Particle Sorting Method And Apparatus

Abstract

A particle sorting method and apparatus for separating minute particles incorporated in a liquid stream in accordance with selected particle parameters. Particles such as biological cells to be separated are incorporated in the inner or central portion of a confined coaxial flow stream which includes an outer cell-free fluid sheath portion. This coaxial flow stream is released through a vibrating nozzle for inspection by one or more cell sensing means for sensing cells in the jet stream immediately downstream of the nozzle. Beam illumination and/or observation of the jet stream for particle sensing is effected outside of the nozzle. Vibration of the nozzle breaks the coaxial jet stream into a series of uniform drops downstream of the cell sensing means, and signals from said means are used to initiate delayed charging pulses applied to preselected cell-containing drops as they break from the vibrating fluid jet. Nozzle vibration is synchronized with the charging pulse to prevent separation of drops from the stream during the drop charging pulse on and off transition times, thereby preventing undesired drop charging. The drops pass between charged deflection plates where the charged drops are deflected into appropriate receptacles. By using a plurality of sensing means such that all cells are sensed by one sensor, and only certain cells, i.e. cells characterized by a particular parameter value, are sensed by another sensor, drops containing both cells characterized by a particular parameter and cells not characterized by that parameter can be sorted away from the receptacle for cells characterized by that particular parameter.

Description

The invention described herein was made in the course of work under a grant or award from the Department of Health, Education and Welfare.

BACKGROUND OF THE INVENTION

This invention relates to a method and apparatus for physically separating particles such as functionally different cell types. Prior cell separation methods are known and include direct fractionation as by centrifugation, column fractionation, electrophoresis, and the like. Among other disadvantages, such methods produce only limited resolution of functionally different cell types. A more closely related cell sorting method described in U.S. Pat. No. 3,380,584, involves passing a suspension of cells through a nozzle to form a liquid stream, employing a sensor to detect cells flowing in said stream as th stream reaches a small nozzle outlet, vibrating the fluid stream to cause it to break into drops, charging the drops which contain desired detected cells, and subjecting the stream to an electric field to deflect the charged drops. However, cell sorters of this type have many shortcomings which substantially restrict their usage. For example, the small diameter nozzle employed for jetting the released fluid is subject to frequent clogging; the sensor windows through which the still-confined stream is illuminated and/or cells are detected are subject to contamination; many drops, including those containing no cells and those containing unwanted cells, must be deflected in order to assure deflection of the drops containing cells characterized by a particular parameter value; and a complex nozzle design is sometimes used for vibrating the jet stream.

It is a general object of this invention to provide an improved particle separating method and apparatus which overcome the above-mentioned difficulties and shortcomings of the prior art devices. A more particular object is to provide a particle separating method and apparatus whereby coupling between the particle sensing means and a fluid stream containing the particles is effected solely through the atmosphere in a manner which eliminates deterioration of such coupling by contamination, and whereby clogging of the jetting nozzle is substantially eliminated.

Another object of this invention is to provide a cell separating method and apparatus of the foregoing type which permit collection of fractions containing large numbers of viable cells highly enriched in a preselected functional type even after multiple passes through the apparatus.

A further object of this invention is to provide a particle separating method and apparatus of the vibrated jet stream type for acurately and precisely imparting the desired charge to the desired drops, and in which separation of drops from the jet stream during the transition time of the drop charging pulses is prevented to avoid charging of drops to unknown and undesired potentials.

A related object is to provide a method and means of this character in which those drops which contain only particles characterized by a particular parameter value are directed along a first path whereas those drops which contain both such particles and particles not characterized by that parameter are directed along another path to keep the same from the fraction directed along the first path.

SUMMARY OF THE INVENTION

The above and other objects and advantages of this invention are achieved by use of a confined fluid flow stream having a central particle-containing portion which is surrounded by a particle-free sheath fluid. This confined coaxial stream is released by jetting the same from a nozzle which is controlably vibrated at an ultrasonic frequency to break the stream into uniform size drops downstream of the nozzle. Particle illuminating and/or sensing means are included for illuminating and/or sensing particles in the emerging jet stream immediately outside the nozzle, said means, being coupled directly to the stream without the use of windows. Drop charging means responsive to the sensing means and connected to the stream act to charge with the desired charge. The drops then pass through an electric field for relative deflection of the drops according to the amount of charge.

In accordance with one embodiment of this invention, means for sensing at least two different particle parameters are employed, along with drop charging circuitry whereby drops which contain only particles characterized by a particular parameter value are charged for travel along one path, whereas those drops which contain both particles characterized by that value and those not characterized by that value are provided with a different charge for travel along another path. This permits separation of the mixed particle-containing drops from those containing only particles characterized by a particular parameter value. Additionally, synchronization of the drop charging circuitry with the nozzle vibration is employed to further assure proper charging of the drops.

