Method for biological cell and particulate analysis

An analytical device for detecting and analyzing the population of neutral or electrically-charged solutes, including providing a light source for producing a collimated beam, providing a solution vessel containing the sample solution to be analyzed, directing the light beam through the sample solution to thereby produce scattered light, a photomultiplier or a photon counter for detecting the scattered light, said photomultiplier being positioned at an angle to receive the scattered light from the collimated beam, an analog-to-digital converter communicating with the photomultiplier, converting the electrical signals output into a digital output, a digital correlator for receiving the digital output and to calculate a time autocorrelation function of the motion of the solutes in the fluid medium, immersing two electrodes in the solution vessel, generating an electrical field between the two electrodes, positioning the electrodes such that a field gradient is created between the electrode, and such that the light beam partially impinges on one of the electrodes to create a heterodyne effect, and an electrical field generator connected to said electrodes, producing an oscillating electrical signals, that can polarize solutes in the solution, the electrical signal generated at a frequency of 0 to 1,000 million hertz.

BACKGROUND OF THE APPLICATION 
This invention relates to an improved method for identifying polarizable 
particles, macromolecules, and biological cells and a device for carrying 
out the method. 
Identification of molecules, particles, and biological cells constitutes a 
major part to their purification, utility, and production. Identifying 
components in solution is equally important for both the biotechnology and 
chemical industries. In the chemical polymerization to manufacture 
commercial polymers and latex particles, for example, knowledge and 
control of the distribution of particle growth and size are critical. The 
situation is at least as complicated for biological organisms, where 
details about protein and cell populations are critical. The terms: 
particles, cells, macromolecules, particulates, and polymers are used here 
interchangeably, unless otherwise noted. 
The present invention describes a device and a method for the 
identification of polarizable particles, macromolecules, and biological 
cells. The invention detects the behavior of dielectric materials in 
non-uniform fields. Dielectric properties are indicative of the 
polarizabilities of the macromolecules, cells, and particles. The 
detection of these properties is accomplished by measuring the modulation 
in the time autocorrelation function, measured by a technique such as 
dynamic light scattering, DLS. Current applications of DLS measure the 
Brownian (random) motion of molecules. Under the influence of a 
non-uniform electric field, polarizable macromolecules undergo 
characteristic motion, called dielectrophoresis. For the sake of clarity, 
a brief introduction to the basis of DLS and dielectrophoresis phenomena 
is presented below. 
In a DLS experiment, a laser light impinges on a solution of macromolecules 
and the intensity of the scattered light is measured at an angle, .theta.. 
A typical apparatus setup for DLS is presented in FIG. 1. The frequency of 
light is Doppler shifted due to the Brownian motion of the scattering 
macromolecules in the scattering volume, defined by the incident and 
scattered beam geometries at their intersection. The frequency shifts are 
related to the diffusion coefficients of the scattering species. Current 
DLS experiments measure the Fourier transform of these frequency shifts as 
the time autocorrelation function of the intensity fluctuations produced 
by molecular motion in solution. Time autocorrelation functions are 
exponential with time constants which are characteristic of the diffusion 
coefficients of the scattering species. From these coefficients, useful 
information can be obtained on the scattering molecules, primarily a 
measure of their size in solution. For a monodisperse system of particles, 
the heterodyne intensity autocorrelation function, C(.tau.), can be 
written as: 
##EQU1## 
where &lt;N&gt; is the average number of particles, .tau. is the delay time used 
to construct C(.tau.), and q is an experimental constant wherein: 
##EQU2## 
Here, n is the refractive index of the solution, .lambda. is the wavelength 
of light and .theta. is the scattering angle. In Equation (1), D is the 
diffusion coefficient, which for a spherical particle is: 
##EQU3## 
where k is the Boltzmann constant, T is the absolute temperature, .eta. is 
the viscosity and r is the particle radius. 
An analysis of the autocorrelation functions using Equations 1-3 can lead 
to the extraction of the diffusion coefficient of the scattering species 
and, hence, a measure of their size. 
In polydisperse systems, the measured autocorrelation function is a sum of 
exponentials (or, for continuous distribution, an integral) representing 
the different species present, 
EQU C(.tau.)=.intg.G(.GAMMA.)e.sup.-.GAMMA..tau. d.GAMMA., (4) 
where .GAMMA.is the exponent in Equation 1, (.i.e..GAMMA.=q.sup.2 D). 
