CA1206559A - Method of and apparatus for detecting change in the breakoff point of a droplet generation system - Google Patents
Method of and apparatus for detecting change in the breakoff point of a droplet generation systemInfo
- Publication number
- CA1206559A CA1206559A CA000421053A CA421053A CA1206559A CA 1206559 A CA1206559 A CA 1206559A CA 000421053 A CA000421053 A CA 000421053A CA 421053 A CA421053 A CA 421053A CA 1206559 A CA1206559 A CA 1206559A
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- Prior art keywords
- stream
- radiation
- jet stream
- amplitude
- undulation
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Links
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- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000002245 particle Substances 0.000 claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 230000005855 radiation Effects 0.000 claims description 45
- 239000006194 liquid suspension Substances 0.000 claims description 7
- 230000006872 improvement Effects 0.000 claims description 6
- 210000004027 cell Anatomy 0.000 description 12
- 239000013078 crystal Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 6
- 230000007246 mechanism Effects 0.000 description 6
- 239000012530 fluid Substances 0.000 description 5
- 238000012937 correction Methods 0.000 description 4
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- 230000003287 optical effect Effects 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- NOQGZXFMHARMLW-UHFFFAOYSA-N Daminozide Chemical compound CN(C)NC(=O)CCC(O)=O NOQGZXFMHARMLW-UHFFFAOYSA-N 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 239000003990 capacitor Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
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- 241000331231 Amorphocerini gen. n. 1 DAD-2008 Species 0.000 description 1
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical compound CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 description 1
- 101100536354 Drosophila melanogaster tant gene Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- PBAYDYUZOSNJGU-UHFFFAOYSA-N chelidonic acid Natural products OC(=O)C1=CC(=O)C=C(C(O)=O)O1 PBAYDYUZOSNJGU-UHFFFAOYSA-N 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- HCTVWSOKIJULET-LQDWTQKMSA-M phenoxymethylpenicillin potassium Chemical compound [K+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)COC1=CC=CC=C1 HCTVWSOKIJULET-LQDWTQKMSA-M 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
-
- 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
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1404—Handling flow, e.g. hydrodynamic focusing
- G01N2015/1406—Control of droplet point
Landscapes
- Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Dispersion Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
ABSTRACT
A particle separator for sorting particles suspended in a liquid according to certain characteristics, including a method of and apparatus for detecting a change in the droplet breakoff point of a liquid jet stream which is subjected to vibrations. The vibrations produce amplitude undulations on the surface of the jet stream. The amplitude of the undulations is monitored or interrogated at a fixed point on the jet stream prior to the breakoff point. A change in amplitude of the undulations at that fixed point produces a signal voltage the value of which is proportional to the amplitude change.
This signal voltage may be used (1) to alert the operator that a change has occurred in the point at which the jet stream is breaking up into droplets, (2) to automatically control the intensity of the vibrations for restoring the amplitude of undulation at that fixed point to its original state, or (3) to automatically disable the sorting portion of the apparatus. Any one or any combination of the foregoing three happenings can be utilized.
A particle separator for sorting particles suspended in a liquid according to certain characteristics, including a method of and apparatus for detecting a change in the droplet breakoff point of a liquid jet stream which is subjected to vibrations. The vibrations produce amplitude undulations on the surface of the jet stream. The amplitude of the undulations is monitored or interrogated at a fixed point on the jet stream prior to the breakoff point. A change in amplitude of the undulations at that fixed point produces a signal voltage the value of which is proportional to the amplitude change.
This signal voltage may be used (1) to alert the operator that a change has occurred in the point at which the jet stream is breaking up into droplets, (2) to automatically control the intensity of the vibrations for restoring the amplitude of undulation at that fixed point to its original state, or (3) to automatically disable the sorting portion of the apparatus. Any one or any combination of the foregoing three happenings can be utilized.
Description
)655~9 This invention relates to apparatus for sorting minute particles in a fluid, and in particular to 3uch apparatus wherein a liquid jet stream containing these particles i5 vibrated to produce undulation3 on the surface of the jet stream and ~ubsequent break-up of the stream into droplets which are then sorted according to the particle characteristics and collected downstream.
