Abstract:
A device for characterizing ex vivo tissue employs a set of independent electrodes that may be used to scan the tissue by moving a voltage gradient across the tissue surface acquiring impedance spectrographs that may be mapped to an image.

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.S. provisional application 61/072,745 filed Apr. 2, 2008 and hereby incorporated by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates generally to instruments for evaluating tissue samples in medical pathology, and in particular to a device that characterizes tissue samples using precise measurements of electrical impedance of the tissue. 
     The diagnosis of cancer and other diseases is often made by the examination of tissue samples removed from the patient during a biopsy or surgical procedure. The tissue sample may be preserved chemically and then stained and sliced into layers that are on an order of one to several cells in thicknesses. These sections are examined by a pathologist who may study these sections under a microscope to reach a conclusion about whether the tissue is cancerous. 
     The above process may take substantial time to complete and therefore an alternate procedure called a “frozen section” may be used that eliminates the step of chemical preservation and encases the specimen in plastic and freezes the specimen. This process can be accomplished in less than an hour, but requires considerable skill. Further the resulting sections provide lower resolution images, and therefore must often be followed by a conventional chemical preservation process described above. 
     In both of these techniques, only small sections of tissue may be analyzed and accordingly many adjacent sections must often be studied to definitively diagnose the disease in an organ. 
     SUMMARY OF THE INVENTION 
     The present invention provides a system that can rapidly assess the electrical impedance spectrum (complex impedance as a function of frequency) of thin but large area tissue samples without sectioning or other preparation. There is currently evidence that impedance characteristics of tissue may provide a method of rapidly distinguishing benign from cancerous tumors. 
     Specifically, the present invention provides an apparatus for tissue sample analysis having a first electrode array providing a surface for receiving an ex vivo tissue sample in abutment with the surface, the surface providing a plurality of electrically independent voltage measurement points and voltage application points. An electronic computer communicates with the first electrode array to control voltage applied to the voltage application points and to read voltages obtained at the voltage measurement points. This electronic computer operates according to a stored program to: (a) establish a voltage gradient among the voltage application points defining a boundary across the first electrode array; (b) sweep the boundary across the first electrode array while the first electrode array is in contact with the tissue sample; (c) monitor the voltage measurement points at the boundary to measure impedance at multiple points along the boundary for each of multiple different locations of the boundary during the sweep; and (d) provide an output characterizing the tissue sample according to the measured impedance at the multiple points. 
     It is thus a feature of at least one embodiment of the invention to provide an accurate method of quickly characterizing relatively large tissue samples over multiple points. By sweeping a voltage gradient across the tissue sample, multiple impedance points can be measured without errors caused by field fringing. 
     The voltage gradient may define sequential first and second boundaries that are mutually substantially perpendicular and the operation of sweeping the boundary across the first electrode array may sweep the first and second boundaries along substantially perpendicular axes. The operation of monitoring the voltage at the measurement points may be repeated for each of the boundaries to measure impedance at each of the multiple points twice, once during a sweep of the first and second boundaries. 
     It is thus a feature of at least one embodiment of the invention to provide multiple measurements and to provide a measurement system that accommodates tissue anisotropy. 
     At least one of the current and voltage at the voltage application points may substantially define a step function over an area of the first electrode array. 
     It is thus a feature of at least one embodiment of the invention to provide a gradient producing a well-characterized current flow through the tissue. 
     The electronic computer may control the voltage applied to the voltage application points to provide a predetermined current through the tissue. 
     It is thus a feature of at least one embodiment of the invention to provide a measurement mode eliminating the need for correction of the measured voltage drops by current flow. 
     Alternatively, the electronic computer may control the voltage applied to the voltage application points independent of the current through the tissue and monitor the current at the voltage application points to measure impedance at the multiple points along the boundary. 
     It is thus a feature of at least one embodiment of the invention to provide a measurement mode allowing simplified application of voltages to the electrodes. 
     The voltage application points and voltage measurement points are electrodes having a surface treated to reduce electrode polarization. 