The nature of the present invention will be more fully apparent and understood from a consideration of the following description in the light of the drawings wherein like reference characters refer to the same items in the several views. In the drawings:

FIGS. 1A and 1B together diagrammatically show a particle separator of the present invention in one embodiment thereof;

FIG. 2 shows a series of waveforms which occur at various points of the circuit during operation of the particle sorter shown in FIGS. 1A and 1B; and

FIG. 3 is a schematic diagram of typical shaping network for shaping the drop charging pulse.

Reference first is made to FIG. 1A wherein there is shown a nozzle assembly generally indicated at 10 from which liquid containing the particles in suspension is jetted downwardly in a coaxial column or stream generally indicated at 12. Pressurized reservoirs 14 and 16 are provided, with reservoir 14 containing a supply of the sample liquid in which the particles to be separated are suspended, while reservoir 16 contains a supply of particle-free sheath fluid. The reservoirs are connected to the nozzle assembly 10 through conduits 18 and 20 and suitable filter elements (not shown). The reservoirs are pressurized as by means of a gas pressure source 22 connected to the reservoirs through adjustable pressure regulators 24 and 26.

The nozzle assembly 10 includes inner and outer coaxially located nozzles 28 and 30 fixedly secured to a mounting block 32 and supplied with fluid from the reservoirs through the conduits 18 and 20, respectively. The structure is such that the particle containing sample fluid from the nozzle 28 is injected within the nozzle 30 into the center of the flowing stream of sheath fluid. By way of example only, each of the nozzles 28 and 30 may have an outlet diameter of say 50 microns, and may be operated at pressure of approximately 12 p.s.i. with a small excess sheath fluid pressure to produce a jet velocity from the nozzle 30 of 10m/sec., a sheath flow rate of 0.02 ml./sec., and a sample material flow rate of 0.002 ml/sec. The coaxial flow stream 12, comprising an inner particle-containing portion 12A and an outer, particle-free sheath portion 12B, emerges from the nozzle 30 in the desired coaxial flow condition. In flowing through this nozzle the inner stream portion 12A is reduced in diameter to approximately 15 microns within which diameter the particles to be separated are confined. It will be understood that whereas a 15 micron diameter nozzle would be plugged frequently if defined by a rigid orifice, the present invention overcomes the problem by the use of the coaxial nozzles of much larger diameter which inherently provide the much narrower inner stream 12A. Coaxial flow systems generally are known, as shown, for example, in an article by P. J. Crossland-Taylor, Nature 171, 37 (1953) and in U.S. Pat. No. 3,649,829.

By vibration of the nozzle assembly 10 in an axial direction, the jet stream 12 is caused to break into drops. To this end a transducer 34 such as a piezoelectric crystal type transducer may be directly attached to the mounting block 32. The transducer is energized by a clock oscillator 36 which is shown connected thereto through two-to-one frequency divider 38, a four-to-one frequency divider 40, a variable phase control circuit 42, and a driver amplifier 44. In the illustrated arrangement a nozzle vibration of 40khz is provided when a clock oscillator having a frequency of 320khz is employed. The resulting velocity modulation of the stream produces small variations in the stream diameter which are amplified by surface tension forces to decompose the jet into very uniform size drops downstream from the nozzle assembly. Therefore, with the above apparatus, and with a nozzle vibration rate of 40khz the jet stream is broken into 40,000 drops per second, drop formation being synchronous with the nozzle vibration. Particle sensing means described below are directly coupled to the jet stream through the atmosphere outside of the nozzle 30 such that modulation of the stream diameter results in modulation of the signal received by the sensing means. To minimize the effect of such modulation in the sensing means, the nozzle vibration amplitude is made as small as possible, consistent with stable and uniform drop formation, and sensing is effected immediately below the nozzle. Formation of drops over a wide range of nozzle vibration frequencies is possible, with the range depending upon the velocity of the fluid stream and diameter thereof, and thus the invention is not limited to the illustrated 40khz vibration frequency.

Sensing of particles in the fluid stream outside of the nozzle is not limited to the use of any particular type of sensing, inasmuch a number of different particle sensing means are known in the prior art. In the illustrated arrangement, two different types of sensors are employed, one being used for the detection of all particles entrained in the fluid stream, while the other is used for the detection of selected particles to be separated. The means for sensing all particles may include a light source 46 such as a helium-neon gas laser operating at, say, 6320A. Illumination from the laser 46 preferably is focused on the inner coaxial portion 12A of the stream by a suitable lens or lens system 48 for highly localized scatter from particles therein. An objective lens or lens system which includes a lens 50 is provided in the beam path of the laser for focusing scatter beams onto the face of a detector 52. A mask 54 which extends over the center of the lens 50 blocks out direct illumination from the laser whereby only laser illumination scattered from illuminated particles in the stream reaches the detector 52. While different types of well known detectors may be used, preferably a photomultiplier detector is employed because of its large amplification. It will be apparent, then, that an output signal is obtained from the detector 52 for each illuminated particle in the stream and that the amplitude of the signal is directly dependent upon particle size.

