US3710933A - Multisensor particle sorter - Google Patents

Multisensor particle sorter Download PDF

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US3710933A
US3710933A US00211473A US3710933DA US3710933A US 3710933 A US3710933 A US 3710933A US 00211473 A US00211473 A US 00211473A US 3710933D A US3710933D A US 3710933DA US 3710933 A US3710933 A US 3710933A
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cells
flow chamber
cell
electrical
inlet tube
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M Fulwyler
J Steinkamp
J Coulter
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US Atomic Energy Commission (AEC)
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/005—Beads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1019—Associating Coulter-counter and optical flow cytometer [OFC]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1024—Counting particles by non-optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1028—Sorting particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/103—Particle shape
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
    • G01N2015/1406—Control of droplet point
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1477—Multiparameters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1486—Counting the particles

Definitions

  • ABSTRACT An apparatus for rapidly and automatically analyzing and sorting minute particles on the basis of certain preselected characteristics. Particles flow in suspension through a flow chamber having multiple sensing means to detect preselected physical or chemical characteristics of each particle and then are jetted between charging electrodes and deflection plates. Signals from the sensors for each particle are compared with preset standards, and those droplets containing particles having characteristics not meeting those standards are automatically charged by the chargingelectrodes.
  • the deflection plates provide a constant electric field which deflects charg'ed droplets away from uncharged droplets, thus sorting particles on the basis of their conformance or nonconformance to standards set for the preselected characteristics.
  • This apparatus is particularly applicable to the rapicl and automatic sorting of biological cells.
  • PAIENIEUJ/III 16 I975 OSCILLATOR SHEET 5 [IF 7 8
  • the -invention described herein was made in the course of, or under, a contract with the U.S. ATOMIC ENERGY COMMISSION. It relates to an apparatus for automatic minute particle analysis and sorting and more particularly to an apparatus wherein the volume, shape, and fluorescence of biological cells in suspension in a continuously flowing fluid are rapidly and automatically measured and analyzed to determine if the cells appear to be normal or abnormal, and cells indicated to be abnormal are physically separated from their normal counterparts.
  • cytological material e.g., for the detection of cancerous or malignant cells
  • screening of cytological material is typically done by a hierarchy of two or more levels of screening. Initially, cell samples are prescreened visually by an observer to search out those that appear to contain abnormal cells. These are then set aside for later examination by a trained cytotechnologist or pathologist who makes the final judgment as to whether the cells are indeed cancerous.
  • this method presently works well, it has a number of disadvantages. It is slow, requires considerable technician time, thus making it costly, and is nonquantitative in that the criteria of abnormality used are largely subjective. Because of the time and cost, it is difficult to apply it to very large populations.
  • apparatus is an outgrowth of that disclosed in U.S. Pat. No. 3,380,584 and allows particle separation on the basis of multiparameter analysis. It is particularly applicable to the analysis and sorting of biological cells.
  • cellular volume, small-angle light scatter, and fluorescence are measured for each cell and compared with preset standards, and cells failing to meet these standardsare separated from cells conforming to the standards.
  • Cell samples stained with an appropriate fluorescent dye are diluted and suspended in physiological saline solution and introduced into a flow chamber on the axis of a moving stream of saline solution which acts as a sheath to confine the cell stream to the central axis of the system.
  • cells flow sequentially through an orifice which serves as a Coulter volume sensor wherein cell volume is electronically measured.
  • the cells flowing in suspension in the saline solution next intersect an argon-ion laser beam.
  • the individual cells scatter light and the dye bound to the cell is excited to fluoresce.
  • the scattered light provides quantitative information on cell size and shape, and the fluorescence is a quantitative measure of any cell constituents to which a fluorescent dye is bound, e.g., DNA content.
  • Small-angle light scatter is measured in the forward direction and fluorescence perpendicular to the cell stream and the laser beam.
  • the cell suspension After passing through the laser beam the cell suspension jets out into air through a coaxially aligned nozzle at the exit end of the flow chamber.
  • a piezoelectric crystal mechanically coupled to the flow chamber is used to produce uniform droplets by regularly disturbing the .emerging liquid jet. Most cells are effectively isolated into single droplets although not all droplets contain cells and certain droplets may contain two or more cells.
  • Droplets containing selected cells are electrically charged and then deflected into a separate receptacle by a static electric field.