As Equation 4 shows, except for a single solute (monodisperse) systems, 
C(.tau.) data will be composed of a sum of exponentials. Analysis of 
multiple exponentials is difficult, even though clever methods have been 
developed for such analyses. The origin of the difficulty is that 
exponentials overlap quite strongly, with no discernible structure 
developed in the resulting functional form. The present invention, in 
addition to furnishing a new tool for the identification of 
macromolecules, also provides a guide to overcoming analysis difficulties 
by giving an estimate of the number of components present. 
DIELECTROPHORESIS 
The familiar phenomenon of electrophoresis results from the interaction of 
charged molecules and particles with an electric field. The field is 
usually uniform and causes a net translational motion of the particles. 
Neutral particles do not exhibit such an effect. The phenomenon of 
dielectrophoresis relates to the motion of particles in non-uniform (DC or 
oscillating) electric fields. This effect is exhibited by polarizable 
macromolecules, even if they are electrically neutral. When a particle or 
a macromolecule is polarizable, the field induces a polarization, or 
charge separation. It must be appreciated that there are various 
mechanisms for inducing polarization. In dielectrophoresis, the spatial 
non-uniformity of the field induces a directed motion toward maximum field 
intensity (positive dielectrophoresis). Positive dielectrophoresis occurs 
when the dielectric constant of the particle is larger than that of the 
medium (see Equation 6), while negative dielectrophoresis occurs when the 
particle dielectric constant is smaller. These effects are depicted 
schematically in FIG. 2. Hence, polarizable macromolecules, such as 
emulsions, suspensions and solutions of DNA and cells, will be affected by 
the field. Macromolecules can be characterized by their motion in response 
to the dielectrophoretic effect. 
The force imposed by dielectrophoresis depends on several factors, 
including the oscillating electric field strength and its frequency, the 
dielectric constant and conductivity of the particle and the suspending 
medium, and the spatial configuration of the electrodes. This force, F, 
can be represented as: 
EQU F=Vg.gradient.E.sup.2 ( 5) 
where V accounts for the particle volume and .gradient.E.sup.2 is an 
expression of the effect of the electric field. It can be seen that the 
force imposed by dielectrophoresis depends on both field strength and on 
the field gradient, as .gradient.E.sup.2 may also be written as 
2E.gradient.E. g is a function that includes the properties of the 
particle and the medium which are relevant to the dielectrophoretic 
effect, namely their electrical permittivities: 
##EQU4## 
Where e*.sub.p and e*.sub.m are the complex permittivities of the particle, 
p, and the medium, m 
The dielectric behavior of biopolymers and cells reflects several modes of 
polarizability. The different modes can be generalized as the polarization 
of water, dissolved biomolecules (proteins, sugars, DNA, etc.), charge 
rearrangement around a membrane, and most characteristically for cells, 
the action of the electrical double layer at the interface of cellular 
membranes where surface charges are localized. The last effect contributes 
a large magnitude to polarization, and is the explanation for the apparent 
extremely high values of dielectric constants observed for biological cell 
suspensions. The existence of the double layer in biological cells makes 
the cells particularly responsive to the effect of dielectrophoresis. 
Variations in the conditions listed above, and in particular the 
distribution of the membrane charges would lead to a change in the double 
layer and, hence, a discernible and unique response to the 
dielectrophoretic force. 
The motion induced upon the macromolecules is peculiar to a particular 
population of macromolecules in solution. The importance of this motion in 
conjunction with the DLS is that the motion is non-random (unlike Brownian 
motion). As previously indicated, it is sensitive to the factors affecting 
the polarizability of the macromolecule to the dielectrophoretic effect is 
the charge distribution on the macromolecule. Changes in cell conditions, 
metabolism, or the infection of cells by invading microorganisms and 
viruses would change the charge distribution inside the cells and on their 
membranes. It is the premise of this invention that changes in cells which 
alters the cells charge distribution would manifest itself in the 
dielectrophoretic effect. The present invention presents a method to 
detect such changes. 
PRIOR ART 
DLS is a widely used technique. There are two techniques that use the more 
familiar effect of electrophoresis in conjunction with DLS. 