In the illustrated arrangement, the detector 52 included in the sensing means for sensing all particles in the stream 12 is used in conjunction with a second sensing means for detecting only particles characterized by a particular parameter value. For purposes of illustration a second particle sensing means of the fluorometer type is shown which includes a high intensity source of illustration 56 which may be in the ultraviolet or blue regions, such as an argon laser having a beam which also is directed onto the center particle-containing portion 12A of the stream through a lens or lens system 58 for the excitation of fluorescent particles contained therein. If desired a combination of spherical and cylindrical lenses for focusing the beam to a narrow ellipse at the intersection with the stream may be used for uniform excitation of cells not on the stream axis. Luminescence from the excited fluorescent particles is directed onto a detector 60 through an object lens or lens system 62 and suitable filter means 63 to pass the fluorescence illumination and block the excitation illumination. In the illustrated arrangement the beam from laser 56 is directed at an angle of substantially 45.degree. from the stream axis, and observation or sensing of luminescence is effected generally radially from the stream at the point of illumination whereby direct light from the laser 56 is prevented from entering the detector 60. Preferably the detector 60 also comprises a photomultiplier having high sensitivity to the relatively faint luminescent pulses as fluorescent particles pass the point of excitation, with the photomultiplier output being dependent upon the fluorescence amplitude of the particles. By direct coupling of the illuminating and detecting means to the unconfined jet stream without the use of windows, window contamination and other window-related coupling problems are eliminated.

Naturally, if fluorescence detection is used as illustrated, particles must be fluorescent under radiation from the laser 56. Treatment of selected particles to render them fluorescent is well known and requires no detailed description. Such treatment, together with a cell sorting method based upon intracellular fluorescence is known as indicated in an article entitled Cell Sorting: Automated Separation of Mammalian Cells as a Function of Intracellular Fluorescence by H. R. Hulett et al. in Science 166, 747 (1969), and no further description is believed to be required.

In the illustrated arrangement, cells contained in th stream 12 are illuminated first by the beam from the laser 46 and then by the beam from the laser 56. It will be seen then that a fluorescent call contained in the stream 12 is first detected by the photodetector 52 and then by the photodetector 60 for the successive production of pulse signals in the scatter and fluorescent channel outputs from the photodetectors. A nonfluorescent cell with result in a scatter channel output pulse only and will be undetected by detector 60 in the fluorescence channel. The scatter and fluorescence channel signal outputs from the photomultipliers 52 and 60 are processed to generate delayed drop charging pulses which, in accordance with another feature of this invention, are applied to an electrode 64 in contact with the electrically conducting (isotonic) sheath fluid within the nozzle assembly 10. The circuity for generating such drop charging pulses is described in greater detail hereinbelow. For present purposes it will be understood that in the illustrated arrangement a charging pulse of one polarity is supplied to the electrode 64 upon detection of a fluorescent particle, an opposite polarity pulse is supplied thereto upon detection of a non-fluorescent particle, and no charge is applied thereto when no particles are detected or when immediately adjacent fluorescent and non-fluorescent particles are detected in the stream.

Drops which break off from the stream while a charging pulse is applied to the electrode will carry a charge dependent upon the polarity and amplitude of such charging pulse. A charging ring or electrode (not shown) may be provided which surrounds the region of drop formation. The charging pulse may be applied to the fluid in the nozzle with the charging ring grounded, or to the charging ring with the fluid jet at ground potential. If the pulse is applied to the fluid, the charging ring is not required, its function being provided by neighboring conductors at ground or other fixed potential, but deflection sensitivity will be somewhat less than if a closely spaced charging ring is used.

After being selectively charged the droplet stream passes through a transverse electrostatic field established as by a pair of parallel deflection plates 66 connected to opposite polarity dc potential sources of, say, .+-.1,000 volts. Uncharged drops pass through the deflecting field between the plates substantially undeflected, to enter a central receptacle 68A. On the other hand, positively charged drops are deflected to the right, as viewed in FIG. 1A, into a receptacle 68B, and negatively charged drops are deflected to the left into the receptacle 68C. In the illustrated arrangement drops which contain cells characterized by a particular parameter value (e.g. drops with one or more fluorescent cells of desired luminescence) and which contain no other cells may be deflected into the receptacle 68C; those drops which contain other cells (e.g. drops with one or more non-fluorescent cells, or with fluorescent cells having a luminescence outside of the desired range) and containing no fluorescent cells of desired luminescence may be deflected into the receptacle 68B; and all drops which contain no cells pass through the electrostatic field undeflected and into the central receptacle 68A. In the illustrated arrangement drops which contain both fluorescent and non-fluorescent cells also pass undeflected into the central receptacle 68A, however, if desired, such drops could be readily supplied with yet another charge for deflection into another receptacle, not shown.