  • An oscilloscope monitors individual signal pulses while a multichannel pulse-height analyzer, printer, and plotter provide and record pulse amplitude distributions representative of cell volume, light scatter, and fluorescence or combinations of these characteristics.
  • a variable delay pulse generator triggered by a single-channel pulse-height analyzer produces droplet charging pulses which are delayed to allow the cell being sorted to travel from the sensing region to the point of droplet formation and charging.
  • FIG. 1 is a block diagram showing the manner in which 'the apparatus of this invention may be used in a cancer screening program.
  • FIG. 2 is a simplified view of the apparatus showing the flow chamber and the charging and deflection plates used to achieve particle sorting.
  • FIG. 3 is an enlarged simplified cut-a-way view of the sensing portion of the flow chamber.
  • FIG. 4 is a detailed cross-sectional view of the flow chamber useful with a preferred embodiment of the invention.
  • FIG. 5 is a block diagram of the optical and electrical elements of a preferred embodiment of the invention.
  • FIG. 6 is a portion of a logic and switching block diagram for the multiparameter signal processing unit indicated in FIG. 5.
  • FIG. 7 is a continuation of the diagram of FIG. 6.
  • the apparatus of this invention may readily be used for rapid and automatic multiparameter analysis and sorting of various types of particles.
  • the size of the. particles analyzed is limited by the size of the Coulter volume sensing orifice. It will be apparent that a limitation on the type of particles that can be analyzed and sorted by this apparatus is that the particles be capable of analysis on the basis of their physical and chemical properties.
  • FIG. 1 Cell samples are prepared for flow system analysis by appropriate dilution, treatment to avoid clumping, staining with fluorescent dyes, etc., as required for the particular form of automated analysis to be used.
  • the cellular parameters measured are cell volume, small-angle light scatter, and fluorescence.
  • the fluorescence measurements depend on the use of biochemically specific stains. Sensors to make these particular measurements are compatible with each other and with electronic sorting of cells.
  • the electronic sorting technique is similar to that described in US Pat. No. 3,380,584.
  • a signal from each sensor. is transmitted to a multiparameter signal processing unit, processed, and coinpared with predetermined criteria of abnormality.
  • the signals obtained from the sensors and representing measured cell characteristics are processed to yield ratios, overlapping ranges, etc., which most effectively describe abnormal cells.
  • the processed signals are electronically compared with specified standards, and the corresponding cell is designated as normal, abnormal, or ambiguous. Once the signals have been obtained, the time required for signal processing and the sorting decision is on the order of 25 users. Classification of a cell as abnormal or ambiguous produces a signal causing a droplet containing that cell to be deflected away from the droplets containing normal cells.
  • Results of analysis of thiscell may be stored separately from data for normal cells of the sample. Sorted abnormal or ambiguous cells are counterstained and held for visual examination by a cytologist. To aid his evaluation of the sorted cells, distributions of the various measured cellular characteristics or combinations of the characteristics of the entire sample or only the abnormal cells underexamination are available from processed data storage.
  • the apparatus of this invention provides both printouts and an oscilloscope display of the data.
  • the specificembodiment disclosed herein is based on the multiparameter analysis of the volume, fluorescence, and small-angle light scatter of individual cells, it will be readily apparent to one of reasonable skill in the art that the analytical and sorting techniques embodied in this invention are readily applicable to other forms of high-speed sensing, and that the electronic and mechanical components of the embodiment described may readily be altered to allow for the measurement of other parameters.
  • the small-angle light scattering sensing may be replaced with sensors capable of detecting light absorption or fluorescence at an additional wavelength.
  • FIGS. 2 and 3 illustrate the basic flow system and sensing region of the apparatus of this invention.
  • An appropriately prepared cell sample is introduced as a continuously flowing suspension into flow chamber 3 through sample entry tube 1 from pressurized reservoir 72.
  • tube 1 Within chamber 3 tube 1 is centered and extends partially through a larger tube 19 which tapers to a nozzle 22 at its lower end.
  • a continuous flow of cell-free liquid, known as a sheath liquid is introduced into tube 19 through sheath input tube 18 from pressurized reservoir 70 and flows coaxially 20 around tube. 1.
  • As the cell stream exits from tube 1 it is reduced in diameter 17 as it obtains the velocity of the sheath liquid. Relative velocities and flow rates are determined by a differential pressure regulator system.