Electrophoretic light scattering has been demonstrated. The method uses 
electrophoresis to induce uniform motion, which modulates the time 
autocorrelation function and discriminates between molecules depending on 
their electrophoretic mobility. Since in electrophoresis only charged 
macromolecules would exhibit migration under the effect of the electric 
field, this technique is limited to detection of macromolecules which 
posses a net charge, and suffers the drawbacks of electrophoresis 
phenomenon, mainly heat generation, electronic polarization, and bubble 
formation due to electrolysis. This method has been used to characterize 
monomer and dimeric bovine serum albumin (BSA) in solution. 
A low frequency field (frequencies &lt;100 Hz) has also been used in a similar 
fashion to electrophoretic light scattering. The low oscillation field, 
called sinusoidal electric field (hence, DLS-SEF) was proposed to avoid 
some of the drawbacks in electrophoretic light scattering. The method has 
the advantages of reducing the Joules heating and the bubble formation as 
well as reducing the electrodic polarization encountered in the DC 
electrophoresis. 
The present invention is phenomenologically different from the prior art. 
In electrophoretic light scattering, as well as DLS-SEF, the 
electrophoretic mobility is the discriminating factor. Both techniques are 
limited to charged molecules, and the limitations of electrophoresis. The 
discriminating factors in the present invention are the dielectric, or 
polarization properties. 
SUMMARY OF THE INVENTION 
The invention comprises a device which consists of a sample cell, 
electrodes for creating a field gradient, a source for creating 
oscillating electrical field with frequency in the radio frequency (RF) 
range, a light source (laser), optics for collimating and guiding of light 
beam, detection components, and data collection, storage, analysis and 
display components. The sample cell, depicted schematically in FIG. 3, 
contains the mixture of the macromolecules to be analyzed. Its walls must 
be transparent to light, and are made of glass or quartz. The cell may be 
immersed in a bath of index-matching fluid to minimize the scattering of 
the laser at the glass-air interface. The electrodes are comprised of 
noble metal, they may be shielded by a suitable insulator, leaving only 
the tips unshielded. The edge of one of the electrodes is displaced from 
the edge of the other electrode so as to create a non-uniform field. The 
electrodes are aligned so that a part of the incident light beam hits the 
tip of an electrode to create a heterodyne mode. The RF source provides 
the electric power needed for an electric field and field gradient 
generation. The light source provides a coherent (laser) beam at a 
wavelength suitable for the mixture to be analyzed, so that components in 
the mixture do not absorb the light and produce heat. The optics consist 
of lenses and mirrors to focus the beam into a small area in the sample 
cell and to convey the scattered light to the detection system. The 
detection system comprises of a photomultiplier tube or a photon counter, 
an analog-to-digital converter to convert the light pulses into digitized 
input, a correlator which uses the digitized input to calculate the time 
autocorrelation function, C(.tau.), which can be stored using a digital 
computer, and analyzed on-line. or thereafter. 
The number of components in a mixture is analyzed by inducing directed 
(non-Brownian) motion in particles using an electric field gradient. This 
amounts to `modulating` the exponential decay time of the Brownian motion 
measured in DLS experiments. The modulation imposes a sinusoidal component 
onto the exponentially decaying autocorrelation function of the form: 
EQU C'(.tau.)=C(.tau.)e.sup.-iq.multidot.v.tau. ( 7) 
where C(.tau.) is the time autocorrelation function for the Brownian motion 
(Equations 1 or 4), i=.sqroot.-1, and v is the instantaneous velocity of 
the particles undergoing directed motion under the applied external field 
in the scattering volume. The prime in C'(.tau.) indicates that a field is 
applied. If the imaginary part is ignored, equation 7 can be written as: 
EQU C'(.tau.)=C(.tau.)cos(q.multidot..v.tau.), (8) 
Analysis of data may be performed by the Fourier transform after removal of 
the component of the spectrum due to the Brownian motion. To detect the 
number of components, or dispersions, in a system, each with its 
characteristic v, the Fourier transform of C'(.tau.) is performed and the 
number of peaks in the transform are related to the number of dispersions 
present. In addition to the determination of the number of components in 
the mixture from the peaks in the transform (v space), the peaks may be 
assigned to species present in the dispersion. Each peak in v space 
(spectrum) under normalized conditions of field strength and gradient 
would give a mobility characteristic of an individual population present. 