Reference now is made to the block diagram showing in FIGS. 1A and 1B of suitable drop charging circuitry for effecting the above-described drop deflection, and to FIG. 2 showing waveforms occuring at various points in the diagram of FIGS. 1A and 1B. The scatter and fluorescent channel outputs from detectors 52 and 60 are supplied to single channel analyzers 74 and 76 through amplifiers 70 and 72, respectively. Analyzers suitable for use in this arrangement are well known and include Ortec Model 406A manufactured by Ortec Inc., 100 Midland Rd., Oad Ridge, Tenn. 37830. Such analyzers simply may comprise discriminators which have an output only when an input signal applied thereto exceeds a lower threshold value and is less than an upper threshold value. In the illustrated analyzers upper and lower limit control 78 and 80, respectively, are included for setting the upper and lower amplitude limits of pulses which will pass therethrough. Alternatively, Schmitt triggers may be used for the analyzers in which case all pulses which exceed a lower threshold value pass therethrough.

A cell contained in the jet stream is detected by scatter illumination received by the photodetector 52 and, if the cell is fluorescent, it also is detected by fluorescent luminescence received by the photodetector 60. Signal output pulses from the detectors, designated 52A and 60A, are shown in FIG. 1A and are included in the waveforms shown in FIG. 2. If the amplified signal pulses fall within the selected amplitude limits of the analyzers 74 and 76 trigger pulses 74A and 76A are generated by the analyzers. The pulse 76A from the fluorescent channel analyzer 76 is used to trigger an adjustable gate pulse generator 82 which generates a squarewave gating pulse of adjustable duration dependent upon the setting of control 84. Generator 82 may simply comprise an adjustable monostable multivibrator.

The pulse signal output 74A from the scatter channel analyzer 74 is fed to a delay unit 86 for delay of the pulse by an adjustable amount, the delayed output pulse being identified as 86A in the drawings. The pulse is delayed for simultaneous occurrence with the gating pulse 82A, preferably for occurrence at substantially the center of the gating pulse. For purposes of illustration, the delay unit 86 is shown comprising a shift register which is clocked at the 320khz rate provided by the output from the clock 36.

The gating pulse outputs 82A and 86A from the pulse gate generator 82 and delay unit 86, respectively, are supplied to a logic unit or network 88 having output lines 89 and 90. The logic network simply may include AND gates 92 and 94, one of which gates 92 supplies an output to line 89 when the input signal 86A from the delay unit 96 is high while the gate pulse signal 82A from the generator 82 is low (i.e. not present). The other gate 94 supplies an output to line 90 when both inputs 82A and 86A thereto are high. It will be understood then that the one gate 92 is enabled in the absence of a fluorescence channel gating signal 82A from the pulse gate generator 82 for passage of a "scatter, not fluorescent" trigger pulse 92A to line 89, whereas the other gate 94 is enabled by the presence of a fluorescence channel gating signal 82A for passage of a "scatter and fluorescent" trigger pulse 94A to the line 90. With this arrangement wherein the gate control pulse 82A controls opening and closing of both gates 92 and 94 in a manner whereby one gate is enabled while the other is disabled the signal pulse 86A is passed through one of the gates 92 and 94 but not through both simultaneously.

The pulse outputs 92A and 94A from the logic unit 88 comprise trigger pulses which are supplied to retriggerable pulse generators 98 and 100 through adjustable delay units 102 and 104, respectively. In the waveforms of FIG. 2 the delayed trigger pulses 102A and 104A from the delay units 102 and 104, respectively, are shown trailing the trigger pulse inputs 92A and 94A supplied thereto. The retriggerable pulse generators 98 and 100 provide drop charging outputs which are amplifier by amplifier 106 and supplied to the drop charging electrode 64 through lead 108.

As described above, the charge carried away by each droplet is determined by the charging voltage supplied to the electrode 64. Also, the drop charging pulses must be supplied at the appropriate time for proper separation of the drops. In the illustrated arrangement the delay units 102 and 104 are adjusted to provide the necessary time delay to allow for travel time of the particle from the point of scatter detection to the point where the stream breaks into drops. With the present arrangement the delay time between observation of a particle and its capture by a separating droplet is predictable to within three drop periods. Such high degree of predictability is due primarily to the uniform velocity of the inner particle containing stream 12A of the coaxial flow jet. That is, across the inner stream 12A the stream velocity is substantially uniform whereby particles anywhere within the cross section of the inner stream travel with the same velocity from the point of observation to the drop separation point of the stream. Adjustment of the shift registers 102 and 104 clocked at 160khz provides for fine delay adjustment of the trigger pulses supplied to the pulse generators 98 and 100. Other well known adjustable delay devices could be used in place of the illustrated shift registers if desired.

