Method and apparatus for diagnosis, detection of cell abnormalities and morphology of living systems

A method and apparatus for dielectric diagnostic analysis of human and non-human cells or tissue functions by measuring the response of the cells or tissue to an applied excitation signal in a time period less than the polarization relaxation time period of a domain group of the cells or tissue under examination.

TECHNICAL FIELD 
The present invention relates to an apparatus and method of diagnostic 
measurement and in particular, a method and apparatus adapted to analyse 
various parameters of living materials and specimens to determine the 
dielectric characteristic of a specimen under test for the purpose of 
diagnosis of the state of the specimen. Depending on the specimen, a wide 
range of states may be susceptible to diagnosis including such as disease 
in plants, animals or humans: the revelation of residual toxins in 
consumer goods from dairy products, meat products, fruit and vegetable 
products, fish, grains and stock feed, oils and other liquids. 
The present invention further helps identify abnormalities and 
transformations in living bodies in their earliest stages, much before the 
clinical appearance of a disease. 
BACKGROUND ART 
At the present state of technology it is well known that the dielectric 
behaviour of such as plant, fruit, animal and human tissue corresponds to 
broad features in their composition and structure. Recent studies have 
revealed that the cell is a highly ordered dynamic entity which acts 
holistically with respect to chemical and physical events within a living 
body, and the existence of domains in the cytoplasm is a general rule. 
These domains are electrically polarised units of ordered, packed 
biopolymers in "biowater". The different organs in a living organism, with 
compartmental similarity and harmonised metabolism, have basic differences 
in domain arrangements which lead to a difference in dielectric responses. 
A disease transformation in a living body which has a viral origin or 
resulting from the action of toxins and other chemicals also changes the 
domain structure and hence the polarisation and dielectric response of the 
tissue or cell. 
A domain is herein defined as a region of a system, or a region of a 
substance, comprising atoms or molecules which can be thought of as a 
single entity; this single entity being responsive to electric or magnetic 
fields and includes such a system having a plurality of these entities. 
Examples of a domain include; a ferroelectric or ferromagnetic domain, a 
cluster of atoms or molecules, an organic cell, a bacterium, a virus, a 
cluster or collection of cells. 
A domain group is a collection of said domains having the same response to 
an electric or magnetic field. 
In the past, precise measurement of parameters of domains were 
inconceivable due to limitations of the instruments. Measurements of 
relative dielectric permittivity, energy dissipation and electrical 
impedance are not possible due to very high values of electrical 
conductance overshadowing real kinetic characteristics. Existing methods 
of measurement are mostly based on impedance bridges, which are inadequate 
at frequencies below 100 Hz due to noise instability, electrode 
polarisation and the time required to obtain balanced conditions. These 
bridges yield relative permittivity, energy dissipation and electrical 
impedance values only at discrete frequencies and therefore each frequency 
setting causes disruption of sequential measurements. The dielectric 
properties of living tissue from bodies will change when they are taken 
out of their natural environment. Dead tissue will show a greater change 
with changes of cell morphology. Conductivity measurement is mostly 
carried out by D.C. electrometers of wide current range, often from 
10.sup.-14 Ampere to a few milliAmpere. This range being covered by 
switching to sequential decade ranges with a mismatch of measured current 
values. A.C. and D.C. measurements require different apparatus, separate 
sample settings and long time switching intervals from one instrument to 
the next. The morphological changes of a cell are much faster, so the 
obtained parameters will refer to different intracellular structures 
resulting in an incorrect correlation between these parameters. Sample 
size limitations sometimes up to a few milligrams reduces electrode 
sensitivity and field noise overshadows the results for fine structural 
studies. 
DISCLOSURE OF INVENTION 
In an effort to ameliorate the disadvantages of the prior art or at least 
to provide a commercially viable alternative to the prior art, the present 
invention proposes a dielectric diagnostic analyser (DDA) and a method of 
diagnosis. 
In a first aspect, the present invention consists in an apparatus adapted 
to perform diagnostic analysis of a specimen having at least one domain 
group as hereinbefore defined, the apparatus comprising: 
excitation generating means to generate a predetermined excitation signal; 
measuring means to measure a response signal of the specimen to the 
predetermined excitation signal: 
electrode means for transmitting and receiving the predetermined excitation 
signal and response signal of the specimen, respectively; 
analysing means arranged to analyse said response signal; and 
switching means adapted to switch the electrode means between the measuring 
means, and excitation generating means, in a time period less than a 
polarization relaxation time period of the at least one domain group in 
the specimen. 