In this invention, these mobilities are called the dielectrophoretic 
mobilities.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In referring to the drawings, and in particular FIG. 4, a schematic is 
shown of one illustrative embodiment of a device to carry out the 
identification of macromolecules and biological cells based on their 
dielectrical properties. The device includes a coherent light source 
(laser) 1, a sample cell 3 which contains the solution under study, 
platinum electrodes 5 and 7 for creating a field gradient, a source 9 for 
creating oscillating electrical field with frequency in the radio 
frequency (RF) range and an RF amplifier, if necessary, a detector or 
photomultiplier 11 positioned at a specified scattering angle, a digital 
correlator 13, and the associated electronics and controls normal to DLS 
measurements, such as those obtained from Brookhaven Co., of Holtsville, 
N.Y. 
The sample cell 3, depicted schematically in FIG. 3, contains the mixture 
of the macromolecules to be analyzed. Its walls must be transparent to 
light, and are preferably made of glass or quartz. The cell may be 
immersed in a bath of index-matching fluid 15 (FIG. 3) to minimize the 
scattering of the laser beam 17 at the glass-air interface. The electrodes 
5 and 7 are comprised of noble metal, such as platinum and may be shielded 
by a suitable insulator, leaving only the tips 19 unshielded. The edge of 
one of the electrodes is displaced from the edge of the other electrode so 
as to create a non-uniform field, as depicted in FIG. 2. The electrodes 
are immersed into the sample cell and aligned so that a part of the 
incident light beam hits an electrode to create a heterodyne mode. The RF 
source 9 provides the electric power needed for an electric field and 
field gradient generation, and may be amplified by using a broad-band RF 
amplifier. The light source provides a coherent (laser) beam 17 at a 
wavelength suitable for the mixture to be analyzed, so that components in 
the mixture do not absorb the light and produce heat. The optics consist 
of lenses 21 (FIG. 4) to focus the beam into a small area in the sample 
cell and to convey the scattered light 23 to the detection system 11. 
The detection system comprises of a photomultiplier tube 11 or a photon 
counter, an analog-to-digital converter 25 to convert the light pulses 
into digitized input, a correlator 13 which uses the digitized input to 
calculate the time autocorrelation function, C(.tau.), described above, 
which can be stored using a computer 27, and analyzed on-line or 
thereafter. 
The procedure for the measurements is affected by immersing the electrodes 
in the sample cell 3 containing the solution under study. The sample cell 
may be surrounded by a bath of refractive index-matching fluid 15, which 
may be used to also control the temperature. An oscillation electrical 
signal (typically sinusoidal) from a function generator is amplified 
through a broad-band RF amplifier and fed to the two electrodes 5 and 7. 
The field gradient is achieved by alignment of the two platinum electrodes 
so that their edges are displaced from each other (as best seen in FIG. 
3), resulting in a non-uniform field. A laser beam 17 (e.g. an Argon ion 
or a He-Ne laser) impinges on the solution in sample cell 3. The laser 
beam also impinges on the uninsulated tip 19 of the electrode 5 to 
generate a heterodyne dynamic light scattering mode as discussed above. A 
DLS spectrum may be collected with no applied field to determine the 
parameters obtainable from the normal DLS measurements (diffusion 
coefficient, hydrodynamic radius). The field is turned on and a frequency 
of the RF field is selected. Measurements in the presence of the field are 
then recorded. The field strength and gradient, the field frequency, and 
the scattering angle may be adjusted to achieve the desirable effect on 
the dispersion under study. 
Analysis of data may be performed by the Fourier transform as follows: The 
component of the spectrum due to the Brownian motion is removed to avoid 
artifacts in the Fourier transform, as the Brownian motion of the 
macromolecules present will be superimposed by the motion under the 
dielectrophoretic effect. This may be accomplished by curve-fitting the 
C'(.tau.) to an exponential function since DLS autocorrelation functions 
from Brownian and other random motions are exponential. This exponential 
fit is subtracted from subsequent C'(.tau.) to give the oscillations due 
to the dielectrophoretic effect. A Fourier transform is then performed on 
the pure oscillations. The peaks in the transform (v space) may be 
assigned to species present in the dispersion. The velocities in the 
Fourier transform may be normalized (using the applied field strength and 
gradient) to assign specific velocities (mobilities) for the species. 
ADVANTAGES OF INVENTION 
The present invention offers a new method for studying particulate matter 
in solution, with distinct advantages, including: 
i. High sensitivity measurements for biological cell suspensions: For 
biological cells, their apparent high dielectric constants would prove 
advantageous to producing large signals. The present invention will 
furnish a `signature` to characterize `normal` or `healthy` populations in 
cases of polymers or biological cells, respectively. 
ii. Applicability to neutral populations, providing a greater leverage in 
characterizing mixed populations of a large class of macromolecules. 