     It is thus a feature of at least one embodiment of the invention to improve the precision of the impedance measurement by reducing electrode polarization effects caused by ion conduction in the tissue. 
     The voltage application points and voltage measurement points are electrodes having a surface adapted not to pierce the tissue. 
     It is thus a feature of at least one embodiment of the invention to provide a measurement technique that does not unduly damage the tissue. 
     The apparatus may also provide a second electrode array like the first electrode array and positionable opposite the first electrode array to sandwich the tissue sample therebetween in contact with the voltage measurement points and voltage application points of the first and second electrode arrays. In this case, the electronic computer also communicates with the second electrode array to provide a spatially corresponding gradient on the second electrode array, to monitor the measurement points at a boundary on the second electrode array to measure impedance at multiple points along the boundary for each of multiple different locations of the boundary during the sweep, and to provide an output characterizing the tissue sample according to the measured impedance at the multiple points on both the first and second electrode array. 
     It is thus a feature of at least one embodiment of the invention to better characterize the impedance through the entire thickness of thin slices of tissue. 
     The gradient boundary may be substantially a line. 
     It is thus a feature of at least one embodiment of the invention to provide a simplified electrode layout and data collection method that reduces current field fringing and distortion. 
     The first and second electrode arrays may be positionable at less than 1 cm separation. 
     It is thus a feature of at least one embodiment of the invention to provide direct measurement of tissue samples without freezing and sectioning. 
     The output may be an image mapping impedance to spatial locations corresponding to the voltage measurement points. 
     It is thus a feature of at least one embodiment of the invention to provide an assessment of tissue samples in which cancer cells, for example, may comprise only a portion. The imaging capability allows comprehensive analysis of larger tissue samples. 
     The output may provide a numeric index characterizing the tissue. 
     It is thus a feature of at least one embodiment of the invention to provide a simple metric characterizing tissue. 
     The electrode array may provide electrodes that are selectively switched by the computer between voltage application points and voltage measurement points, and the electronic computer controls the voltage application points and voltage measurement points so that a pair of voltage measurement points are separated by the boundary and are flanked by voltage application points. Alternatively, the electrode array may provide electrodes that may be simultaneously voltage application points and voltage measurement points. 
     It is thus a feature of at least one embodiment of the invention to permit either four-lead or two-lead type resistance measurements. 
     The electronic computer may first measure impedance by controlling the voltage application points and voltage measurement points so that a pair of voltage measurement points are separated by the boundary and are flanked by voltage application points in a four-lead impedance measurement, and may second measure impedance by controlling the voltage application points and voltage measurement points to be combined in a two-lead impedance measurement. The electronic computer may in this case evaluate the difference between the four-lead and two-lead impedance measurements to deduce electrode polarization. 
     It is thus a feature of at least one embodiment of the invention to provide a method of characterizing the effects of electrical polarization to correct the output characterizing the tissue sample or provide a warning to the user if polarization effects are substantial. 
     These particular features and advantages may apply to only some embodiments falling within the claims and thus do not define the scope of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of an impedance measuring device according to the present invention having an electrode array connected to a laptop computer or the like, the elements together providing tissue analysis; 
         FIG. 2  is a block diagram of the invention of  FIG. 1  showing the components of the electrode array and the associated computer which may also be incorporated into one housing; 
         FIG. 3  is a fragmentary schematic diagram showing electrical interconnection of multiple electrodes of the electrode array to voltage measurement and voltage application points using a multiplexer/demultiplexer; 
         FIG. 4  is a flow chart of the program executed by the computer of  FIGS. 1 ,  2 , and  3  for providing impedance scans; 
         FIGS. 5   a - d  are top plan views of one electrode array of the impedance measuring device as operated according to the program of  FIG. 4  showing voltage zones generating a scanned measurement boundary across the electrode array and hence across the tissue sample proximate to the electrode array; 
         FIG. 6  is a top plan view similar to  FIGS. 5  showing fringing current fields avoided by the present scanning system within tissue sample; 
         FIG. 7  is a fragmentary cross-sectional view taken along line  7 - 7  of  FIG. 1  showing electrical connections for a four-lead impedance measurement using scanning pattern of  FIG. 5 ; 
         FIG. 8  is a figure similar to that of  FIG. 7  showing a two-lead measurement; 
         FIG. 9  is a sample impedance spectrogram produced by the present invention; 
         FIG. 10  is an example output display produced by the present invention providing an image of the tissue impedance together with quantitative and spectrographic data localized to a cursor location in the image; 
         FIG. 11  is a simplified representation of electrical polarization effects occurring in a two-lead measurement; and 
         FIG. 12  is a figure similar to that of  FIG. 11  showing a reduction in electrical polarization effects in a four-lead measurement. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Referring now to  FIG. 1 , the impedance measuring apparatus  10  of the present invention may employ a tissue sample unit  12  and associated computer  14 , the latter providing display and program input capabilities as will be described below. In alternative embodiments, it will be understood that the computer  14  functions may be incorporated into the tissue sample unit  12 . 
