signature. Furthermore, the cellular structure seems to be common to all basic life forms and celestial bodies, regardless of their content and composition. That such a structure is preferred by Nature is also because of the physical origin of either electromagnetic force or gravitational force, and optimization of geometrical arrangement. The functionality of a cellular life consists of electromagnetic operations at the atomic and molecular level by controlling largely the behavior of cell membrane. A cell death is usually accompanied by the destruction of the membrane that eliminates the cellular structure. This functionality of cellular life should remain similar for all basic life forms, although the molecules of the cell membrane and nucleus can be entirely different from one life form to another. Thus it should be a valid universal assumption that a basic unit of life has a cellular structure being acted on by electromagnetic force, as a necessity of life. Therefore, studies of the electromagnetic properties of cell-containing media, including cellular-structured media, multi-cellular organisms and microorganism- bearing porous media, should provide means to distinguish life from non-life, to differentiate life forms, and improve our ability for searching for life by measuring and classifying the dielectric signatures of matter and life.

This is the fundamental reason why impedance spectroscopy has been actively used for classifying a variety of tissues and organs for medical applications. It is Maxwell himself that initiated the field now called dielectric spectroscopy and impedance spectroscopy. Impedance measurements gave the first direct evidence of the existence of the membranes surrounding living cells and provided the first good estimate of the nanometer (nm) thickness of the membranes. Since then, the electrical properties of biological materials have been studied over the last 100 years for biomedical, medical, and public health applications. Most recently, High-transition temperature (Tc) Superconducting Quantum Interference Devices (SQUID) have been used to improve the accuracy of dielectric measurements of living cell suspensions in the frequency range of direct current (dc) to 100 kHz. However, the need for both theory and techniques to study life-bearing sediments and rocks has never been raised as a fundamental and strategic inquiry as discussed herein. Existing subsurface and downhole remote sensing methods developed for oil industry and geoscience applications are impossible for direct imaging of life in subsurface formation, including downhole nuclear magnetic resonance (NMR) or magnetic resonance imaging (MRI) and other downhole ultrasonic, electrical, and nuclear logging tools or devices.

Because of their large membrane effects on dielectric properties, living microorganisms of concentration about 1011 cells/ml have been detected in liquid cultures. The dielectric constant of bacteria and tissues changes from 107 to 100 as frequency varies from 0 Hz to 10 GHz . On the other hand, the dielectric constants of water and minerals are about 80 and 5 respectively and they are relatively frequency independent as illustrated in FIG. 1. Therefore, a key to the success of detecting subsurface living microorganisms can be to exploit their unique dispersion signature over a wide bandwidth of the electromagnetic spectrum. However, the complexity involves the existence of rock matrix and pore volume amount available for life-bearing fluid. Embodiments of the present invention include deploying a GHz dielectric tool to acquire high-resolution (cm-scale) estimates of both porosity and hydrate concentration in a research well in, for example, the Northern Canadian permafrost region.

Although at the GHz range, the difference between dielectric constant of hydrate (3.1) and that of rock matrix (5) is small, the cm-scale estimates of hydrate concentration from dielectric measurements are quite accurate compared with laboratory measurements and other downhole data as illustrated in FIGS. 2A-2C. The overall trend of the 1.1 GHz dielectric logging device estimated hydrate saturation agrees very well with the low resolution resistivity estimates. The high-resolution structures revealed by the dielectric log data are consistent with the cm-scale formation microimage (FMI) image structures. The existing dielectric devices in the GHz range so far offer little clue on the presence of microorganisms in natural environments. Nevertheless, the successful field deployment of the dielectric logging device for high-resolution gas hydrate estimation encourages the expectation that downhole dielectric logging tool at lower frequency range can be successfully used to obtain continuous records of bio-signatures, microorganism and even their concentration in natural environments. FIGS. 3A-3C illustrate a living cell and an electric model of a living cell. Maxwell's equations can be used to analyze the effects of all possible motions of a living cell in rocks on electromagnetic wave fields, which includes the rotational motion utilized by NMR and MRI. A first step includes the mathematical definition of the electrical properties of the membrane, nucleus and cell wall, and uses them to determine a signature of a living cell. FIGS. 3B and 3C illustrate a cell with no applied electrical field and a cell under applied electric field, respectively.

A second step includes identification of microbes and their transport in sediments and rocks using porosity. FIGS. 4A-4D illustrate a field scale, Darcy scale, pore scale and sub- pore/interfacial scale images of the microbes and their transport in sediments and rocks, respectively.

The following are the constitutive relations used in the methods of the embodiments to analyze data:

D = ε_QεE

ε « - ε ~-ι . <y , σ _* = ιωε_oε _* ωε₀ _* . _* ε = {\ -φ)ε_s +φε_f ... 3 wherein ε_f ^* is related to cell properties

D, E are the electric displacement field and electric field , respectively

£₀,ε,σ are the vacuum electric permittivity, complex dielectric constant, and conductivity, respectively ε ,ε, ,ε_rσ

are the effective complex dielectric constants and effective conductivity respectively, and ω,φ are the angular frequency, and porosity

Conductivity is a good indicator of porosity and dielectric constant is a good indicator of bacterium and its concentration, if the porosity is known.