Macromolecules that are neutral (but polarizable) will still experience 
motion under the field. This method may be used to resolve neutral 
particle distributions and biological cells at their isoelectric points. 
iii. The frequency dependence of the dielectrophoretic mobility provides an 
important variable in the present invention The frequency of the applied 
field may be used to selectively enhance the mobility of certain 
populations in solution mixtures. Biological cells show some 
characteristic response to the field frequency. It has been shown that 
some cells respond specifically to certain frequencies of the field, 
experiencing greater effect from the field at those frequencies. Since the 
state of a normal biological cell corresponds to a certain configuration 
of charges inside and on the cell surface, the present invention can be 
used to probe a biological cell and indicate changes in its state. 
iv. The present invention furnishes a fast means (seconds to minutes) to 
study dielectrophoretic behavior. 
v. Resolution improvement: DLS has limited resolving power of components in 
solution due to the strong overlap of the autocorrelation functions as 
discussed earlier. The enhancement of the resolving power is due to adding 
structure to the composite autocorrelation functions. There are at least 
three factors that contribute to resolution in the present invention: 
First, the difference in the dielectric constant of the species present, 
as apparent from Equations 5 and 6. As mentioned earlier, this effect 
would be particularly pronounced for biological cells. Studies on live and 
dead cells have shown that the two populations are affected differently by 
dielectrophoresis. Second, the difference in volume between the species 
present as indicated by Equation 3. In Example 2 below, measurements 
indicate that neutral latex particles 2.9.mu. and 4.1.mu. in diameters can 
be easily identified. Third, the frequency of the applied field. As 
indicated from earlier discussion, depending on the mechanism of 
polarizability, different populations would `resonate` with specific 
frequencies. 
vi. Low heat evolution, as no or insignificant electrolysis would occur, as 
the electrodes can be isolated from contact with solutions. 
vii. Elimination of electrolysis gas bubbles and electrodic polarization, 
due to the use of high frequency oscillating fields. 
A potential limitation is a clustering of particles or cells, called `pearl 
chain formation` which occurs when the field strength is larger than a 
certain threshold value. Pearl chains can potentially complicate the 
interpretation of the results, as they may form their own peaks in the 
Fourier transform. The chains are large and their formation time is 
usually longer than the measurement time. Since they are large, they are 
susceptible to precipitation. The formation of pearl chains may be avoided 
altogether by applying a field lower than the threshold value which is 
required for their formation. Another limitation in biological cell 
suspensions may arise due to the high conductivity of the solution, which 
may cause heat generation. This may be remedied by the use of certain 
buffers, electrodes, insulation and geometry. 
In referring to the drawings, FIG. 1 is a schematic depiction of a dynamic 
light scattering spectrometer, which consists of a laser light source 1 
that provides a coherent beam 17. The beam 17 enters a sample cell 3 which 
contains a sample 29 to be analyzed. Scattered light 23 at an angle 
.theta. leaves the cell 3 and enters a photomultiplier tube 11. The 
photomultiplier 11 converts the scattered light 23 into voltage pulses. 
The voltage pulses are transferred to an analog-to-digital (A-D) converter 
25 used by a digital correlator 13. The A-D converter 25 converts the 
voltage pulses into digital output and the digital correlator 13 uses the 
digital output to calculate the time autocorrelation function C(.tau.). 
The result of the autocorrelation is stored in a computer 27 where the 
data may be displayed and analyzed. 
FIG. 2 diagramatically shows the dielectrophoretic effect when an 
electrical field, represented by the curved lines, is made to be 
non-uniform by virtue of the configuration of the electrodes, 5 and 7. In 
the top panel A a neutral but polarizable macromolecule M is subjected to 
the field which polarizes the macromolecule and subjects the macromolecule 
to experience a higher force on its end facing the negative electrode. 
This forces the macromolecule toward the cathode. Upon a reversal of the 
polarity of the field, as shown in the lower panel B, the macromolecule M 
will experience a similar effect, but will not change its direction of 
motion albeit the reversal in the polarity of the applied field. 