In the illustrated embodiment, the retriggerable pulse generators 98 and 100 provide equal duration and opposite polarity drop charging pulse outputs to the amplifier 106; the generator 98 having a positive pulse output 98A and the generator 100 having a negative pulse output 100A. The amplifier output may comprise, for example, a positive 100 volt or negative 100 volt pulse depending upon the polarity of the input signal from a pulse generator. The amplifier output remains at a zero level with no input signals and during simultaneous application of positive and negative signals from the pulse generators 98 and 100. The above voltage values are given for purposes of example only, it being understood that different drop deflection in the electrostatic field requires only that drops be provided with different charges, no particular charge differences being required.

The instrument accuracy is such that an observed particle predictably will appear in one of three successive drops. Therefore, a charging pulse of sufficient duration to charge three drops is employed to ensure separation of the desired particle. With a nozzle vibration of 40,000khz as shown, a drop is produced every 25 microseconds, and a drop charging pulse width of 75 microseconds is used for charging three drops. The amplifier 106 may include a shaping network to modify the rise and fall times of the charging pulse to correct a tendency that otherwise exists for the first and last drops in a sequence charged by a rectangular pulse to be incorrectly charged. A typical shaping network 108 and resulting pulse waveforms are shown in FIG. 3. Obviously, if instrument tolerances, variations, drift and the like permitted, then a drop charging time sufficient to charge only two successive drops, or a single drop could be employed.

A drop breaking from the stream will carry with it a charge which is proportional to the potential on electrode 64 when the drop separates from the stream. If a drop breaks off from the jet stream during the transition time of the drop charge pulse, either during the leading or trailing edge of the pulse, such drop will be charged to some intermediate value between zero and the desired full charge, and upon passing between the deflection plates 66, will be deflected some intermediate amount proportional to the reduced charge thereon. In accordance with another feature of this invention, on and off transitions of the drop charging pulse are synchronized with the drop formation means whereby such transitions occur only intermediate the formation of drops and not when drops separate from the stream. Such synchronization in the present arrangement is provided by use of synchronization signals to the pulse generators 98 and 100. The variable phase control unit 43 included in the transducer drive cirucit is adjusted for proper timing of drop formation with the drop charging pulse such that on or off transitions of the charging pulse occur only intermediate formation of drops and not when the drop breaks from the stream. Proper charging of drops is thereby ensured by such synchronization.

For ease in operation of the instrument it is desirable that means be provided for direct observation by the operator of the jet for drop formation and drop deflection. For observation of undeflected drops there is provided a light source 112 such as a light emitting diode which is energized by the 40khz transducer driver and pulse generator synchronizing signal applied thereto over lead 114 through a one-to-four frequency divider 116 and driver amplifier 118. The stroboscopic illumination of the stream by the diode permits the viewing thereof through a suitable microscope not shown. Deflected drops are illuminated by use of a stroboscopic light 120 which is energized by the drop charging pulses supplied thereto over line 122 through a time delay unit 124. With this stroboscopic arrangement deflected drops are illuminated for viewing through a suitable microscope, not shown.

Although the operation of the particle sorter is believed to be apparent from the above description, a brief description of the operation thereof with reference to the waveforms of FIG. 2 will be made. These waveforms illustrate the following operating conditions: (A) detection and sorting of a nonfluorescent particle, (B) detection and sorting of a fluorescent particle, (C) detection and sorting of closely adjacent fluorescent and nonfluorescent particles in the stream, and (D) detection and sorting of closely adjacent particles of like type in the stream, e.g. adjacent fluorescent particles.

A nonfluorescent particle observed by the scatter light detector 52 produces no luminescence and so passes undetected by the fluorescence detector 60 as illustrated by the group (A) waveforms of FIG. 2. If the amplified signal pulse 52A is within selected limits it passes through the single channel analyzer 74 emerging therefrom as signal pulse 74A which is applied to the logic network 88 through the delay unit 86. With no output from the gate pulse generator 82 because of no fluorescence channel signal the gate 92 is enabled, while gate 94 is disabled, for passage of the signal through the gate 92 and onto line 89 as pulse 92A. The pulse is fed through delay unit 102 and supplied as a delayed trigger pulse 102A to condition the pulse generator 98 for generation of a drop charging pulse 98A. As seen by the waveforms, the leading edge of the drop charging pulse 98A is initiated by the first synchronizing pulse 40A to occur following the delayed trigger pulse 102A. Such operation may be provided, for example, by the inclusion of a flip-flop in the input to the retriggerable pulse generator which is set by the delayed trigger pulse to allow the next synchronization pulse to initiate the generation of the drop charging pulse. Similar synchronizing means may be employed by the retriggerable pulse generator 100. The amplified drop charging pulse 106A is applied to the stream through electrode 64 for supplying drops which break from the stream with the desired charge. Synchronization of the ulse generator 98 with the vibration of the nozzle assembly ensures uniform charging of the drops formed during the drop charging pulse. The uniformly charged drops are uniformly deflected as they pass the deflection plates 66, with the group of drops positively charged by the pulse 106A being deflected to the right as viewed in FIG. 1A into receptacle 68B.