Preferably, the excitation generating means is the source of the 
predetermined excitation signal and may be an electrometer or a frequency 
bridge adapted to generate a predetermined signal. In one form of the 
invention the measuring means compares an electrometer or a frequency 
bridge arranged to measure responses received at the electrode means as 
the response signals of the system. 
Typically the analysing means comprises an electronic computer, 
electrometer and frequency bridge arranged to analyse the response 
signals, received at the electrode means and the computer has a display 
for displaying a diagnostic result. Preferably the switching means is also 
controlled by the computer which allows switching of the electrode means 
between the excitation means and the measuring means at times less than 
the smallest relaxation time, of the polarized domain group, to be 
measured. 
In an embodiment of this invention the electrode means is in the form of a 
suction cup electrode, a pinch electrode, a thermocontrolled electrode or 
any combination of two or more similar electrodes. 
In a second aspect, the present invention provides a method of diagnostic 
analysis comprising; 
applying a predetermined first excitation signal to a specimen having at 
least one domain group, as hereinbefore defined, so as to elicit a 
response from the domain group within the specimen; 
analysing the response from the domain group to determine the maximum 
response of each domain group; and 
comparing said maximum response to a maximum response of a control 
specimen. 
Preferably the first excitation signal is a ramp function voltage sweep or 
a time rate of change of voltage, and the response from the domain, in the 
domain group of the specimen, is measured as a change in a current flow 
through the specimen over time. 
Typically the point of maximum response is at the threshold polarization 
voltage of each domain group and is representative of a maxima in the 
polarization of each domain group of the specimen. 
Preferably a control specimen is any specimen, analogous to the specimen to 
be diagnosed and considered to be the statistical norm of that specimen. 
In an alternative form of the second aspect of the present invention, the 
first excitation signal is a frequency dependent applied voltage and the 
response from the domains is measured so as to allow the determination of 
dielectric permittivity, and dissipation energy, of each domain group. In 
this form the point of maximum response of each domain group is determined 
by a local maxima in the dielectric permittivity or a local minima in the 
dissipation energy of that domain group. 
In a third aspect, the present invention provides a method of diagnostic 
analysis comprising: 
all the steps of the second aspect of the present invention as well as; 
applying a second excitation signal corresponding to a signal value at, or 
near, the point of maximum response of each domain group to elicit a 
further response in each domain group; and 
detecting the variation and length of said further response upon removal of 
the second excitation signal. 
Preferably the second excitation signal is applied in the absence of the 
first excitation signal, and the further response is measured upon removal 
of the second excitation signal while each domain is relaxing to its 
natural state. 
Typically the detecting of the variation and length of the further response 
occurs within the time in which the domains in each group relax to the 
state they were in before the second excitation signal was applied.

BEST MODES 
FIG. 1 shows an embodiment of the first aspect of the present invention, 
which comprises a switching means 97 connected to a frequency bridge 95, 
an electrometer 96 and a computer unit 91 via appropriately shielded 
cables. The computer unit 91 is also connected to a keyboard 92, a display 
monitor 93 and a printer unit 94 in the usual way to provide a computer 
system. The electrometer 96 and frequency bridge 95 are also connected to 
the computer unit 91, such that an operator can through the use of the 
keyboard 92 instruct the computer unit 91 to change the settings on the 
electrometer 96 or the frequency bridge 95. Preferably, the electrometer 
96 and the frequency bridge 95 has the additional option of changing the 
settings manually. The computer unit 91 can be programmed to receive input 
signals, from the electrometer 96 and the frequency bridge 95, which can 
be analysed by means of dedicated software programmes such as Intel's IEEE 
488 and then to output the resulting analysis on the display monitor 93 or 
printer 94. 