     In the embodiment shown, the tissue sample unit  12  includes a base portion  16  having a well  81  exposing at its bottom a first planar electrode array  20  comprised of perpendicular and rectilinear rows and columns of electrodes  22  electrically isolated from each other by intervening channels. The well  18  is sized to receive an unprocessed tissue sample  24  typically several millimeters thick and no more than 1 cm thick (measured perpendicularly to the surface of the array  20 ) and having a height and width (measured along the surface of the array  20 ) of less than approximately 4 cm. The electrode array  20  is sized so that the tissue sample  24  may lie within the boundaries described by the limits of the electrodes  22 , thus contacting the electrodes  22  over its entire surface. 
     A second electrode array  26  may be positioned on a carrier  28  fitting within the well  18  so that an interface between the walls of the carrier  28  and walls of the well  18  serve to align the second electrode array  26  with the first electrode array  20  such that each electrode  22  of the first and second electrode arrays  20  and  26  are aligned in opposition about the tissue sample  24 . In this configuration, the electrode array  20  contacts an underside of the tissue sample  24  and the electrode array  26  contacts the top of the tissue sample  24  to sandwich the tissue sample  24  there between. 
     A flexible conductor  30  may communicate between the electrode array  26  and the base portion  16  so that electrical signals associate with all the electrodes  22  can be brought together within the base portion  16 . 
     Electrical signals to and from each electrode  22  are processed by multiplexer/demultiplexer circuitry within the base portion  16  as will be described and may be conveyed through a connector  32  on the base portion  16  via a cable  34  such as a USB cable to computer  14 . The computer  14  may include a display  35  displaying an image  36  of the tissue sample  24  as will be described and may provide for an input device  38  such as a keyboard for inputting data. 
     Referring now to  FIG. 2 , a USB interface circuit  40  in the sample unit  12  connects via the USB cable  34  with interface circuit  44  in the computer  14 . The interface circuit  44  may in turn attach to an internal computer bus  46  also communicating with the display  35  and input device  38  as well as an internal microprocessor  50  executing a stored program  52  contained in a memory  54 . In one embodiment, as described above, the components of the sample unit  12 , the interface circuits  40  and  44 , the microprocessor  50 , and the memory  54  may all be contained in a common housing  55 . 
     Referring now to  FIG. 3 , as mentioned, each of the electrode arrays  20  and  26  provides a set of electrodes  22 . These electrodes  22  provide an outer surface facing the tissue sample  24  having a raised tissue-contacting portion  53  increasing the contact area between the electrode  22  and the tissue sample  24 . The surface of the electrodes  22  contacting the tissue sample  24  may be treated to reduce electrical polarization, for example, with a silver/silver chloride coating of the type used in ECG electrodes or a platinum black application according to techniques well known in the art. These coatings serve to reduce spurious voltage measurements caused by the accumulation at the electrodes of charged ions such as form a principal conduction path within the tissue. 