  • sheath liquid and cells in suspension be essentially laminar in nature to avoidturbulence effects as the sheath liquid and cell suspension pass through volume sensing orifice 21 in nozzle 22.
  • the pressure differential between sheath liquid 20 and the cell stream is adjusted to provide a cell stream 28 through orifice 21 having a diameter such that most cells pass through one at a time.
  • Orifice 21 serves as a Coulter volume sensing orifice in which the impedance is changed in accordance with the volume of the cell passing through.
  • the laminar flowing sheath liquid acts not only to control the size of the cell stream passing through orifice 21 but also to center it within the orifice, thus substantially reducing electric field edge effects affecting the volume sensing.
  • cell stream 28 intersects a beam 4 of intense light.
  • Flow chamber 3 is provided with an entry port 35 through which this beam may be focused on the cell stream.
  • Ports 36 serve as exits for fluorescence 9 and light scatter 11 produced as the result of the interaction 16 of the light with a single cell as the cells flow sequentially in cell stream 28.
  • Light beam 4 intersects cell stream 28 at an angle of approximately 90. Fluorescence is given off in all directions but is only measured in a cone 9 extending at right angles to the plane ofintersection of cell stream 28 and light beam 4. These 90 angles are critical only insofar as they serve to simplify the optical measurements involved.
  • Cell stream 28 jets from flow chamber 3 through exit nozzle 26. It is essential that orifice 21 be properly aligned with nozzle 26. Misalignment may result in interference with the optical measurements because of turbulence and will affect the proper timing by which measurements associated with a particular cell are translated into a sorting signal because the cells lose their sequential and equal spacing within cell stream 28. It is also necessary that the cell stream and its surrounding sheath liquid leave flow chamber 3 at a sufficiently high velocity to form a jet 10. Because of the pressure drop associated with orifice 21 an additional source of sheath liquid is required to provide a sufficient pressure in region 30 for an adequate jet to form. This second sheath liquid input 25 occurs through tube 54 which is connected to sheath input tube 12.
  • Tube 12 is in turn connected to pressurized sheath liquid reservoir 71.
  • the input 25 is sufficiently far from cell stream 28 that it does not introduce any effect on the cell flow aside from increasing the velocity through nozzle 26.
  • Sheath liquid input 25 has the additional advantage of allowing the flow chamber to be flushed of accumulated gases. Because the volume sensing involves the use of electrical current there is a tendency for electrolytic dissociation to occur in the liquids present in flow chamber 3.
  • the dimensions of the flow chamber are sufficiently large that the gas bubbles resulting from such dissociation rise to the top of the flow chamber and may be temporarily stored there without disrupting the optical or electrical sensing. However, it is desirable that some means be present to periodically flush any collected, gases from the system. This is readily accomplished by means of input tube 12. When flushing is required, a valve in flushing outlet tube 2 is opened and additional sheath liquid is added to flow chamber 3 through inlet tube 12.
  • a vibration means 31 is coupled to flow chamber 3 by means of coupling rod 32. Vibrations imparted to flow chamber 3 produce minute disturbances or bunching 29 on jet 10. By producing these disturbances at a proper frequency, determined by the jet diameter and velocity, they are made to grow in amplitude by surface tension until jet 10 is broken into evenly spaced, uniformly sized droplets 13. In this way cells in suspension are isolated into liquid droplets.
  • the manner in which vibration means 31 is coupled to flow chamber 3 is not critical except that the vibration frequency must be kept relatively constant. Typically, a piezoelectric crystal is used as the vibration source, but other means may readily be used.
  • the droplets 13 thus produced pass between charging electrodes 5 where those droplets determined to contain abnormal cells on the basis of an analysis of the volume, light scatter, and fluorescence of each cell are charged. Droplets indicated to contain normal cells are not charged. This particular sequence is followed on the assumption that most droplets will contain normal cells and hence the simplest approach is to charge only the droplets containing abnormal cells. The reverse procedure can just as readily be adopted, however.
  • the droplets then pass through an electrostatic field between deflection plates 8. Under the influence of this field charged droplets 14 are deflected into separate receptacles 7 than the uncharged particles 15.
  • FIG. 4 is a detailed cross-sectional view of a flow chamber adapted for use with this invention.