The switching means 97 further having connections via a plurality of 
electrically shielded conducting cables to three electrode devices. The 
computer unit 91 is programmed to instruct the switch means 97 to switch 
between any one of the three electrode devices. The first electrode device 
as illustrated in FIG. 1 and FIG. 2 is a suction cup electrode 118 which 
comprises an excitation electrode 114 to induce a current in a tissue 
specimen 117, a measuring electrode 115 to measure the response signals of 
the specimen 117 resulting from the excitation induced by the excitation 
electrode 114, a guard electrode 116 to prevent unwanted surface currents 
reaching the measuring electrode 115, and a suction device 112 connected 
to the suction cup electrode 118 by way of an airflow link 113 to the air 
passage channel 125 of the suction cup 118. The suction device 112 is used 
to adjust the pressure within the suction cup electrode 118, so that not 
only does the cup adhere to the specimen but the contact pressure between 
the specimen 117 and the electrodes (i.e. the excitation electrode 114, 
the guard electrode 116 and measuring electrode 115) can be adjusted to an 
optimum pressure. The optimum pressure between the electrodes and specimen 
is obtained from a local maximum value of the dielectric permittivity in a 
hysteresis plot, as shown in FIG. 5. The excitation and measuring 
electrodes 114 and 115, respectively, are set to the optimum pressure 
before diagnostic measurements are obtained. The second electrode device 
illustrated in FIG. 1 and FIG. 3 is hereinafter referred to as the pinch 
electrode 101 which comprises an excitation electrode 100, mounted on one 
jaw of a pair of pincers 136. while the guard 98 and measuring electrode 
99 are mounted on the opposite jaw of the pair of pincers 136. At the 
other end of the pair of pincers 136, a spring 132, an adjusting screw 
mechanism 131 and a micrometer measuring gauge 130 are arranged to adjust 
and measure the distance between the excitation electrode 100 and the 
measuring electrode 99 at the jaw end of the pair of pincers 136. A 
specimen 102 is pinched between the electrodes at the jaw end and a force 
between the jaws is applied by the adjustment of the screw mechanisms 131 
and spring until the desired distance is read off the gauge reflecting the 
distance between the excitation electrode 98 and the measuring electrode 
99 at the jaw end sandwiching the specimen 102 between the electrodes. 
The third electrode device illustrated by FIG. 1 and FIG. 4 is hereinafter 
referred to as the thermocontrolled electrode 105 which comprises a first 
piston 141, of electrically conductive material to function as the 
excitation electrode 108, and fits within a first teflon cylinder 147 so 
that it protrudes from both ends. The said first teflon cylinder 147 has, 
a guard electrode 107 which wraps around one end of the outer surface of 
the cylinder 147 and an electromagnetic shield 144 which wraps around the 
other end of the outer surface of the teflon cylinder 147. 
A second teflon cylinder 149 substantially similar to the first teflon 
cylinder 147, has a second piston 142 functioning as the measuring 
electrode 106. Piston 142 is allowed to slide in and out of the cylinder 
149 by means of an adjusting nut 145 located at the end of the piston 142 
which protrudes from second teflon cylinder 149 nearest to the 
electromagnetic shield 144. A cap 146 placed over the nut 145 stops it 
from turning at will. The guard electrode 107 on the second teflon 
cylinder 149 extends beyond the end of the cylinder 149. The two teflon 
cylinders 147, 149 slide, with some frictional force, into a third 
cylinder so that the guard electrodes 107 meet, leaving a gap between the 
excitation electrode 108 and the measuring electrode 106 to fit a specimen 
112. The adjusting nut 145 can then be used to change the distance between 
the gap. The third cylinder being a thermocontrolled jacket 148 with two 
ports 150 so that fluid can be pumped in or out, at a predetermined 
temperature, to thermally control the specimen 112. The thermocontrolled 
jacket 148 is connected to a thermocontrol unit 110 (seen in FIG. 1) by 
means of tubing to the ports 150. The thermocontrol unit 110 being capable 
of adjusting the flow rate and temperature of the fluid within the jacket 
148. The thermocontrol unit 110 further having a feedback cable 109 to the 
computer unit 91, so that the flow rate and temperature of the fluid can 
be set or monitored. 
The second aspect of the present invention comprises a method of diagnostic 
analysis of the human body or specimen under test. The following 
parameters can be measured directly, or indirectly by way of calculations; 
current, voltage, specific surface conductance, specific volume 
conductance, domain relaxation time constants, capacitance, inductance, 
relative permittivity, impedance, reactance and dissipation factor at 
different frequencies and temperatures. 
FIG. 6 is a schematic diagram of the equivalent electric circuit for the 
resistive, capacitive and inductive processes in the intracellular 
morphology based on the known concepts of domain structures. 
The embodiment of apparatus of the first aspect of this invention 
hereinbefore described, enables the measurement of various parameters by 
exciting the intracellular domains and measuring those parameters within 
the relaxation time periods of the domains to thereby ameliorate the 
problem of electrode polarisation obscuring the measurements. Values of 
these parameters are therefore revealed by measuring these parameters 
during the relaxation cycle after excitation. 
By way of example only, we will demonstrate how the diagnostic results are 
obtained, for the induction of cancer in a Wistar rat, using the pinch 
electrode 101 hereinbefore described. 