Reference now is made to the group (B) waveforms of FIG. 2 for operation of the sorter in response to observation of a fluorescent particle. Such a particle scatters light from the source 46 which is detected by detector 52, and fluoresces in the presence of the beam from source 56 which fluorescence is detected by detector 60. Amplified scatter and fluorescence channel signals 52A and 60A of selected amplitude from the analyzers or discriminators 74 and 76 are supplied to the delay unit 86 and gate pulse generator 82, respectively. The delayed pulse 86A from the delay unit 86 occurs during the gate pulse output 82A from the generator 82 whereby the logic network 88 is conditioned for gating the signal pulse over the "scatter and fluorescent" line 90 to the delay unit 104. The output 104A from the delay unit 104 conditions the pulse generator 100 for production of a delayed negative drop charging pulse 100A which is amplified by amplifier 106 and supplied over line 110 as drop charging pulse 106A to the electrode 64 for negatively charging the stream. Drops breaking off during application of the drop charging pulse are subsequently deflected to the left as viewed in FIG. 1A as they pass the deflection plates 66 to enter the receptacle 68C. Again synchronization of the pulse generator 100 with the nozzle vibration provided by transducer 34 ensures against partial drop charging during on and off transition of the charging pulse.

During operation, one or more non-fluorescent cells may be lcoated closely adjacent a fluorescent cell in the jet stream so as to be captured in the same or adjacent drops as the stream breaks up. With the novel arrangement of this invention any such drops which may contain both fluorescent and non-fluorescent cells are neither deflected into the receptacle 68C for fluorescent cells nor into the receptacle 68B for non-fluorescent cells. Instead a zero drop charging potential is applied during detachment from the stream of drops containing both fluorescent and non-fluorescent cells whereby such drops pass undeflected through the electrostatic field and into the receptacle 68A.

The group (C) waveforms of FIG. 2 illustrate the condition wherein detection of a nonfluorescent cell is immediately followed by detection of a fluorescent cell in the jet stream. Two adjacent scatter channel signals 52A are shown followed by a fluorescent channel signal 60A, and if their amplitudes are within the selected ranges of the analyzers 74 and 76 two signal pulses 74A-1 and 74A-2 are obtained from the analyzer 74 and a single signal pulse 76A which follows in time is obtained from the analyzer 76. A gate control pulse 82A is triggered by the pulse 76A, and the two signals 74A-1 and 74A-2 are delayed by delay unit 86 having output signal pulses 86A-1 and 86A-2. The first signal pulse 86A-1 which occurs before the gate control pulse 82A is gated through the gate 92 of logic unit 88 and emerges as trigger pulse 92A. The following pulse 86A-2 however occurs during the gate control pulse 82A for passage through gate 94 to emerge therefrom as trigger pulse 94. Following equal time delays in delay units 102 and 104 the delayed trigger pulses 102A and 104A therefrom condition pulse generators 98 and 100 for generation of positive and negative drop charging pulses 98A and 100A, respectively, which are applied to the amplifier 106. The amplifier 106 includes means, i.e. a summing junction, for combining the separate input signals such that the amplifier output remains at a zero level during application of equal potential an opposite polarity signals applied thereto. Therefore, in the operation illustrated by the group (C) waveforms the drop charge signal rom the amplifier 106 comprises a positive pulse portion 106A-1 during the time only the input pulse 98A is applied thereto, a zero level pulse portion 106A-2 while both input pulses 98A and 100A are applied thereto, and a negative pulse portion 106A-3 while only the input signal 100A is applied thereto. Of the drops charged the detected non-fluorescent cell will be contained in either the drop charged during pulse portion 106A-1 or 106A-2, and the detected fluorescent cell will be contained in either the drop charged during pulse portion 106A-2 or 106A-3. Of the five drops formed during the illustrated drop charging signal the first two positive charged drops are deflected into receptacle 68B, the next uncharged drop is undeflected to enter receptacle 68A, and the last two negative charged drops are deflected into receptacle 68C.