     Each electrode  22  may be electrically connected to a solid-state single-pole, double-throw switch  51  that may in turn connect the electrode  22  alternately to a voltage measurement point  56  or to a voltage application point  58  under the control of an electrical switching signal  60 . The voltage measurement point  56  may connect to an input of high impedance amplifier  62  to produce a measurement signal  64 . The voltage application point  58  connects to the output of a buffer amplifier/sample and hold circuit  66  receiving a voltage command  68 . The output of the buffer amplifier/sample and hold circuit  66  passes through a current sensor  70  measuring the current flowing into or out of the voltage application point  58  to produce a current signal  72 . 
     The elements  51 ,  62 ,  66 , and  70  are duplicated for each of the electrodes  22  and exchange respective signals  60 ,  64 ,  68 , and  72  with a microcontroller  76  via an analog multiplexer/demultiplexer  74 , which switches among the electrodes  22  according to address signal  61  generated by the microcontroller  76  so that one electrode  22  may be written to or read. The microcontroller  76  may implement the USB interface  40  providing communication to the computer  14  through cable  34 . 
     Referring now to  FIG. 5   a , the computer  14  and tissue sample unit  12  operating in tandem, control the electrodes  22  to impose different voltage patterns thereacross. For example, during a columns scan, electrodes  22  in a first set of columns forming a zone  80  may connect to a first voltage V 1  (the first two columns on the right as depicted) and the electrodes  22  in a second set of columns forming a zone  82  may connect to a second voltage V 2  different from the first voltage (the last four columns on the left as depicted). This voltage pattern creates a voltage difference across two center columns  84  and  86  between the zone  80  and zone  82  which provides sensing columns S 1  and S 2  flanking a column-aligned boundary  90 . 
     By manipulating the signals  60  and  68 , the boundary  90  may be moved from left to right (as shown in  FIG. 5   b ). In a complete four-lead columns scan, the boundary  90  can start as far over as the interface between the second and third columns from the left and move to the interface between second and third column from the right being positioned momentarily in between each column At each position of the boundary  90 , a voltage difference may be measured across the boundary  90  by electrodes  22  of sensing columns S 1  and S 2  being the mathematical difference between the voltage measured at an electrode  22  in sensing column Si and an electrode  22  in the same row in sensing column S 2 . 
     For each pair of electrodes  22  and computed voltage drop, a corresponding measurement of current is obtained and an impedance value deduced at the intersection between that row and the boundary  90 . As the boundary  90  moves from left to right, impedance measurements may be made at multiple row and boundary locations over the two-dimensional surface of the arrays  20  and  26 . 
     Referring momentarily to  FIG. 6 , current passing between electrodes  22  on either side of the boundary  90  within the sensor zones S 1  and S 2  may with reasonable precision be considered equal to the current passing through the tissue sample  24  in a line between those electrodes because the voltage pattern of electrodes in the zones  80  and  82  provide essentially planar opposed electrical fields, this geometry eliminating fringing current flow  94  except at the very edge rows removed from the tissue sample  24 . By keeping the tissue sample  24  smaller than the size of the array  20 , the approximation of linear current flow between electrodes  22  is good throughout the entire tissue sample  24  and therefore current in the tissue between each electrode pair can be approximated by the average of the current measured flowing out of and into the electrodes  22  of that pair. 
     Referring now to  FIGS. 5   c  and  5   d , the process of scanning the boundary  90  may be repeated with a new boundary  96  oriented horizontally and thus parallel to the rows. In this case, the voltages of V 1  and V 2  cover contiguous blocks of rows on either side of horizontally oriented sensor regions S 1  and S 2  as shown in  FIG. 5   b . The boundary  96  may be scanned from top to bottom of the arrays to obtain impedance measurements at pairs of vertically flanking electrodes  22  for each of the columns. Impedance values obtained during this scan maybe recorded separately or combined with the values contained during the horizontal scan. 
     While the preferred embodiment uses a boundary  96  that is substantially linear extending across the electrode array  20 , it will be understood that variations on this are possible including, for example, a boundary that encloses a small surface on the electrode array and that is scanned, for example, in a raster pattern. Such a boundary would not provide the benefits of eliminating the effects of fringing fields as will be described but could be useful in exploring other aspects of the impedance at the surface. 