The following embodiments provide an understanding of the complexity resulting from sediment structure and verify the possibility that the dielectric property can indeed be used to characterize accurately the physical signatures of microorganisms present in sediments and rocks.

Embodiments include methods to determine the dielectric signature of Escherichia coli (E. coli) in liquid cultures of various concentrations. Published results on dielectric measurements of cell suspensions are restricted to bacterial density up to about 10¹¹ cells/ml. However, estimated bacterial density in subsurface sediments and rocks usually ranges from 10⁶-10⁹ cells/ml. Thus, embodiments include methods and systems to measure the dielectric properties of cultured E. coli solution with varying density from 0.0 to 10¹¹ cells/ml in culture medium. E. coli is an appropriate bacterium because it has been used in nearly all studies on dielectric measurements of cell suspensions. These embodiments determine the lowest discernable limit of the dielectric signatures of E. coli in solution.

Alternate embodiments include methods to measure the dielectric properties of E. coli liquid cultures in glass bead sediments and as such E. coli mixed with glass beads. The E. coli density varies from 0.0 to 10ⁿ cells/ml in culture medium as in previously described embodiments. The glass beads are used instead of natural sediments because commercially available glass beads have well-defined dielectric properties. Using glass beads to simulate sediments enable accurate theoretical analysis of the measurement results by eliminating many possible unknown factors associated with natural sediments such as mineral composition. The porosity of the glass bead sediments are also measured for each embodiment. These measurements determine the lowest discernable limit of the dielectric signatures of E. coli-like bacteria in unconsolidated sediments or sedimentary rocks of intermediate porosity range.

Further embodiments include methods to determine the dielectric signatures of deep- sea bacteria in glass bead sediments with various concentrations. E. coli is replaced by isolates of deep-sea basalt samples with varying density from 0.0 to lθ" cells/ml in culture medium in these embodiments.

Measurements are taken at room temperature and pressure. Measurements can also be made of other microorganisms, natural sediments and rocks with varying salinity, and at varying temperatures and pressures to simulate possible extreme environments. Embodiments include measurements of microorganisms at varying temperatures being taken in dry sediments and low-porosity rocks deprived of water with spores of some isolates obtained from basalt.

FIG. 5 graphically illustrates a comparison of theoretical modeling results with experimental data on E. coli. The theoretical model is in very good agreement with experimental data for E. coli suspension.

FIGS. 6A and 6B graphically illustrate the strong effects of bacterium concentration on dielectric measurements in accordance with embodiments of the present invention. Dielectric constant is a good and perhaps the only unique signature of living cells in the natural environment.

FIGS. 7A-7D illustrate graphically the strong effect of porosity on dielectric constant and conductivity in accordance with an embodiment of the present invention. FIGS. 8A-8C graphically illustrate model results for signature of living bacterium in sediments and porous rocks in accordance with an embodiment of the present invention.

For accurate bio-probe and bio-logging, high-resolution porosity is needed. To detect in situ life in porous rocks a four-electrode multi-frequency impedance spectrometer can be used as illustrated in FIG. 9. The multi-electrode, multi-frequency impedance spectrometer device 460 includes in an embodiment at least four electrodes. Two electrodes serve as current electrodes 464 while two are voltage electrodes 466. The electrodes provide inputs to the electronic unit 462 of the device 460 where the data signals are converted, for example, to a digital format and further processed. A processor 468 may receive inputs from the multi- frequency impedance spectrometer device 460 as well as inputs from an electromagnetic propagation tool in the, for example, GHz frequency range (for example, 1.1 GHz) and a density tool. These inputs are processed and provide measurements indicative of the cell concentration. Intermediate and final measurements and processed values can be displayed on the display device 470. The display device may be a cathode ray tube (CRT), a liquid crystal display (LCD), or the like, for displaying information to a user. The processor 468 includes an operating system and can include of one or more processors executing machine- readable instructions obtained, at least in part, from a memory device. The memory device stores information and instructions in machine readable form for execution by the processor 468 to perform data analysis on the inputs of the multi-frequency impedance spectrometer device, the electromagnetic propagation tool/device and the density tool/device to result in a value indicative of cell concentration. The sequence of instructions can include, in an embodiment, the steps of the method illustrated in FIG. 10.