FIG. 7 is a printout of two graphs for the current versus voltage applied 
to a Wistar rat, the right hand side graph being an enlargement of a 
section of the curves on the left hand side graph. The curve 301 
represents the results of a test on the tongue tissue of a healthy Wistar 
rat and the curve 302 is a test of the same rat where the tongue was 
treated with a known carcinogen and cancer was allowed to develop. The 
diagnosis of cancer follows a series of steps; 
In a first step, the initial rate of change of voltage "v" (hereinafter 
called the voltage sweep rate) and the distance "d" between the excitation 
electrode 100 and the measuring electrode 99 are assumed. A test run is 
performed to obtain the current versus voltage graph similar to that of 
FIG. 7. Numerical data is obtained from the test run and substituted into 
the following equation to obtain a new voltage sweep rate, and a new 
electrode spacing amongst other parameters. 
##EQU1## 
where I(B) is the current of the function B and B=E-E.sub.TPV. E the 
electric field strength, E.sub.TPV is the electric field at the threshold 
polarisation voltage; 
"d" the distance between electrodes: 
".upsilon." the voltage sweep rate in V/S: 
"R" the total resistance of the specimen; 
.tau..sub.o is the domain relaxation time constant and .tau.=.tau..sub.o 
exp[U/k T], where U is the activation energy and T the absolute 
temperature: 
"A" is the constant of "softness" which is inversely proportional to the 
piezomodulus of the polarising unit (domain, cell, etc.). 
The test is then set up to the new voltage sweep, the new electrode spacing 
and the other parameters, to be run again. This first step is repeated 
until all of the parameters in the above equations converge to their 
correct values which are determined when the values stop changing 
substantially after each iteration. Finally, a test run with the correct 
values is performed and the threshold polarisation voltage relating to 
each domain group, indicated on the curves in FIG. 7 by the local maxima, 
is obtained. On these curves a local maxima or humps of a domain group 
having a threshold polarisation voltage of less than 1 volt is indicative 
of some abnormality. 
In a second step the relative permittivity (FIG. 8) and the dissipation 
factor (FIG. 9) is obtained as a function of the frequency of the applied 
voltage. In FIGS. 8 and 9 the curve marked 201 is the result of the 
measurements of a healthy Wistar rat, the curve marked 202 is the result 
of a Wistar rat with an ulcer and curve 203 is a Wistar rat with cancer 
which is indicated by the local maxima or hump 204 in the curve. 
In a third step the specimen is excited or charged to the threshold 
polarisation voltage for each domain independently and allowed to 
discharge. During this discharge cycle measurements of the discharge 
current versus time are obtained and analysed to reveal a relaxation time 
for each domain. A computer software program designed to analyse the 
relaxation times for each domain is based on the evaluation of the 
following equations: 
EQU I(t)=I.sub.o +I.sub.1 exp[-(t/.tau..sub.1)]+I.sub.2 exp[-(t/.tau..sub.2)]+. 
. . +I.sub.n exp[-(t/.tau..sub.n)], 
where I.sub.n is the current amplitude and .tau..sub.n is the relaxation 
time constant for the n.sup.th polarised domain group. The computer 
software program cross-checks the results of the relaxation time constants 
by a Fourier analysis (as an example of the Fourier analysis the 
dissipation factor D for the thigh muscle of a Wistar rat, see FIG. 12) 
based on the equation: 
##EQU2## 
where C.sub.n is the capacitance of the n.sup.th domain group, which is 
related to the current amplitude I.sub.n and the applied voltage "V" by 
C.sub.n =I.sub.n .tau..sub.n /V. C.sub..varies. is the sum of the 
capacitance of each domain group and .omega. is the angular frequency 
(2.pi.f) for "f" the frequency of the applied voltage "V". D(.omega.) is 
the energy dissipation as a function of the angular frequency. 
The experimental determination of the natural frequency of each domain and 
hence the relaxation time constants, is obtained by the computer software 
program IEEE 488 from Intel via the measured parameters of the dielectric 
permittivity and frequency on the basis of the following equations: 
EQU .epsilon.(.omega.)=.epsilon..sub.r1 (.omega.)+. . . +.epsilon..sub.rn 
(.omega.)+.epsilon..sub.i1 (.omega.)+. . . +.epsilon..sub.in (.omega.) 
where 
##EQU3## 
L. C and R are electrical parameters of the equivalent circuit (FIG. 6) 
correspond to the electromechanical coupling (piezoelectric like) within 
the domains in living cell cytoplasm or between living cells in organisms. 