As noted above the pulse generators 98 and 100 are retriggerable such that drop charging pulses 106A of extended duration may be generated during detection of closely adjacent fluorescent cells or non-fluorescent cells. In the group (D) waveforms of FIG. 2 the detection of two fluorescent cells following one another in less than 75 microseconds time is shown. The photodetector pulse output signals 52A and 60A are processed in the manner described above and provide a pair of trigger pulses 104A which are supplied to the retriggerable pulse generator 100. The first pulse conditions the generator 100 for triggering by a synchronizing pulse and the second pulse reconditions the same to extend the drop charge pulse period from the generator without interruption.

While the invention has been described above in connection with various embodiments thereof, various changes and modifications will suggest themselves to those skilled in this art. For example, illumination of the stream for scatter and fluorescent sensing is not limited to the use of laser beams. Other illumination sources such as an arc lamp with suitable beam filtering and directing means may be used for illumination as the desired frequency or frequencies. When plural sensing means are employed, as illustrated, the order of illumination and sensing of particles is not critical. For example fluorescent sensing may precede scatter sensing where these two sensing means are employed. Further, if desired, the same stream cross sectional area may be illuminated by both illuminating means for simultaneous sensing of particles. Also, as noted above, other types of particle sensing means such as those utilizing radiant energy absorption, radioactivity, electrical conductivity and the like may be employed. Also, because drop separation is dependent upon the differences in drop charge and not upon the absolute values thereof, it will be apparent that different drop charging voltages than those described may be employed.

If desired, the photodetector outputs may be supplied to multichannel pulse height analyzers to obtain the pulse height spectrum of the observed signals. Also, event counters 130 and 132 may be included for counting the "scatter not fluorescent" and "scatter and fluorescent" pulses from the logic network. Information obtained by such analyzers and counters may be utilized for checking instrument accuracy, particle counting, and the like. Additionally, a single illuminating means with a plurality of detector means may be used in which the separate detectors are supplied through beam splitting means and which detectors are responsive to different portions of the spectrum. The ratios of such signals may be used to trigger drop pulse generators.

As noted above, proper operation of the separator requires the use of the proper signal delay in an amount related to the transient time of the particle from the point of observation of the scatter beam to the point where the drop breaks off from the jet. One factor in this delay is the particle velocity which, if desired, could be measured by any suitable means and the delay units 102 and 104 adjusted accordingly. Numerous methods of measuring fluid flow velocity are known which could be used, including means employing the Doppler shift of the scatter light. Also, although the invention has been illustrated in terms of method and apparatus wherein a coaxial flow stream having an inner stream portion of particle containing fluid and an outer stream portion of sheath fluid is jetted from a nozzle and broken into drops, by nozzle vibration, the principles of synchronizing the drop charging pulse period with the nozzle vibration, coupling the particle detecting stream to the unconfined stream without the use of windows, and charging the drops in such a manner to prevent those drops which contain both fluorescent and non-fluorescent cells from being deflected the same amount as those drops which contain only fluorescent cells or only non-fluorescent cells, are also applicable to arrangements wherein a stream without sheath fluid is employed. It is intended that the above and other such changes and modifications shall fall within the spirit and scope of the invention as defined in the appended claims.

Claims

We claim:

1. A method of separating certain particles from others contained in a fluid stream comprising,

producing a coaxial flow stream having an inner stream portion of particle containing fluid and an outer stream portion of sheath fluid,

jetting the coaxial flow fluid stream,

modulating the jet stream velocity in the direction of the stream axis to break the stream into discrete drops,

detecting certain particles in the inner stream portion of the coaxial flow stream,

charging the jet stream while drops containing detected particles break from the jet stream to supply such drops with an electrical charge,

synchronizing the charging with the modulating of the jet stream to ensure against parallel drop charging during on and off transitions of the drop charging pulse, and

deflecting the charged drops in an amount related to the drop charge.

2. The method of particle separation as defined in claim 1 wherein the coaxial flow stream is jetted into the atmosphere, and including detecting directly through the atmosphere desired particles in the inner stream portion of the jet stream as said stream travels through the atmosphere.

3. The method of particle separation as defined in claim 1 wherein said certain particles detected are those characterized by a particular parameter value, the method also including

detecting other particles in the inner stream portion of the coaxial flow stream which are characterized by other parameter values,

charging the jet stream by use of different drop charging pulse while drops containing detected particles characterized by other parameter values break from the stream to supply such drops with a different drop charge, and

modifying charging of the jet stream while drops containing both particles characterized by a particular parameter and those characterized by other parameters break from the stream for a different deflection of such drops.

4. A method of separating certain particles from others contained in a jet stream comprising,

modulating the jet stream velocity in the direction of the stream axis to break the stream into discrete drops,

detecting certain particles in the jet stream,

charging drops containing detected particles during a drop charging pulse period,

synchronizing the drop charging pulse period with the modulating of the jet stream to prevent drops breaking from the stream during drop charging on and off transitions, and

deflecting charged drops in an amount related to the drop charge thereon.