     Referring now to  FIG. 7 , the above measurements may occur simultaneously on the upper electrode array  26  and the lower electrode array  20 . Thus, driving electrodes  22   a  and  22   b  on opposite sides of the tissue sample  24  on one side of the boundary  90  (or  96 ) removed from the boundary  90  (or  96 ) by sensing electrodes  22   e  and  22   f  may be given the same electrical potential (V 1 ), and electrodes  22   c  and  22   d  on opposite sides of the tissue sample  24  and symmetrically offset from the electrodes  22   a  and  22   b  about the boundary  90  (or  96 ) may be given the second electrical potential (V 2 ) to establish a virtual voltage source  100  therebetween implemented by a combination of buffer amplifier/sample and hold circuits  66 . Current between the electrodes  22   a  and  22   b  and electrodes  22   c  and  22   d  may be measured as indicated by virtual current sensor  102  implemented by a combination of current sensors  70 . 
     Voltages measured at electrodes  22   e  and  22   f  on one side of the boundary  90  (or  96 ) may be averaged and subtracted from voltages measured at electrodes  22   g  and  22   h  on the other side of the boundary  90  (or  96 ) to produce a voltage difference or voltage drop  101  across the boundary  90  (or  96 ). The voltage drop together with the current measured at virtual current sensor  102  yields an impedance measurement. For thin tissue samples  24 , the electrodes  22  on both sides of the tissue sample  24  promote a uniformity of current flow and a measurement that is sensitive to impedance throughout the thickness of the tissue sample  24 . 
     Referring now to  FIG. 8 , the above description has been that of a four-lead measurement. The present invention may also operate in the two-lead mode that may be useful for measuring impedances at the edges of the arrays  20  and  26  wherefore contiguous electrodes are not readily obtained, or in other modes, for example between electrodes of the arrays  20  and  26 . As will described further below, the two-lead mode may also be used to provide an estimation of electrical potential artifacts occurring in the measurement of currents through tissue to improve the accuracy of the impedance measurement. In the two-lead measurement mode, only four electrodes  22   a - 22   d  are needed for an impedance measurement in contrast to the eight electrodes  22   a - 22   h  shown in  FIG. 7 . 
     As shown in  FIG. 8 , in a two-lead measurement scan electrodes  22   a  and  22   b  are positioned on opposite sides of the tissue sample  24  on one side of the boundary  90  (or  96 ) which is scanned horizontally and vertically as described above. The electrodes  22   a  and  22   b  may be connected to a first potential V 1  provided by virtual voltage source  100 . Similarly, electrodes  22   c  and  22   d  on opposite sides of the tissue sample  244  and on the other side of the boundary  90  (or  96 ) may be connected to the second potential V 2  of voltage source  100  with the current therebetween being measured by virtual current sensor  102 . 
     The voltage difference between the pair of electrodes  22   a ,  22   b  and the pair of electrodes  22   c ,  22   d , may be measured directly by a virtual voltmeter  104  implemented by a measurement of the signals  68  driving the corresponding buffer amplifier/sample and hold circuits  66 . The impedance may be then calculated as the ratio of the voltage of virtual voltmeter  104  to the current measured by virtual current sensor  102 . 
     In the preferred embodiment, the invention measures complex impedance using voltages V 1  and V 2  that create alternating currents through the tissue sample  24  over a range of frequencies from 10 Hz to 1 MHz. The complex impedance at each frequency creates an impedance spectrum  106  as shown in  FIG. 9  indicating, for example, the magnitude of the impedance as a function of frequency or the real and imaginary parts of the impedance (not shown) or phase angle as a function of frequency to yield a complete understanding of the impedance of the tissue. 
