WO1979000834A1

WO1979000834A1 - System for analyzing chemical and biological samples

Info

Publication number: WO1979000834A1
Application number: PCT/US1979/000194
Authority: WO
Inventors: S Hahn
Original assignee: High Stoy Tech
Priority date: 1978-03-24
Filing date: 1979-03-23
Publication date: 1979-10-18
Also published as: IL56943A0; EP0015937A1

Abstract

A method and apparatus are provided for analyzing chemical and biological samples, comprising at least one slide (12, 14) having an electrically conductive surface (16, 18) thereof; a sample (24) to be analyzed; means (10, 20) to induce an electro-static field in said sample to cause particles within said sample to selectively re-orient themselves or migrate to a new position; and means (22) to analyze said migrating particles. In one embodiment an electrical field activates a nematic fluid stain (47) for identification of particles within a sample. By altering the electrical characteristics of a sample and passing an electrical waveform through the sample, the resultant change in waveform is indicative of the sample's constituency.

Description

SYSTEM FOR ANALYZING CHEMICAL AND BIOLOGICAL SAMPLES

BACKGROUND OF THE INVENTION:

The present invention relates generally to a small particle analysis system and, in particular, to a system wherein biological or other colloidal specimens are placed on the specially designed microscope slides described hereinafter and subjected to an electrostatic field and analyzed using a conventional optical microscope or a conventional computer analysis system. The specimen examined with this system need not be stained, may be stained with conventional optical dyes, or, in certain instances, may be stained with special substances which are electrostatically active. The special microscope slides on which the specimens are placed form part of an electrostatic system which activates, changes orientation, or otherwise cause movement of particles or cells within the specimen and to permit viewing or analysis thereof either automatically or with a conventional optical microscope.

The interpretation and analysis of specimens, particularly biological specimens, using a conventional light microscope has sometimes proven difficult due to the subjectivity of the human observer involved. In particular, recognition of subtle optical variations is often difficult since biological specimens are optically complex and variable. Many attempts have been made in recent years to automate biological analysis, however, in many instances, this has proven difficult due, in part, to problems associated with the preparation of the biological specimen itself. Furthermore, chemical staining of slides is of times somewhat of a "black art" with technique varying from researcher to researcher.

OMPI . IPO In most instances, biological specimens are placed or mounted on a transparent glass microscope slide and chemically stained prior to processing. To effect chemic staining, well-known dyes or stains such as, for example, Wright's Stain and Giemsa Stain, are used and then the mo is "fixed" to the slide. The biological specimens uptake the dye in varying ways and when observed optically, rend pathological information to the observer.

Traditionally, using a standard optical microscope, the technician scans the slide looking through the microscope and manually counts cell types or looks for specific pathology. Automated systems analyze specimens either in a slide system using a substage which is digitally moved or in a flow through system where the sample is subjected to a series of light beams such as, for example laser beams, while looking for parameters such as absorba transmission and reflectance. Software is programmed to look for certain optical combinations or certain limits. Such automated systems are capable of analyzing a large number of slides or sample streams in short periods of ti and then generate data or histograms which may prove usef in establishing patterns and trends.

Sample preparation methodology, on the other hand, is somewhat less clearly defined. The problems in sample preparation are numerous and varied and include an inabil to repeatedly optically stain a sample in the same or similar way; problems with repeatability using stains of different batches due to factors such as chemical composi tion and purity differences of the stain, the amount of stain used, the skill of the technician, the temperature of the environment, the length of the fixing cycles and the appearance of any debris in the sample, etc. The presence of these variables make it sometimes difficult t repeatedly and reliably interpret optical data. This problem is further compounded by the fact that in any given cell population, a wide variety of different cells may occur, each with a subtle variation in physical and optical parameters.

Some attempts have been made to replace optical stain systems heretofore used with systems employing non-optical substances to permit examination by non-optical means. For example, radioactive tracer elements have been used to stain specimens for subsequent analysis with a scintillation counter. Difficulties, however, have been encountered in such systems with respect to resolution and system complexity.

Still another approach to sample analysis has been an electrophoresis systems.which measures the velocity of particulate or chemical constituents therein when the sample is subjected to an electric current. For example, such systems can measure the membrane potentials of a protein cell or the so called zeta potentials of a colloid and compare it to a known or standard measurement for purposes of identification. Studies have shown that cells have a specific electrical potential known generally as their membrane potential which varies with biological conditions of the particular cell. Similarly, particles in colloidal suspension have a specific electrical potential referred to as their zeta potential. Since different biological or chemical samples have different particle potentials, a system which is capable of inducing and measuring this potential is thus capable of sorting and identifying such cells or particles.