In the equations above: 
##EQU4## 
The natural frequency is referred to the inductance "L.sub.in ", 
capacitance "C.sub.in " and resistance "R.sub.in " interrelation of the 
domain following the equivalent electrical circuit in FIG. 6. The 
resistivity "R" relates to the resistance of each domain group, and 
.delta. relates to the piezoelectric constant. The relative permittivity 
".epsilon.(.omega.)" is described in the above equation as a function of 
the angular frequency, noting that in the equations, the subscript "n" 
relates to the n.sup.th domain group. 
FIG. 10 is an example of a discharge current versus time curve for the 
domain structure in a Wistar rat thigh muscle, however, at the top right 
hand corner of the figure is a table of relaxation time constants, with 
corresponding current values and "Q" or charge values for each of four 
domain structures of the cytoplasm. If the "Q"-values of the discharge 
processes sum up to give the corresponding value calculated from the input 
polarisation current, then the test has been successful and the relaxation 
times of each domain structure correctly reflect the dielectric 
characteristics of the specimen. These relaxation times are then compared 
to average relaxation times for a healthy specimen, similar to the table 
in FIG. 11. If relaxation time constants of the Wistar rat of FIG. 10 are 
far removed from the values indicated by the table in FIG. 11 then we can 
surmise with very good probability that there is an abnormality. The 
abnormality in this case for the Wistar rat of FIG. 10 was cancer. 
The third step of the diagnostic procedure is performed within a period 
less than or equal to the relaxation time period for the domain and 
preferably within the time frame before any substantial change to the 
intracellular morphology of the cells of the specimen under test. In the 
preferred embodiment of the present invention all three steps would be 
achieved in a few relaxation time cycles. 
The DDA as hereinbefore described in the embodiments make possible the 
recording of the dielectric parameters of tissue samples with minimal 
invasion. As the domains in cytoplasm are vulnerable to spontaneous 
ordering, rearrangements or disruption by slight changes, for example by 
temperature, the simultaneous measurements of parameters make possible the 
analysis of these changes with reference to the same intracellular 
morphology. 
FIGS. 13(a)-(f) relate to the changes of polarisation in the tongue tissue 
with a change in temperature and FIG. 13(f) shows a comparison of the 
minute energies required during heating below 41.degree. C. 
FIG. 13(e) illustrates the irreversible process that occurs to the 
dielectric parameters and hence to living tissue (in this case tongue 
tissue of a Wistar rat), before and after heating the tissue to 
temperatures above 42.5 degrees Celsius. The process of heating the tissue 
above a certain temperature "cooks" the tissue. This "cooking" process 
changes the state of the dielectric parameters of the tissue, compared to 
the tissue undergoing chemical "fixation" (chemicals such as Kryofix are 
generally used for optical studies of cellular morphology) which preserves 
the tissue. These changes in the dielectric parameters are shown in part 
in FIG. 14. 
FIG. 14 is a table showing some dielectric parameters of various tissue 
samples of rat organs, averaged over two rats, and a comparison of these 
parameters for fresh or "fixated" tissue. 
Industrial Applicability 
The dielectric diagnostic analyser (DDA) as described in the embodiments of 
the present invention also provides a non-invasive, or at least minimally 
invasive, technique to diagnose changes in the fine structure in cell 
cytoplasm with respect to the complexity of chemical context. cellular 
packing, disease transformation and reveal the action of preservation 
(e.g., Kryofix) and staining (e.g., Haematoxylin) on tissue. 
It will be appreciated by a person skilled in the relevant art that this 
method of diagnostic testing can be applied to any specimen or substance 
where a domain type structure within cells can be defined including any 
Maxwell-Wagner system. To study ultrafine structure and intracellular 
kinetic parameters of cells, including the cell cytoplasm, tissue, organs, 
the body's metabolic processes, the detection of disease and disease 
transformation at the onset of said disease including the differentiation 
of diseases having or not having a viral origin. 
The method herein described provides a diagnostic tool which can be adapted 
to imaging techniques, similar to medical imaging. This diagnostic method 
and apparatus can be adapted to animals in animal husbandry, plants in 
agriculture, environmental diagnostics of bacteria and algae in waterways 
and to chemical analysis of effluent amongst other fields of use. 
Typically the diagnostic method hereinbefore described is well suited to 
the analysis of the presence or absence of toxins and other chemicals in 
specimens such as dairy products, vegetables, meat, fruit, fish, grains, 
oils, seeds and stock feed products, soil, water as well as viral diseases 
in plants, animals or human bodies. 
It will be appreciated by persons skilled in the art that numerous 
variations and/or modifications may be made to the invention as shown in 
the specific embodiments without departing from the spirit or scope of the 
invention as broadly described. The present embodiments are, therefore, to 
be considered in all respects as illustrative and not restrictive.