  • Particles in suspension flow into the chamber through tube 1.
  • This tube which also serves as one electrode for the Coulter volume sensor, may be formed from any suitably conductive material.
  • this tube is formed from platinum-rhodium alloy to avoid corrosion problems resulting from the use of physiological saline solution as both the carrier liquid for biological cells and the sheath liquidsurrounding the carrier liquid.
  • Tube 1 is aligned coaxially within a larger tube 19 by means of a guiding star 46 and is provided with a nozzle 47.
  • This nozzle may be composed of platinum, but there is no requirement that this be so.
  • a noncorrosive material such as an appropriate plastic will serve as well.
  • Tube 19 ends in a nozzle 22 in which Coulter volume sensing orifice 21 is emplaced.
  • Tube 19 may be of any nonconductive material such as glass.
  • volume sensing orifice 21 is in a sapphire insert 34 bonded to a glass tube 19. Sapphire is used because of its ready availability, but insert 34 may consist of any suitable nonconductive material in which an appropriate orifice can be produced.
  • Sheath liquid is introduced into tube 19 through inlet tube 18. Sheath liquid also enters cuvette 23 through inlet tube 12 and flushing tube 54. Flushing tube 54 also serves as the second electrode for the Coulter volume sensor so that both tube 54 and tube 12 must be of suitably conducting material.
  • tube 12 is composed of platinum and tube 54 of platinum-rhodium alloy. Flushing outlet tube 2 connecting to the interior region 30 of cuvette 23 is provided for the removal of any gases that may be produced in region 30.
  • outlet tube 2 is provided with a valve; however, if desired, sheath liquid may be continuously flowed from flushing tube 54 through cuvette 23 and out outlet tube 2.
  • Cuvette 23 which is open at the top, may be made of any material that is an insulator and is transparent to light at the wavelengths used for light beam 4.
  • cuvette 23 is composed of quartz, primarily because a quartz cuvette of the desired size is readily available commercially. Centered in the base of cuvette 23 is an opening 58 through which nozzle 26 extends. To minimize wall effects on the velocity of the cells being jetted from nozzle 26, it extends nearly to plane A-A in'which the optical measurements on the individual cellsare made. This extension also allows volume sensing orifice 21 to be more easily aligned with orifice 59 in nozzle 26.
  • Cuvette 23 is surrounded by a body shell 24 of metal.
  • Shell 24 serves to protect cuvette 23 and also to shield the Coulter volume sensor from outside electronic noise.
  • At the base of shell 24 is sealing end plate 56 and nozzle retaining end plate 57.
  • End plate 56' has a circular opening 60 centered in it through which the extended portion of nozzle 26 is passed.
  • Nozzle 26 is secured by threading it into central opening 61 in end plate 57.
  • draw nut 55 Attached to the upper portion of body shell 24 is draw nut 55. Threaded into draw nut 55 is enclosure cap 52. Cuvette 23 is sealed closed within shell 24 and cap 52 by means of O-ring 48 located in well 62 in end plate 56 and gasket 51 adjacent to cap 52. Enclosure cap 52 extends partially into cuvette 23 to form well 63. At the base of well 63 is centered a circular opening 64 through which tube 19 extends. Angular adjusting seal gland 44 threads into well 63 until it is flush with the top of enclosure cap 52. Gland 44 has centered within it truncated conical shaped well 65 having lip 66 near its top. Well 65 has a circular opening 67 centered in its base through which tube 19 passes.
  • sealing collar 43 Around the upper end of tube 19 and attached to it is sealing collar 43. Atop sealing collar 43 and tube 19 is tube connector and positioner 41. Positioner 41 has a channel 68 through it by which tube 1 enters tube 19 and is coaxially aligned in the upper portion of tube 19. Sheath liquid inlet tube 18 also enters positioner 41 and by means of channel 69 passes sheath liquid into tube 49. Inlet tubes 1 and 18 are surrounded by a shielding tube 40 which serves to prevent the introduction of electrical noise into the flow chamber through either tube 1 or tube 18. Shielding tube 40 and positioner 41 are held in place on top of tube 19 by means of connector compression cap 42 which threads onto'sealing collar 43. .O-ring 50 provides a seal between collar 43 and positioner 4].