Heretofore, two types of methods were used to measure membrane or zeta potential—a direct and an indirect method. The direct method entails the insertion of a pair of needlelike microelectrodes into the cell or particle and then measure its electrical characteristics. An example of such a direct analysis is described in an article entitled "Membrane Potential of Mitochondria Measured with Microelectrodes" which appeared in Science, Vol. 195, dated 4 March 1977, at page 898-899. Due to the size of such cells, generally in the micron range, an the fact that their membranes must be pierced, such a measurement is extremely difficult to make and the risks of contamination or traumatic shock to the specimen are quite high.

The indirect methods heretofore used generally relied on flow through type technique wherein the particles were suspended in a fluid which was then passed through an analyzing chamber upon the introduction of an electrical current which flows through the fluid. Examples of such systems are the Zeta-Meter manufactured by Pen Kem Corporation of Croton-on-Hudson, New York, and the Cytopherometer of Carl Zeiss. See also the discussion of the zeta potential which appeared in the March 9, 1964 edition of Chemical Processing entitled "Fast Accurate Measurement of Zeta Potential Proves Boon to Processes". All of these systems directly introduce an electric curre into the specimen and measure the resultant velocity of t moving particles (electrolytic migration) . With particle velocity known, mathematical treatment can then be applie to develop potential information. The disadvantages of such systems include the fact that as the current is induced in the sample or specimen, the sample resistance causes the sample to heat up, thus altering the character istics of the specimen. For example, the velocity of a cell or particle is dependent upon a given fluid viscosit any change in the viscosity can affect the results. Additionally, the electrical current may have an adverse affect on biological specimens. Furthermore, such a system may contaminate the sample since metallic electrod have to be introduced into the fluidized sample to permit the generation of an electrical current. In addition, the size of the samples or specimens analyzed with these systems tend to be rather large, i.e. 20cc to 30cc and processing time can become quite long. Against the foregoing background, it is a primary objective of the present invention to provide an analytic system capable of measuring particle charge including the cell membrane or zeta potentials of a given specimen.

It is another object of the present invention to provide an analytic system capable of measuring the membrane or zeta potential of a given specimen using an indirect approach wherein no electrical current is passed through the specimen.

It is another objective of the present invention to provide an electrical capacitance analysis system for analyzing biological and small particle samples.

It is yet still another objective of the present invention to provide an electrical capacitance analysis system wherein specimens are placed between microscope slides and an electrical capacitance is induced therein.

It is still another objective of the present invention to provide a sample staining technique which is based on usage of nematic fluids.

SUMMARY OF THE PRESENT INVENTION

To the establishment of the foregoing objects and advantages, the present invention briefly comprises method and apparatus for analyzing chemical and biological specimers comprised of at least one transparent substrate having at least one conductive surface thereof; a sample to be analyzed; means processing the sample by inducing an electric field in the sample to cause particles within said sample to selectively migrate to a new position or re-orient themselves; and means to analyze said processed sample. BRIEF DESCRIPTION OF THE DRAWINGS:

The foregoing and still other objects and advantages of the present invention will be more apparent from the following detailed explanation of the invention in connection with the accompanying drawings wherein:

Fig. 1 is an exploded view of a pair of slides and a schematic showing of an electrical circuit connected thereto;

Fig. 2 is a side elevational view of the slides of Fig. 1 in juxtaposition and a schematic showing of an electrical circuit connected thereto;

Fig. 3 is an exploded view of a pair of slides provided with electrically conductive areas, a specimen under test and an insulator member;

Fig. 4 is a side elevational view of the members of Fig. in assembled condition;

Fig. 5 is a partially sectioned elevational view of a pair of slides provided with a well for receiving a sample;

Fig. 6 is a top plan view of the upper slide of Fig. 5;

Fig. 7 is a top plan view of the lower slide Of Fig. 5;

Fig. 8 is a bottom plan view of the lower slide of Fig. 5;

Fig. 9 is a side elevational view of a slide system connected to a power supply;

Fig. 10 is a schematic showing of the position of charged particles without a voltage applied to the system of Fig. Fig. 11 is a schematic showing of the position of charged particles with a DC potential applied to the system of Fig. 10;

Fig. 12 is a schematic showing a slide system in which the AC transfer characteristics of the sample are measured;

Figs. 13-14 are a schematic showing of slide systems employing nematic stains in the sample wherein in Fig. 13 there is no potential applied to the sample and in Fig. 14 there is a potential applied; and

Fig. 15 is a schematic showing of a system of this invention employing a ladder type electrode arrangement.