5. The method of particle separation as defined in claim 4 wherein said jet stream travels through the atmosphere, and including detecting directly through the atmosphere particles in the jet stream as said stream travels through the atmosphere.

6. The method of particle separation as defined in claim 4 including detecting other particles in the jet stream in addition to detecting said certain particles therein,

charging the jet stream by use of a different drop charging pulse while drops containing other detected particles break from the stream to supply such drops with a different drop charge, and

modifying charging of the jet stream while drops containing both detected certain and other particles break from the stream for a different deflection of such drops.

7. A method of separating certain particles from others contained in a stream which is jetted from a nozzle into the atmosphere comprising, modulating the jet stream to break the stream into discrete drops at a distance from the nozzle,

detecting directly through the atmosphere certain particles in the jet stream while the stream travels through the atmosphere before the stream into discrete drops,

charging drops containing detected certain particles, and

deflecting charged drops in accordance with the drop charged thereon.

8. The method of separating particles as defined in claim 7 including synchronizing the charging of drops with the modulating of the jet stream.

9. The method of separating particles as defined in claim 7 including,

detecting other particles in the jet stream,

charging drops containing detected other particles to another drop charge, and

modifying charging of drops containing both certain and other detected particles.

10. A method of separating certain particles from others contained in a jet stream comprising,

modulating the jet stream to break the same into discrete drops,

detecting certain particles in the jet stream,

detecting other particles in the jet stream,

providing drops containing detected certain particles with a first drop charge, those containing detected other particles with a second drop charge, and those containing both detected certain and other particles with a third drop charge, and

separating drops according to their drop charge.

11. The method of particle separation as defined in claim 10 wherein the jet stream travels through the atmosphere before breaking into discrete drops, and including employing first and second particle detecting means to detect said certain particles and said other particles directly through the atmosphere as the stream travels through the atmosphere before breaking into discrete drops.

12. The method of particle separation as defined in claim 10 including synchronizing the drop charging with the modulating of the jet means to assure against partial drop charging.

13. Apparatus for separating particles contained in a fluid comprising,

means including a nozzle assembly for producing a stream of particle containing fluid,

means for jetting the stream from the nozzle assembly,

means for modulating the velocity of the stream to break the jet stream into discrete drops,

detector means for detecting particles in the stream,

pulse charging means responsive to the detector means for charging selected drops,

means for synchronizing the pulse charging means and modulating means to ensure against partial drop charging during on and off transitions of the pulse charging means, and

means for deflecting charged drops an amount related to the drop charge.

14. The apparatus as defined in claim 13 wherein the stream is jetted into the atmosphere, and the detector means are positioned outside the jetting means where the jet stream travels through the atmosphere.

15. The apparatus as defined in claim 13 wherein

particles characterized by a particular parameter are detected by said detector means, said apparatus including

second detector means for detecting substantially all particles in the stream, including particles characterized by said particular parameter and those not so characterized,

said pulse charging means being responsive to both said detector and second detector means for charging drops containing particles characterized by said particular parameter to a drop charge of one value, for charging drops containing particles not characterized by said particular parameter to another value, and for modifying the drop charge for drops containing both particles characterized by said parameter and those not so characterized.

16. The apparatus as defined in claim 13 wherein a coaxial flow stream having an inner stream portion of particle containing fluid and an outer portion of sheath fluid is produced by said stream producing means, which coaxial flow stream is jetted into the atmosphere by said jetting means.

17. A method of separating certain particles from others contained in a fluid stream comprising,

jetting the fluid stream,

detecting certain particles in the fluid stream,

modulating the fluid stream velocity in the direction of the stream axis to break the stream into discrete drops,

charging drops containing detected particles during drop charging pulse periods,

synchronizing the drop charging pulse period with the modulating of the fluid stream to prevent drops breaking from the stream during drop charging on and off transitions, and

deflecting charged drops in an amount related to the drop charge thereon.

18. The method of particle separation defined in claim 17 wherein said fluid stream comprises a coaxial flow stream having an inner stream portion of particle containing fluid and an outer stream portion of sheath fluid.

19. The method of particle separation defined in claim 17 wherein the fluid stream is jetted into the atmosphere, and wherein the step of detecting certain particles in the fluid stream is effected at a point along the jet stream travel through the atmosphere before the stream breaks into discrete drops.

20. The method of particle separation defined in claim 17 wherein said certain particles detected are those characterized by a particular parameter value, the method also including,

detecting other particles in the fluid stream which are characterized by other parameter values,

charging the jet stream by use of a different drop charging pulse while drops containing detected particles characterized by other parameter values break from the stream to supply such drops with a different drop charge, and

modifying charging of the jet stream while drops containing both particles characterized by a particular parameter and those characterized by other parameters break from the stream for a different deflection of such drops.