FIG. 10 illustrates a method 500 to calculate the in-situ signatures of living micro¬ organisms in sediments and rocks. The method includes the step 502 for collecting data from a plurality of electrodes in a device such as illustrated in Fig. 9 at a plurality of frequencies. The frequencies can include, without limitation, 100, 500, Ik, 5k, 10k, 100k, IM, 5M, 1OM and 32 MHz. Data is also collected from an electromagnetic propagation device, for example, in the GHz frequency range (for example, 1.1 GHz) per step 504 and per step 506 from a density device. The data collected in step 502 is processed to generate an effective measured complex dielectric constant (ε* measured) per step 508. The data collected per step 504 is processed to provide a measure of propagation time (tpl) and measured attenuation (a ) of the sinusoidal electromagnetic wave traveling from the transmitter to the receivers per step 512 and forms an input to step 514 along with the output of the density data per step 506, to determine a porosity measure (ø) in step 514. The density tool also provides a measure of the effective dielectric constant of the solid matrix ε] such as rock. A value indicative of the effective dielectric constant of a fluid mix (water and microorganisms cells) ( ε_f ^* ) is determined per a cell model such as illustrated in FIGS. 6A and 6B per step 510 f^* forms an input into step 516 which provides for the calculation of the effective complex dielectric constant (ε*) using the values ø,

, and ε_f ^* . It is then determined if the effective complex dielectric constant (ε*) is equal to ε* measured in step 518. If ε* is not equal to ε* measured the process is iteratively repeated by using values of ε_f ^* from the models.

If it is determined, per step 518, that the value of ε* is equal to ε* measured then the final value of ε_f ^* from the cell model is identified per step 520. The following step 522 provides for the values indicative of cell concentration (p) and porosity (ø) as provided by step 514. The value indicative of cell concentration is derived from a model that relates the ε_f" to the cell concentration of a particular microorganism and as such, can include lookup tables from a plurality of different models for different micro-organisms.

In view of the wide variety of embodiments to which the principles of the present invention can be applied, it should be understood that the illustrated embodiments are exemplary only, and should not be taken as limiting the scope of the present invention. For example, the steps of the flow diagrams may be taken in sequences other than those described, and more or fewer elements may be used in the diagrams. While various elements of the embodiments have been described as being implemented in software, other embodiments in hardware of firmware implementations may alternatively be used, and vice- versa.

It will be apparent to those of ordinary skill in the art that methods involved in the systems and methods for detecting living organisms in sediments and rocks may be embodied in a computer program product that includes a computer usable medium. For example, such a computer usable medium can include a readable memory device, such as, a hard drive device, a CD-ROM, a DVD-ROM, or a computer diskette, having computer readable program code segments stored thereon. The computer readable medium can also include a communications or transmission medium, such as, a bus or a communications link, either optical, wired, or wireless having program code segments carried thereon as digital or analog data signals.

All aspects, modifications, and embodiments that come within the scope and spirit of the following claims and equivalents thereto are claimed as the invention.

Claims

What is claimed is:

1. A method for determining in-situ micro-organisms in a solid matrix including sediments and rocks, comprising the steps of: collecting data indicative of an effective measured complex dielectric constant (ε^* measured) using a multi-frequency impedance spectrometer; collecting data indicative of propagation time and attenuation of a sinusoidal electromagnetic wave using an electromagnetic propagation device; collecting data indicative of the porosity and effective dielectric constant of the solid matrix (£^•*); calculating a value indicative of an effective complex dielectric constant (ε*) using the effective dielectric constant of the solid matrix ( ε^* ) and an effective dielectric constant of a fluid mix comprising micro-organisms ( ε_f ^* ) determined from a theoretical model and the porosity; comparing the values of the effective complex dielectric constant (ε*) measured and calculated ε*; and determining a cell concentration of the micro-organisms from the value of ε_f ^* when ε* is equal to ε* measured.

2. The method of claim 1, wherein the step of collecting data indicative of the effective measured complex dielectric constant (ε* measured) comprises collecting data from a plurality of electrodes at a plurality of frequencies.

3. The method of claim 2, wherein the plurality of frequencies include at least 100Hz, 500Hz, IKHz, 5KHz, 10KHz, 100KHz, IMHz, 5MHz, 10MHz and 32MHz.

4. The method of claim 1 , wherein the step of collecting data indicative of propagation time and attenuation comprises using the electromagnetic propagation device operating in the gigahertz (GHz) range.

5. The method of claim 1, further comprising collecting data indicative of a density measurement, the density measurement determining the porosity and effective dielectric constant of the solid matrix ε^* .

6. The method of claim 1, wherein the step of determining the cell concentration of the micro-organisms comprises examination of lookup tables from a plurality of theoretical models for a plurality of micro-organisms.

7. A system for determining in-situ micro-organisms in a solid matrix including sediments and rocks, comprising: a multi-frequency impedance spectrometer to provide data indicative of an effective measured complex dielectric constant; an electromagnetic propagation device; and a processor for processing data provided by at least the multi-frequency impedance spectrometer and the electromagnetic propagation device to generate values indicative of cell concentration of the micro-organisms.

8. The system of claim 7, further comprising a display device for displaying at least one of intermediary measurements, final measurements and processed values.

9. The system of claim 7, further comprising a memory device to store data and processing instructions.

10. The system of claim 7, further comprising a density device.

11. The system of claim 7, wherein the multi-frequency impedance spectrometer device includes at least four electrodes.

12. The system of claim 11 , wherein at least two electrodes are current electrodes and two electrodes are voltage electrodes.