DESCRIPTION OF THE PREFERRED EMBODIMENTS:

The present invention constitutes a system for analyzing and identifying charged particles such as are found in biological samples involving cells, colloidal suspensions and other fluid samples. All of these samples contain some form of charged particle (for example, cell membrane potential or zeta potentials) or particles whose behavior is altered in the presence of electric fields. The sample involved is placed in a special microscope slide system and then subjected to an electric field (DC or AC) in order to create movement of the particles or changes of state within the particles. A microscope is then used to observe the change, or, alternatively, an automated, computerized method of determining sample change may also be employed.

In Fig. 1 there is shown the basic preferred form of the invention utilizing a DC power supply 10 and employing electrostatic energy for the movement of the particles. Two standard glass or transparent plastic microscope slides 12,14 are employed, each of which has, on one

OMPI ». WIPO surface, an optically clear, etched conductive electrode 16,18 composed of tin oxide or similar material. The position of these optically clear conductive electrodes is such that when the two slides are pressed together, the circular upper electrode 16 is physically directly on top of the circular lower electrode 18. These two electrodes 16,18 are then connected to a stable, high voltage DC supply 10 whose output can be precision controlled by a voltage divider 20 and the voltage appearing across the slide system is measured by a voltmeter 22. It can be seen from Fig. 1 that when these two slides are pressed together and connected to a DC pow supply, a capacitor is formed with the slides 12,14 as we as any material between the slides, forming part of the dielectric. A drop of the sample 24 to be examined is placed on the bottom slide and the slides are then presse together, as shown in Fig. 2, resulting in a very thin fi of sample 24 sandwiched between the two microsope slides 12,14 and subjected to the electrostatic field existing between the conductive electrodes on the top and the bottom of the slides.

A small capacitor will form between the two conductive circles facing each other. It is irrelevant as to the extent to which the sample spreads along the surface between the two slides since this excess of sample does not form part of the capacitance system. The same pressu is to be applied to each test in order to keep uniform the thickness of the sample film. It will be noted that the conductive leads to the capacitor plates are not opposite each other and, accordingly, do not form part of the capacitor.

When a controlled DC voltage is applied to the electrodes the particles within the sample will begin to move upward, providing that the polarity of their own charge is opposite to the polarity of the upper electrode. The

O , W electrostatic force which is exerted on the charged particles is determined by a number of complex parameters including voltage, dielectric constants of the materials used, thickness of the slides, amplitude of the particle charge, fluid viscosity and others. Furthermore, the velocity with which the particle moves towards the oppositely charged electrode is also dependent upon numerous factors including voltage impressed across the system, distances involved, sample viscosity, etc. It will be understood that the velocity of charged particle movement could be increased if the voltage across the system is increased or if the electrodes are brought closer to the particles. Consequently, another embodiment of this system is shown in Figs. 3 and 4 where the two optically clear conductive electrodes 34,35 are etched on the microscope slides 32,33 on the side thereof adjacent to the sample. Since this system is a capacitor system, the flow of current must be prevented and, accordingly, a very thin insulator such as a 0.001" thick mylar sheet 30 is placed between the sample 36 and the upper slide. Consequently, as shown in Figs. 3 and 4, the sample in this particular embodiment of the invention electrically becomes a part of the bottom electrode 35, the mylar sheet 30 serves as the dielectric, and the upper electrode 34 attracts the oppositely charged particles through the mylar sheet. The movement of the particles are studied by means of microscope M.

Figs. 1, 2, 3, and 4 disclose versions of this invention in which a droplet of the sample is formed into a thin film (a few microns thick) when the two slides are squeezed together. Another embodiment of this invention is shown in Figs. 5-8 which uses the optically clear conductive electrode slide system the same as previously described. However, in this embodiment of the invention the bottom slide contains a small well 40 in which the drop of sample is placed. In this embodiment of the invention the drop is subjected to electro¬ static forces rather than the thin film previously described. The actual operation of the system using DC potentials is shown in Fig. 9. The sample is placed between the two slides which are then squeezed together forming a thin fi as previously discussed. (In the well type slide, the entire sample remains in the well.) The system is then connected to a precision DC power supply 21 whose output can be controlled and measured in a precise fashion. Thi slide sandwich is then placed into a standard optical microscope which is focused in such a manner that the foc point of the microscope falls on the top surface of the film or the well sample formed within the sandwich slide system. Point F. When no voltage is applied, as shown in Fig. 10, the charged particles P are evenly distributed throughout the sample or may even be at rest within the sample film favoring the bottom slide. As soon as a DC potential is applied to the system, a capacitor is formed and an electrostatic field is generated within -the sandwi This field causes particles P to move upward toward the upper slide providing that their charge is opposite to th charge of the upper electrode. The velocity with which these particles move is of course dependent on the factors which have previously been discussed and include power su voltage and dielectric properties of the sandwich. After short period of time, certain charged particles will migr into the focal point F of the microscope where they can b visually analyzed by the observer. Consequently, using t technique, samples containing charged particles which are distributed at random can be sorted and analyzed by struc ing an electrostatic slide system as shown.