Apparatus of the foregoing kind may be referred to as flow cytometric sorting systems and are used in the medical research and diagnostic field for the rapid analysis of blood cells and other biological cells. Systems for cell separation and soreing are described in U.S. patents 4,038,556; 3,963,606; 3,710,933 and 3,380,584, in SCIENCE, Vol. 198, pages 149-157, published October 14, 1977, and in the references cited therein.
U.S. patent 4,038,S56 discloses a me~hod of and apparatus for the simultaneous optical mea~urement of several characteristics of each particle of a group of small particles while the particles are suspended in a liquid.
U.S. patent 3,963,606 discloses a particle separator for separating particle~ in a liquid according to certain characteristics including a device for adjusting an electrical delay to be equal to the time between the emergence of a particle from a jet forming aperture to the breakoff point.
U.S. patent 3,710,933 disclos2s an apparatus for automatically analyzing and sorting minute particle~ suspended in a liquid on the basis of certain preselected characteri~tics.
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U.S. patent 3,380,584 discloses a particle separator in which electrical pulses cause an acoustic coupler driver to vibrate the fluid which contains the particles.
The SCIENCE article, Vol. 198, pages 149-157 discloses a flow cytometer. Fluorescence from biological cells within a fluid stream is measured at the intersection with a laser beam. Droplets containing cells of interest are sorted out of the fluid tream. To the extent that it might be necessary to understand fully the techniques involved and ~he teachings of the invention herein, the above patents are incorporated herein for reference.
A major problem in u~ing the cell sorter systems wherein a jet stream subjected to vibrations breaks off into droplets îs ascereaining if a change has occurred in the point at which the jet stream i8 breaking into droplets. If the precise instant of breakoff with relation to the sense point change3, then the instrument cea3es to be a cell sorter and become~ a water or saline solution sorter of unknown cell content. An even wor~e undesired result is that the unwanted particles are qorted when there i~ such a change in the breakoff point. Such a change i3 often due to changes in the mechanical coupling coefficients, such as air bubbles entering the flow ~hamber and partial plug5 of the jet stream exit orifice.
Optical sensitivities, such a~ the presence of undesired light, render impractical the use of an illuminating strobe source to observe the breakoff point on the jet stream while the system is taking data or sorting the cells. It is at this time of 80rting that a monitor i8 most needed.
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An object of the present invention is to sense in a droplet generation sy~tem a change in the amplitude of the undulations at a fixed point on the surface of a liquid suspension jet stream prior to the droplet breakcff point, and to indicate that such a change has occurred.
Another object is to automatically disable the ~orting function in a particle analy~er and sorting system when there is a significant change in the droplet breakoff point.
~ A further object is to utilize the information derived from a change in the amplitude of undulation at a fixed point on the surface of a jet stream to restore the level of the amplitude of undulation at that fixed point to its original state.
Other objects will appear from a reading of the detailed description herein.
The liquid jet stream containing the minute particle3 in suspen~ion is driven at the frequency at which it is desired to generate droplet~, nor~ally 20 to 40 KC depending on the diameter of the jet exit orifice; for example, 32 KC frequency for a 76 micron jet exit orifice diameter and 40 KC frequency for a 50-60 micron jet exit orifice diameter. The jet is vibrated by introduciag a small disturbance, as from a driven piezo-electric crystal, at the exit orifice of the jet stream. This disturbance is in the form of an undulation or a ~tanding wave on the surface of the jet stream and this undulation grow~ as the jet advances and causes the breakoff of droplets downstream. The amplitude of the disturbance or undulation at any point along the jet stream is a function of the distance of that point from the point of droplet breakoff and this a~pect is ~2~J6~
described in the report oF Richard G. Sweet, SEL-64-004, ~arch 1964, of the StanEord Laboratories of the Stanford University. As here described, the amplitude of the undulation at a fixed point is monitored. A change in this amplitude at that fixed point indicates a change in the breakoEf point. In a prefer~ed embodiment herein, a laser beam, used as the illumination source in the optical cell sorting systems described in the foregoing U.S. patents, is utilized as a concentrated source of light rays in the monitoring scheme. The interrogating 1aser-beam strikes the jet stream at a fixed point nnd then the laser beam scatters to provide a lobe pattern of scattered light that i9 in the same plane as the laser beam and is perpendicular to the jet stream.