  • Located equidistant around seal gland 44 are four adjusting lock screws 45 (only one of which is shown in 1 FIG. 4). These lock screws 45 provide a ready means by which orifice 21 in the end of tube 19 may be aligned with orifice 59 in nozzle 26. As indicated earlier in this specification, it is essential to the proper functioning of this apparatus that these two orifices by accurately aligned.
  • FIG. 5 A block diagram of the electrical and optical elements of an embodiment of this invention useful in the rapid, automatic analysis and sorting of abnormal from normal cells is given in FIG. 5.
  • a piezoelectric crystal coupled to the flow chamber serves as the source of vibration for producing droplets at the desired frequency.
  • the light source used in this embodiment is an argonion laser which is appropriately focused to intersect the cell stream in the flow chamber.
  • the light beam entering the flow chamber normally has an elliptical cross section to aid in the analysis of the cell structure by means of the resultant light scatter and fluorescence.
  • the laser beam is optically shaped such that it has an elliptical cross section as the laser beamcell stream intersection.
  • the elliptical cross section improves ease in operation (alignment), increases signal strength improving resolution, and allows characterization of cell structure (nuclear-to-cytoplasm ratio) and doublet discrimination. Doublets are two cells passing through the flow chamber in contact with each other. To the volume sensor they appear as one abnormally large cell. To avoid receiving erroneous data from the against.
  • An argon-ion laser is used as the light source because cancerous cells usually contain substantially more DNA than do normal cells, and the fluorescence of an excited Feulgen dye biochemically bound to the DNA in a cell is a quantitative indication of the DNA present in the cell.
  • the argon-ion laser emits light at a wavelength suitable for exciting this dye to fluoresce. Pulse of fluorescence coming from the flow chamber as the result of the interaction of the light beam with the cells are focused by a lens system on a pinhole and then onto a photomultiplier tube. The signal from this photomultiplier tube is amplified and fed into a multiparameter signal processing unit.
  • the signal produced by the passage of individual cells through the Coulter volume sensing orifice has the advantage of already being electrical in nature so that all that is required of it is to amplify it and feed it also into the multiparameter signal processing unit.
  • the multiparameter. signal processing unit processes these input signals, compares them with certain preset standards, and then provides three types of output signal.
  • One signal is transmitted to a multichannel pulse height analyzer which in turn provides digital printouts, a pulse height analyzer display, and histograms of the data obtained from the multiparameter signal processing.
  • the signals from the processing unit may also be directly monitored by means of an oscilloscope display.
  • an output from the processing unit is passed through a single channel analyzer and separation logics and droplet charging generator to provide droplet charging pulses which act to separate selected cells from the cell stream. It is apparent that time delay means are used in conjunction with the multiparameter signal processing unit to coordinate all sensor signals with a particular cell.
  • the signals received by the multiparameter processing unit can be processed in various ways to modify their dependence on the measured property. For example, a signal proportional to cell volume (r") can be processed to make it linearly proportional to cell radius (r) or to area (r Because only one piece of data is produced by each sensor for each cell, the amount of information to be processed is small, and the requirements placed on the electronics are not great. By using a two-dimensional pulse-height analyzer, a two parameter frequency distribution of cells can be obtained. Threeor more parameter analysis requires the data capacity ofa small computer. As an alternative to storage of all information, logical restrictions can be imposed on the analysis scheme, thus lessening the electronics requirement.
  • the processed signals from several sensors can be combined as ratios (or sums, differences, etc.) and the frequency distribution of the combination determined among a population.
  • ratios or sums, differences, etc.
  • a ratio of the processed signals can be formed to obtain a distribution of RNA density among a population of cells.
  • a type of nuclear-to-cytoplasmic ratio is given by using a.
  • nucleus-specific fluorescent stain to give a measure of nuclear volume and total cell volume measurement by scattered light or by the Coulter sensor.
  • the multiparameter signal processing unit is the central electronic processing unit for single parameter analysis, ratio computing, serial or sequential analysis and subsequent cell sorting.
  • the signal processing unit serves as a central analog electronics computing interface between the sensors, the multichannel pulse height analyzer, and the cell separator control (single channel analyzer, separation logics, droplet charging generator).
  • the signal processing unit also provides x and; y outputs for a dual parameter pulse height analyzer.
  • Amplified signal (0.4 to 8.0 V) pulses from the Coulter or cell volume (CV), light scatter (LS), and fluorescence (FL) sensors are fed directly to the processing unit.