Hereinabove there has been described a slide system for t analysis of charged particles using a DC power supply. Y another embodiment of the invention is a system in which DC power supply is not used. An embodiment of this inven is shown in Fig. 12 where the sample is placed into the sl system as previously discussed. However, in this embodime an AC signal 49 consisting of a sine wave, a square wave. or some other complex AC wave shape, is injected into the system. This causes the system to look like an AC fed capacitor in series with the load resistor 51. The AC fed capacitor consisting of the slides 12 and 14 with their conductive electrodes 18 and 16 and the sample 47 which forms part of the dielectric system. An oscilloscope 53, or similar measuring device, is then used to observe the AC transfer characteristics of the sample. Consequently, internal conditions and changes in the sample could be observed, for example, if the input signal was a sine wave 49 which was distorted into a pulse 55. In this manner, a number of electrical parameters of the sample can be measured, including the sample's impedance, biological changes when subjected to pulses, ability to produce harmonic distortion in sine wave inputs, to name only a few. Such an AC transfer technique can be applied to a wide variety of biological and chemical samples, and staining may or may not be used.

Another embodiment of this invention requires an AC power supply, but in this embodiment the sample must be stained by a special stain which alters the electrical character¬ istics of the sample. A typical stain that would achieve this effect is composed of a nematic fluid (liquid crystals— Schiff bases) including but not limited to compounds as marketed by Eastman Kodak, Rochester, New York, such as Methoxybenzylidene-p-butylaniline; p-ethoxybenzylidene-p- aminobenzonitri (PEBAB) ; and anisylidene-p-aminophenylacetate (APAPA) . The molecules of such compounds are in a particular molecular arrangement and alignment in the absence of an AC field. As soon as an AC field is impressed across such compounds, the molecules lose their ordered alignment and give the nematic fluid an opaque appearance. Such nematic fluids (commonly termed "liquid crystals") have found widespread use in indicia displays in wristwatches and electronic instruments to indicate digits and alphanumerics. As previously stated, and as Fig. 13 shows, a sample 47 i stained with such a nematic fluid and then placed into th sandwich slide system. Using the microscope previously described, the sample 47 will be randomly clear or trans¬ lucent as in Fig. 13 when no AC voltage is applied across the system. In Fig. 14 an AC voltage is shown applied across the system. Those cells or particles which are prone to selectively uptake the nematic stain become opaque within the sample itself. Using such a technique, the optical microscope M can then be used to identify those particles which as a result of staining have taken up the nematic fluid.

With reference to Figs. 1-11 there has been discussed the application of DC fields to fluid samples. In all of these instances, where the sample was within a DC field, the motion of the particles was in a vertical direction. That is, when a DC field is applied the particles migrate from the bottom slide to the upper slide in the event tha their charge is opposite to the charge of the upper electrode.

An additional embodiment of the invention shown in Fig. 1 utilizes a slide where the motion of the charged particle is in a horizontal direction. In this system a series of optically clear conductive coatings are etched in the for of stripes 61, 62, 63, 64 on the bottom slide 60. Each o these lines are connected to a different DC potential provided by a voltage divider 65 connected across a high voltage DC power supply 66. The sample 67 is placed on a mylar sheet 68 at the end of the slide nearest the lowest potential electrode. As soon as the DC power is turned o the particles begin to move toward the oppositely charged series of electrodes. Over a period of time a sorting of the particles will take place dependent upon their own charge as well as the potential gradient formed by the slide system. At the end of such time period the slide

OM

/j. WIP system can be scanned with a microscope in the horizontal plane to examine the various populations of charged particles which have gathered at the various potential gradient points.

The system of the present invention may be applied to a number of applications and is useful for medical or bio- medical diagnostic techniques such as, for example, the analysis for sickle cell anemia, determination of male/ female cells in animal husbandry and in pathological fields such as cancer detection. In the chemical industry, the system has applications for checking the efficacy of colloidal suspensions and in the analysis of bond integrity by measuring the degrees of coagulation. Additionally, the system can be used for analyzing chlorinated water.