As is well known, thi~ scattered laser light is modulated by the surface undulations on ehe jet. Heretofore, this modulation has been considered to be an undesirable phenomenon when cell light scatter is being measured and has usually been blocked out in the in~trument.
The present apparatus detects such modulation by positioning a photo diode in the plane of laser light scatter, hereinafter also referred to a~ the scattering plane. The output of the photo diode is converted to a voltage which may be used (1) to alert the operator either visually or audibly that a change has occurred in the point at which the jet stream is breaking up into droplets, ~) to automatically control the intensity of the vibrations applied to the jet for restoring the amplitude of undulation at the point on the jet at which it is being monitored, or (3) to automatically disable the apparatus. Any one or any combination of the foregoing happenings can be utilized itl the method or apparatus here described.
If desired, a secondary source o concentrated light rays other than the laser beam can be used to impinge on or interrogate the jet ~65iS~
stream at a point closer to the droplet breakoff point where the undulations are larger and, therefore, where the signal obtained therefrom i~ larger. In thi~ case, the photo diode would be judiciously positioned to receive the light scattered or reflected from the jet.
A feature of this disclosure is the sorting disabling circuitry which includes a fast change detector responsive to a rectified output whose D.C. level i9 proportional to th~ percentage ~odulstion of the beam of concentrated light ray~ (laser beam, for e~ample) as affected by the undulations on the surface of the jet stream. This detector controls a relay driver latchin~ mechanism which acts to disable the sorting function of the particle analyzer whenever there i8 a significant change in the amplitude of undulation on the jet stream.
This circuitry i9 provided with an operator reset device and an alar~
which, if desired, can indicate the type of change ba~ed on the polarity of the output from the fast change detector.
Another feature i9 an automatic gain control (AGC) loop ~ystem for varying the intensity of the vibrations applied by the pie~o-electric crystal to the jet stream, for 310w changes in the amplitude of modula~ion. The aforementioned rectified output serves a~ a control voltage for the AGC loop. Thi3 loop system varies the power which is applied to drive the piezo-electric crystal. As a result, fine, a~ distinguished from coarse, amplitude corrections on the surface of the jet stream i9 obtainable. An arm-disable control for the AGC syste~ enable ;.nitial ~etting of the droplet breakoff point.
~Q6~9 More particularly in accordance with one aspect of the invention there is provided, a method for use in a droplet ~eneration system wherein a liquid jet steam is produced and vibrations applied thereto in order to produce undul~tions on the surface of said stream and subseguent breakup of the jet stream into droplets which are collected downstream, said stream having a breakpoint region, and sensing the undulation on the ~urface of said jet stream at a location on said stream prior to the breakoff point of said stream into droplets, said method comprising the step of: controlling said vibrations as a function of the amplitude of the undulation at said location.
In accordance with the second aspect of the inventlon there is provided, in an apparatu~ for analyzing and sorting particles suspended in a liquid9 said apparatus including first means for producin~ a jet stram from said liquid suspension, second means for controlling the position of the breakoff point, vibrating mesns for vibrating said jet stream to produce an undulation on the surface of said stream and subsequent breakup of said stream into droplets for collection downstream, said stream havin~ a breakpoint region, radiation means for providin~ a beam of radiation to interrogate a portion of said jet stream at a location thereon prior to the brea~point re~ion by impingin~ said beam thereon, the improvement comprising: sensing means, coupled to said second means, responsive to radiation from said radiation means which interrotages said portion of said stream at said location, for providing an output which is a function of the amplitude of undulation at said location on said portion of said jet stream.
In accordance with a third aspect of the invention there is provided, in an apparatus for analyzin~ and sorting particles suspended in a liquid, said apparatus including first means for producing a jet stream from ~aid liquid suspension, second means for controlling the position of the breakoff point, vibratinæ means for vibratin~ said jet stream to produce an undulation on the surface of said stream and subsequent breakup of said stream into droplets for collection downstream, ssid stream having a breakpoint region, radiation means for providin~ a beam of radiation to interrogate a portion of said jet stream by impinging said beam thereon, the improvement comprising: sensing means, coupled to said second means, responsive to radiation from said radiation means which interrogates said - 6a -~2~5~i~
portion of said stream, for providing an output to said second means whichis proportional to the amplitude Oe undulation at said portion of said jet stream.