  • the unit has separate inputs for the volume, light scatter and fluorescence signals. If desired, asecond wavelength fluorescence signal, such as red, may be substituted for the light scatter input, or wide angle light scatter substituted for the fluorescence input.
  • the processing unit is divided into three sections: input conditions, signal processing, and output routing.
  • the Input Condition section (shown in FIG. 6) consists of Operation Mode and CV-FL/LS Delay selector switches.
  • the eight position operation mode switch allows signal parameter cellular analysis, i.e., CV, LS, and FL, and dual parameter analysis of cells, i.e., CV and FL, CV and no FL, CV and LS, LS and FL, and LS and no FL.
  • CV-FL/LS Delay switch is used to set the proper CV to FL/LS signal delay. This delay is on the order of psec. The delay need be only used in the dual parameter analysis mode when Coulter volume is to be analyzed.
  • the Signal Processing section consists 'of Ratio and Serial (Input and Analyze) Analysis selector switches (see FIG. 7).
  • a six position ratio selector switch allows the following ratios to be computed: CV/FL, CV/LS, FL/CV, FL/LS, LS/CV, and LS/FL. It is mandatory that the operation mode and ratio selector switch coincide, e.g., operation mode selector in the LS and FL position and ratio selector in either the LS/FL or FL/LS ratio position. It is also important that the CV to FL/LS signal delay be used if ratios containing C V are to be computed.
  • the serial or sequential analysis section consists of input and analyze selector switches (four positions each).
  • the serial analysis input selector switch selects either CV, FL, LS, or Ratio signals to be inputed to an external single channel pulse height analyzer (Serial SCA). If the signal amplitude falls within a variable width (0.4 to 8.0 V) preset SCA window, and SCA trigger pulse is produced and is returned to the signal processing unit gating on the serial analysis analyze linear gate, thus allowing either the CV, FL, LS, or Ratio signal to be analyzed, as determined by the Serial analysis analyze selector switch.
  • Serial SCA single channel pulse height analyzer
  • Both the Serial analysis input and analyze selector positions must correspond to the operation mode, CV-FL/LS delay, and ratio selector switches whenever required, e.g., Serial analysis input CV, Serial analysis analyze FL, Operation mode CV and FL, CV- FL/LS delay z psec, and ratio off.
  • the Output Routing section (see FIG. 7) consists of pulse height analyzer (PHA) input, separator input, and dual parameter analyzer input selector switches.
  • the FHA input can select single parameters (CV, LS, or FL), ratios, serial analysis input and analyze parame-
  • the signal processing unit serves as a gated-signalters to be routed externally to a multichannel pulse height analyzer, whereas, the separator input selector can route single parameters, ratios, or serial analysis analyze signals to the separator control.
  • the dual parameter pulse height analyzer selector switch provides outputs of CV -FL CV,-LS,, LS,-FL,,, CV,-Ratio',' FL ,Ratio ,and LS Ratio ,where the x and y subscripts refer to the x and y axes of the dual parameter PHA. By interchanging the x and y axis inputs the above can be inverted.
  • OPERATIONAL SEQUENCE Cell populations to be tested are first stained (Fluorescent Feulgen, etc.) are placed in aqueous suspension, such as normal saline. Fixed or unfixed cells can be measured. Prior to placing the cell suspension in the cell reservoir, it is filtered through a 60-70 micron nylon mesh screen to remove large debris and clumps. The electronics are in a standby condition, system pressurized, the laser turned on. The system is aligned and adjusted prior to cell measurements. If cell sorting is desired, the droplet generator oscillator-amplifier which electrically drives the piezoelectric crystal (or equivalent) transducer must be turned on.
  • aqueous suspension such as normal saline. Fixed or unfixed cells can be measured. Prior to placing the cell suspension in the cell reservoir, it is filtered through a 60-70 micron nylon mesh screen to remove large debris and clumps. The electronics are in a standby condition, system pressurized, the laser turned on. The system is aligned and adjusted prior to cell
  • Droplet formation is checked by illuminating the emerging liquid jet near the flow chamber with a strobe light or equivalent light source.
  • the strobe light is synchroflashed with respect to the oscillator frequency.
  • Droplet formation can then 'be viewed using a microscope.