The system of this invention is adapted to process unstained specimens, as well as those stained with traditional optical dyes and with substances whose electrical characteristics change in the presence of an electric charge.

Thus, it has been shown that the particles are charged to varying degrees permitting the particles to move up or down or horizontally or to be re-oriented responsive to an electrostatic field. By altering the electrical character¬ istics particles may be differentiated by means of an oscilloscope or other electronic measuring means.

Having thus described the invention with particular reference to the preferred forms thereof, it will be obvious that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

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Claims

WHAT IS CLAIMED IS:

1. Apparatus for the analysis of specimens containing particles of matter responsive to electrical potentials comprising:

(a) a first substrate;

(b) a first electrically conductive member carried by said substrate;

(c) a second electrically conductive member positioned above said substrate and spaced therefrom providing a cavity or surface for receiving a specimen to be analyzed, the specimen is part of the dielectric of the capacitor formed by the first and second electrically conductive members, the specimen being supported on said first substrate;

(d) means connected to said electrically conductive members for creating an electric potential or field between said conductive members for physically altering the specimen; and

(e) means to analyze the specimen.

2. The apparatus of Claim 1 wherein the first electrically conductive member is on the surface of the said substrate.

3. The apparatus of Claim 1 wherein the second electrically conductive member is transparent.

4. The apparatus of Claim 3 wherein the second electrically conductive member is carried by a transparent substrate.

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5. The apparatus of Claim 1 wherein said first substrate has a well for receiving a specimen.

6. The apparatus of Claim 1 wherein said means for creating an electric potential generates signals of a repetitive shape and includes means for detecting and displaying said signals after passing through the specimens.

7. The apparatus of Claim 1 including an electrically non- conductive member interposed between said first and second electrically conductive members.

8. The apparatus of Claim 3 wherein said substrate is composed of glass and said second conductive member is comprised of tin oxide.

9. The apparatus of Claim 1 wherein the first electrically conductive member is composed of a plurality of electrically, isolated elements connected to said means for creating a potential and wherein said last named means provides a higher potential to each successive element.

10. Apparatus for analyzing specimens containing particles susceptible to response to electrical potentials, said apparatus comprising:

a pair of substrates in juxtaposition to each other, the opposing faces having electrically conductive surfaces;

a specimen to -be analyzed positioned between said opposing surfaces; means for applying an electrical potential to said electrically conductive surfaces whereby an electric differential will exist between said surfaces so as to cau particles within said specimen to selectively re-orient; a

means to analyze said re-oriented particles.

11. The apparatus of Claim 10 wherein said substrates are composed of glass and said conductive surface is comprised of tin oxide.

12. The apparatus of Claim 1 wherein said means to analyz comprises a microscope focused at the resulting re-oriente particles.

13. The apparatus of Claim 10 wherein the particles of said specimen are stained with a dye.

14. The apparatus of Claim 10 wherein the particles of said specimen are stained with an electrically active compound.

15. The apparatus of Claim 14 wherein said electrically active compound is a nematic fluid.

16. Apparatus for analyzing chemical and biological samples containing particles responsive to electric potential, said apparatus comprising:

at least two slides in juxtaposition to one another, each of said slides containing on their inner surface at least one electrode in electrical connection with an external power source; sa ple to be analyzed, said sample adapted to be placed between said electrodes;

means to introduce an electrical charge to said electrodes to cause particles within said sample to migrate to a new position; and

means to analyze said migrating particles.

17. The apparatus of Claim 16 wherein a DC current is introduced into said electrodes.

18. The apparatus of Claim 16 wherein an AC signal is introduced -in said sample.

19. The apparatus of Claim 18 wherein said AC signal is a harmonically rich wave.

20. The method of analyzing biological and chemical samples containing particles responsive to electrical signals, said method comprising:

mounting a sample on an electrically conductive slide sandwich structure;

inducting an electrical charge in said structure to cause particles within said sample to re-orient; and

analyzing the resulting re-oriented particles.

21. The method of Claim 20 wherein the particles are stained to render them electrically responsive.

22. The method of Claim 21 wherein said particles are stained with a nematic fluid.

23. The method of Claim 20 wherein said particles are subjected to a DC potential.

24. The method of Claim 20 wherein said particles are subjected to an AC signal.

25. The method of Claim 24 wherein the AC transfer characteristics of the particles are determined.

26. The method of Claim 24 wherein the AC signal is a harmonically rich wave.