The configuration of the sample cell 3 and electrodes 5 and 7 are shown in 
FIG. 3. The sample cell includes a sample holder made of transparent 
material which, in turn, is placed in another clear container 31. The 
container 31 is preferably filled with an index-matching fluid 15 and is 
covered with an insulating cover 33. The cover 33 also serves to support 
the insertion of two insulated platinum electrodes 5 and 7. The electrodes 
5 and 7 are connected via a connector 35 to a source 9 of an oscillating 
electrical voltage, which is conducted to the solution via the uninsulated 
tips 19 of the electrodes, which are of different lengths to generate a 
non-uniform field. The incident light beam 17 partially hits the 
uninsulated tip 19 of one of the electrodes to create heterodyne effect. 
Scattered light 23, at an angle .theta., can be collected and analyzed as 
in FIG. 1. 
FIG. 4 schematically shows the configuration of a device to carry out the 
identification of macromolecules and biological cells based on their 
dielectrical properties according to the present invention. The device 
includes a laser light source 1 which emits a beam 17. The laser beam 17 
is passed through a focusing lens 21 and into the sample solution, 
contained in a sample cell 3 made of transparent material. The sample cell 
3 is placed in another clear container 31 which may be filled with an 
index-matching fluid 15 and covered with an insulating cover 33. The cover 
33 also serves to support the insertion of two insulated platinum 
electrodes 5 and 7 which have uninsulated tips 19. The electrodes 5 and 7 
are connected via a connector 35 to a source 9 of oscillating electrical 
voltage, which is conducted to the sample solution via the uninsulated 
tips of the electrodes. The electrode tips are of different lengths to 
generate a non-uniform field when the light beam 17 impinges on the 
electrode tips. Where the incident light beam 17 partially hits the 
uninsulated tip 19 of one of the electrodes a heterodyne effect is created 
and scattered light 23 exits sample cell 3 at an angle .theta.from the 
emitted beam 17. 
The scattered light 23 enters a photomultiplier tube 11 which converts the 
scattered light into voltage pulses. The output from the photomultiplier 
tube 11 is received by an analog-to-digital (A-D) converter 25 which 
converts the signal from the photomultiplier tube 11 into a digital 
output. The digital output from the A-D converter 25 is received by a 
digital correlator 13 which then calculates the time autocorrelation 
functions C(.tau.) and C'(.tau.). The data from the autocorrelation 
functions are then stored in a computer 27 where the data may be analyzed 
and displayed. 
FIG. 5 is a chart which plots the autocorrelation function C'(.tau.) 
against time shift .tau.for different voltages which are applied to a 
2.times.10.sup.-6 gram/ml sample of 0.98 micron latex sample obtained 
from Polyscience Corporation. The scattering angle .theta. was at 
90.degree., the sample temperature was 23.degree.C, and the laser beam 26 
was produced by an Argon ion laser operating at 488 nm. The graph of FIG. 
5 plots the autocorrelation function against time with various voltages 
(0V to 50V) being passed through the electrodes. The frequency of the 
oscillating sinusoidal field was 350 kHz frequency. The inset (FIG. 5A) 
plots the Fourier transform of the data from FIG. 5, and shows a 
predominant single peak representing the velocity of the 0.98micron latex 
particles under the field. 
FIG. 6 is a graph similar to the graph of FIG. 5 of the autocorrelation 
function C'(.tau.) against time for dielectrophoretic DLS on 4.1 micron 
latex sample using the device of FIGS. 2 and 4. The inset (FIG. 6A) shows 
the Fourier transform of the data. The graph of FIG. 6A shows a 
predominant single peak representing the velocity of the particles under 
the field. 
FIG. 7 is a graph showing data on a mixture of 4.1.mu. and 2.98.mu. latex 
samples which were diluted to 2.times.10.sup.-5 gram/mi. The remaining 
conditions were similar to those of Example I. The device of FIGS. 3 and 4 
was used under a field of 50 volt/cm. The data collected was treated by 
removing the component due to the Brownian motion by fitting the data to a 
single exponential, and subtracting the fit from C'(.tau.) to yield only 
the oscillations due to the field effect. The results of the Fourier 
transform are displayed in the inset (FIG. 7A). The Fourier transform 
shows two predominant peaks representing the two populations of particles 
present. 