  • droplet formation can be adjusted by varying the voltage and frequency applied to the piezoelectric crystal. Typical values are 15 volts RMS (sinusoidal) at 40 to 50 kHz.
  • the droplet charging electrode is placed astride the point of droplet formation (separation) about /16 inch below the flow chamber to ensure maximum droplet charging.
  • Typical charging pulses are 50 volts for 100-200 psec.
  • the electrostatic deflection plates are located 2 to 3 inches below the flow chamber and spaced about V; to 36 inches apart. A differential of kV do. is normally applied to the deflection plates.
  • a sample collection beaker or appropriate collection system is placed 8 to 9 inches from the flow chamber exit side and is slightly offset from the main jet (uncharged) so as to only collect the deflected droplets (charged). If it is not desired to sort out cells, then the above procedure can be omitted.
  • Suspended cells are placed in a pressurized (23.4 psi) cell reservoir.
  • Pressurized sheath fluid No.-l (24.0 psi) and sheath fluid No. 2 (20.0 psi) are turned on and proper droplet formation achieved if sorting is desired.
  • Sheath fluid No. l with no cell stream has a flow rate of 0.3 ml/min.
  • Sheath fluid No. 2 flow rate is approximately 3.9 ml/min.
  • the total flow rate exiting the 86 p. diameter exit nozzle is thus 4.2 ml/min.
  • the cell reservoir pressure of 23.4 psi corresponds to a cell stream flow rate of about 0.08 ml/min.
  • the cell' stream flow rate can easily be adjusted from O to 0.3 mllmintlOOpercent) by adjusting the cell reservoir pressure relative to sheath No. 2 reservoir pressure 0.2 psi),holding sheath No. l reservoir pressure fixed.
  • Sheath No. 1 pressure relative to sheath No. 2 pressure normally remains fixed, but can be varied if desired. Increasing sheath No. 1 pressure relative to sheath No. 2
  • a particle free sheath flows coaxially around the sample inlet tube and serves to centrally align the cell stream as it passes through the volume sensing orifice, thus improving the resolution of cell volume and fluorescence/light scatter measurements. Typical d.c.
  • aperture currents flowing from the volume signal electrode through the orifice can be adjusted from 0.05 to 1.0 mA.
  • the aperture current and amplifier gain can be adjusted to give volume signal pulses (0.4 to 8.0 V) which in turn are fed to the CV input of the multiparameter signal processing unit.
  • Typical volume signal risetime is about 20 usec with pulse widths of 40 usec.
  • Typical time delays between initiation of the Coulter volume and fluorescence/light scatter pulse are in the order of -180 usec.
  • Fluorescence and light scatter electro-optical pulses are amplified (0.4 to 8.0 V) and fed to their respective inputs on the signal processing unit.
  • Typical risetimes are in the order of l-2 p.sec with pulse widths of about 5 usec.
  • a second particle free sheath liquid (sheath No. 2) of normal saline serves to reduce the effect of the pressure drop created by the Coulter sensing orifice.
  • the cell exits the flow chamber via the exit nozzle contained in a liquid droplet which can subsequently be separated.
  • the approximate time delay between cell sensing and droplet formation is in the order of 1400 usec.
  • the processing unit serves as an interface between the multichannel pulse height analyzer, cell separation logics and droplet charging generator, and
  • the signal processing unit must be properly set up as previously discussed, depending upon the requirements for each experimental run.
  • the multichannel pulse height analyzer would be used first to display frequency distribution histograms of single parameters (volume,.light scatter, and fluorescence)'ratios of parameters, or possibly to serially analyze parameters, e.g., analyze the fluorescence for a given cell volume range, etc.
  • the dual parameter analyzer could also be used to analyze various dual parameter frequency distribution histograms that might be needed. From either or both the multichannel PHA and Dual Parameter PHA displays it isipossible to pick out abnormalities from various histograms, e.g., an abnormally large nuclear-to-cell volume ratio.
  • the cell separation logics-droplet charging generator can be set up to physically sort out those cells exhibiting questionable properties for microscopic examination and identification.
  • the lower and upper threshold level of the single channel pulse height analyzer (SCA) is set to accept pulse amplitudes (ratios, etc.) from cells exhibiting abnormal characteristics.
  • the SCA then triggers the droplet charging generator which produces a delayed (1400 usec) droplet charging pulse (50 V peak for 100-200 usec).