     Referring to  FIG. 10 , the data of each impedance spectrum  106  may be mapped to pixels  108  of an image  36  having a location corresponding to the measurement points of the impedance. Particular values or classifications of the impedance spectrum  106  may be used to determine the color or grayscale of the pixels  108 . Alternatively, a frequency peak or local maxima in the spectrum  106  may be mapped to a particular color of the pixels  108  to yield a color image having a qualitative representation of the changes in the tissue sample over its area in the area of the arrays  20  and  26 . Optionally the user may manipulate a cursor  109  over the image  36  linked to windows  110  and  112  providing respectively a numeric value reflecting one or more important parameter of the frequency spectrum  106  and the frequency spectrum  106  itself. General statistical metrics, for example a likelihood of the tissue being cancerous or percentages of cancerous tissue, may be presented in a window  111 . Analysis of impedance spectrums is described in “Correction Of Electrode Polarization Contributions to the Dielectric Properties of Normal and Cancerous Breast Tissues at Audio/Radiofrequencies” Stoneman, M. R. et al. Phys. Med. Biol. 52 (2007) 6589-6604, hereby incorporated by reference. 
     Referring now to  FIG. 11 , the present invention contemplates that multiple scans of the tissue sample  24  may be completed, first in a two-lead mode as described with respect to  FIG. 8 , and second in a four-lead mode as described with respect to  FIG. 7 . As shown in  FIG. 11 , the two-lead mode has the problem of being sensitive to lead impedance and contact resistance because of the necessity of current flow through electrodes  22   a  and  22   b  which also serve simultaneously as the measurement points. In addition, to the extent that the conduction of electricity in the tissue sample  24  is by means of ions  114 , electrical potentials may build up at the electrodes  22   a  or  22   b  corrupting the measurement of voltage drop thus adversely affecting the impedance measurement. 
     In contrast, as shown in  FIG. 12 , in the four-lead mode, electrodes  22   a  and  22   b  used for voltage application can be separated from electrodes  22   c  and  22   d  used for a measurement of voltage drop. This measurement of voltage at electrodes  22   c  and  22   d  can be conducted with very little current flow (eliminating the effect of the impedance or contact resistance). Similarly, this reduced current flow at electrodes  22   c  and  22   d  eliminates or reduces problems caused by the accumulation of ions  114 . The present invention contemplates that both of these measurements may be used and the difference between the measurements in areas where both measurements are made may be used to provide a calibration factor indicating offset caused by electrical potential that can be used to correct the impedance measurements, for example, using the two-lead mode, near the edges of the arrays  20  and  26 . 
     Referring now to  FIG. 4 , in operation then, the present invention may begin by the placement of tissue sample  24  in the tissue sample unit  12  as shown in  FIG. 1  sandwiched between upper array  26  and lower array  20  as indicated by process block  120 . A horizontal scan may be conducted per process block  122  as shown in  FIGS. 5   a  and  5   b  using the two-lead and four-lead modes. At each different scan location (defined by the position of the boundary  90 ), as indicated by process block  124 , a set of impedance measurements may be made at each row for a range of different frequencies to establish spectrum  106  for each row at the particular boundary location. 
     At succeeding block  126  and  128 , a vertical scan may be conducted and frequency measurements made in a manner analogous to process blocks  122  and  124 . 
     At process block  130 , the program may collect the measured information of current and voltage obtained during the scans and calculate impedance parameters at points centered between each pair of sensing electrodes. Each of these impedance parameters may be in the form of a spectrum and associated with a particular coordinate on the arrays  20  and  26 . 
     The collected impedance data for corresponding points taken in different scan directions may be combined, for example by averaging, and the impedance measurements over the arrays may be further processed, for example, by spatial filtering or the like. 
     Optionally, at process block  132  the impedance measurements of process block  130  may be corrected by compensating the two-lead measurements contributing to the calculation of impedance for electrical polarization deduced as described above. 
     At process block  134  tissue scores may be calculated reducing the data to simple scales or dimensions, for example, an arbitrary scale from 1 to 10 indicating a likelihood of cancer or the percentage of the measurements indicating a likelihood of cancer. 
     Finally, at process block  136  an output such as shown in  FIGS. 9  or  10  may be provided. 
     The present invention has been described in terms of the preferred embodiment, and it is recognized that equivalents, alternatives, and modifications, aside from those expressly stated, are possible and within the scope of the appending claims.