FIG. 8 graphs the autocorrelation function against .tau. for 
dielectrophoretic DLS carried out on a Baker's yeast cell suspension using 
the device of FIG. 4 under the conditions of example below. The sample was 
diluted to 2.times.10.sup.-3 % solid, using the device described in FIG. 4 
and Example 4, with other conditions being similar to those in FIG. 5. The 
inset (FIG. 8A) shows the Fourier transform of the data. The Fourier 
transform produced a multiplicity of peaks which are stipulated to 
represent the velocity of the different species of yeast cells under the 
field. 
EXAMPLE 1 
A sample of 0.98 micron latex particles, obtained from Polyscience 
Corporation, was diluted to a 2.times.10.sup.-6 gram/ml concentration 
with distilled water. The sample was placed in the sample cell, as 
described in FIG. 4. The sample cell was placed in another glass container 
which was filled with fluid possessing a refractive index matching that of 
glass fluid container. A hole drilled in the cover served as a support of 
two insulated platinum electrodes. The electrodes were connected to the 
output of an electrical wave generator after amplification using a 
broad-band radio frequency amplifier. The tips of the electrodes were 
uninsulated and were placed staggered (different lengths) to generate a 
non-uniform field. An argon ion laser provided a beam at 448 nm which was 
focused on the sample using lens. The. beam was positioned to partially 
impinge on the uninsulated tip of one of the electrodes to create a 
heterodyne effect. Scattered light at 90.degree. was collected via a 
photomultiplier and was converted into digital output using an 
analog-to-digital converter. A digital correlator was used to calculate 
the time autocorrelation functions. The data were analyzed using a 
personal computer. The normalized heterodyne autocorrelation functions am 
displayed in FIG. 5. The top curve shows the function with no field 
application (i.e. 0V), while the lower oscillating curves show the effect 
of the application of a 350 kHz, with varying strength, as noted in FIG. 
5. The number of oscillations increased with increasing field strength, as 
predicted by Equation 8. The analysis was performed on the autocorrelation 
function collected with the application of 80 volts/cm field. The analysis 
was performed after removing the component corresponding to the and was 
accomplished by fitting C(.tau.) to a single exponential and subtraction 
the fit from C'(.tau.). A predominant single peak representing the 
velocity of the 0.98 micron latex particles under the filed is displayed 
in the inset (FIG. 5A). A single peak is predicted since the sample 
contains mainly monosized particles. 
EXAMPLE 2 
A sample of 4.1 micron latex particles was diluted to a 1.times.10.sup.-6 
gram/ml concentration. Data were collected and analyzed using conditions 
similar to those described in Example 1. FIG. 6 presents the data with no 
application of electrical field (i.e. 0V) in the top curve, and with the 
application of field with different intensities in the remaining curves. 
The inset (FIG. 6A) to FIG. 6 represents the Fourier transform of data, 
carried out as described in Example 1, and shows a predominant single peak 
representing the velocity of the 4.1 micron latex particles under the 
field. 
EXAMPLE 3 
A mixture of 2.98 and 4.1 micron latex beads (Polyscience) were prepared at 
a 1:1 ratio to give a final concentration of 2.times.10.sup.-5 gram/ml in 
each component. Measurements and analysis were carried out on the mixture 
as explained in Example 1. FIG. 7 shows the resulting normalized spectra 
from the mixture. Preliminary Fourier transform shows two distinct peaks 
in velocity domain, shown in the inset (FIG. 7A) to FIG. 7. The peaks 
represent the velocity of the 2.98 and 4.1 micron latex particles under 
the field. 
EXAMPLE 4 
A 0.5 gram sample of baker's yeast was suspended in 100 ml water. 
Measurement was carded out as explained in Example 1. FIG. 8 shows the 
resulting spectrum from the mixture collected at 80 Volts/cm of applied 
field. Analyzing the data as explained in Example 1 shows numerous peaks 
in velocity domain (FIG. 8A). 
The resolution enhancement of the peaks in discovering these populations 
would not be possible without the application of the non-uniform field as 
described in this invention. The multiplicity of peaks are stipulated to 
represent the velocity of the different species of yeast cells under the 
field. 
The foregoing summary, description and drawing, and description of the 
preferred embodiments, in addition to a variety of examples that define 
the use and application of this invention, have been previously reviewed. 
Variation or modifications to the subject matter of this invention, may be 
envisioned by those skilled in the art. Such variations or modifications, 
if within the spirit of this invention, are intended to be encompassed 
within the scope of any claims to patent protection issuing upon this 
development. The description as set forth herein is done so for 
illustration purposes only.