  • the optimum operating condition wouldbe that in which all cells pass singly through the volume sensing orifice and light beam and only one cell is caught in each droplet. As a practical matter, this is most difficult to achieve.
  • the cells are frequently widely spaced in the cell stream such that numerous droplets contain no cells. This presents no particular problem; however, when two cells are in actual contact (thus forming a doublet) or so closely spaced that the sensors cannot discriminate between them, then sensing data are received which indicate abnormal cells and a sort signal goes out. If, as is likely in the usual population of cells, the doublet is composed merely of two normal cells, this serves to dilute the purity of the sorted sample.
  • An apparatus for rapidly analyzing and sorting minute particles on the basis of preselected characteristics or combinations of preselected characteristics which comprises a. a flow chamber,
  • electrical deflecting means whereby charged droplets are deflected to a separate receptacle from that for uncharged droplets.
  • said flow chamber contains a. means whereby said particles are made to pass along a narrow stream of fluid, said siteam passing through a first region having a Coulter volume sensing orifice wherein the change of impedance produced by the passage of each particle is measured and a second region, termed a viewing region, wherein said stream intersects a beam of light from said high-intensity light source,
  • the apparatus of claim 3 having means for delaying the analog electrical signal produced by the passage of each particle through said volume sensing orifice and correlating it with the analog electrical signals produced for that same particle by the optical sensing means.
  • optical sensing means consist of a photodiode and a photomultiplier tube.
  • said means for causing said particles to pass along a narrow stream of fluid consists of a. a sample inlet tube for introducing said particles in suspension in said fluid into said flow chamber, said sample inlet tube extending substantially into said flow chamber, and
  • a first sheath liquid inlet tube concentrically surrounding said' sample inlet tube and extending somewhat beyond it into said flow chamber, said first sheath liquid inlet tube having a nozzle at its lower end in which is located said volume sensing orifice, whereby said particles on leaving said sample inlet tube are surrounded by a coaxial laminar flow of sheath liquid, said sheath liquid having a velocity sufficiently high to narrow the flow of particles in suspension to a stream of desired diameter and surround said stream substantially coaxially as it passes through said volume sensing orifice.
  • sample inlet tube serves as one electrode for said volume sensing orifice and said second sheath liquid inlet tube serves as the second electrode for said orifice.
  • said means for periodically disturbing said jet consists of a piezoelectric crystal coupled to said flow chamber and oscillated at a desired frequency.
  • said alignment means consists of a plurality of adjusting screws uniformly spaced around said first sheath liquid inlet outside said flow chamber whereby said first sheath inlet tube is rotated about an axis located partially within said reservoir.
  • An apparatus for rapidly sorting biological cells failing to meet preset standards of normality from cells meeting such standards by imparting an electrical charge to fluid droplets containing the abnormal cells and passing the charged droplets through a static electrical field whereby the charged droplets are deflected into a separate receptacle from that of uncharged droplets containing normal cells which comprises in combination a. means for introducing a suspension of cells in fluid meansfor generating a first electrical signal proportional to the volume of each cell as it passes through said first region of said channel in said flow chamber,
  • laser means for illuminating each cell as it passes through said second region of said channel, meansfor measuring the scatter in the laser light as it emerges from said first viewing port and generating a second electrical signal proportional to the amount of scatter
  • electrical delay means whereby said sort signal is used to activate the electrical charging means adjacent to the jet path at a time when the cell determined to be abnormal is in 'the droplet separation zone, said charging means remaining inactivated unless said sort signal is received, and
  • electrical deflecting means whereby charged droplets are deflected to a separate receptacle from that for uncharged droplets.

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DE2261695A1 (de) 1973-06-28
NO138305B (no) 1978-05-02
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NL7216901A (enrdf_load_stackoverflow) 1973-06-26
SE388278B (sv) 1976-09-27
FR2164898A1 (enrdf_load_stackoverflow) 1973-08-03
AU5020272A (en) 1974-06-20
NO138305C (no) 1978-08-09
BE793185A (fr) 1973-04-16
CA971913A (en) 1975-07-29
GB1380756A (en) 1975-01-15
JPS4874292A (enrdf_load_stackoverflow) 1973-10-06
DE2261695C2 (de) 1987-04-09
AU468637B2 (en) 1974-06-20

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