In accordance with a fourth aspect of the invention there is provided, in an apparatus for analyzing and sorting particles suspended in a liquid, said apparatus including first means for producing a jet stream from said liquid suspension, second means for controlling the position of the breakoff point, vibrating means for vibrating said jet stream to produce an undulation on the surface of said stream and subsequent breakup oE said stream into droplets at a breakoff point for collecting downstream, said stream having a breakpoint region, radiation means for impinging a beam of radiation upon a portion of said ~et stream to thereby scatter said radiation impingine thereon, the improvement comprising: sensing means, coupled to said second means, responsive to radiation scattered by said stream, for providing an output to said second means which is a function of the magnitude of the sensed scattered radiation.
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_, By way of example only illustrative embodiments of the invention now will be described with reference to the accompanying drawings in which:
Fi8. 1 ia a block diagra~ showing how the electrical elements of the invention are coupled to a known type of particle analyzer and sorter;
Fig. 2 6hows the electrical circuitry of the fa~t change detector, the relay driver latching mechani~m and the alarm circuit3 of the diagram of Fig. l;
Pig. 3 ~hows diagra~matically the electrical circuitry for converting the signal obtained from the photo diode to a receified D.C. which is proportional to the modulation of the light rays by the surface undulations on the jet stream; and Figs. 4 to 8 illustrate in more detail the electrical circuitry of the blocks shown in Fig. 3.
Fig, 9 illustrates in side elevation, to scale, the breakup of a regularly disturbed fluid jet stream;
Fig. 10 ia an enlarged side elevation view o~ the non-electronic portions of the system components to the left of the dot-d~sh line 10 of the block diagram of Fi8. 1 and the aense means 44;
Fig. 11 schematically illustrates the laser light being scattered fro~ the surface of the jet ~tream 16 and bein8 detected by the sense means 44 of the system shown in Figs. 1 and 10;
Fig. 12 is an enlarged, simpliEied, view of the laser beam impinging upon the sense point of the iet stream, in the X-Y
scattering plane of Fig. ll, with the jet streamis ~inimum diameter shown in dot-dash, of the syate~ shown in Figs. 1 and 10;
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Fig. 13 is an enlarged, simplified, view of the laser beam impingin~ upon the sense point of the jet stream viewed in the X-Z
plane of Fig. ll, when the jet stream is at its maximum diameter at the sense point, of the system shown in Figs. l and l0; and Fig. 14 is an enlarged, simplified, view of the laser beam impinging upon the sense point of the jet stream viewed in the X-Z
plane of Fig. ll, when the jet stream is at its minimum diameter at the sense point of the system shown in Figs. l and l0.
Throughout the figures of the drawings the same parts are designated by the same reference numerals.
The block diagram of Fig. l is divided into two parts by a dot-dash line l0. System COmpOQentS to the left of the dot-dash line are those which normally exist in a known type particle analyzer and sorting system, sometimes referred to as a flow cytometric sorting system. One such known sorting system is found in the TPS and EPICS
series of instruments manufactured and sold by Coulter Electronics, Inc. of Hialeah, Florida 33010. Only those componenes of the particle analyzer and sorter have been shown which are necessary to explain the operation of the present invention. System components eO the right of the dot-dash line l0 comprise parts of the present invention which have been added and couple to the known particle analyzer and sorter for achieving the objects of the invention. It should be noted that the automatic gain control (AGC) feature and the disabling feature involving a relay driver latching mechanism of the invention are inserted at two locations into the normal signal paths of the particle analyzer and sorting systems a~ will appear in more detail hereinafter.
The known particle analy~er and sorting system shown to the left of the dot-dash line 10 will now be briefly described. It includes a flow chamber 12 into which a saline solution (normally 13 p.s.i.g) is introduced unde~ pressure and exits through a small orifice 14 (diameter ranges from 50 u to 200 u, depending upon the application of the system) to form 8 liquid jet stream 16 all of which i~ referred to as a first mean3 for producing a jet stream. The sample ta suspension of minute particles, ~uch as blood cells or biological cells) i8 introduced into the flow chamber 12 through a tube 18. Below the exit orifice 14 and above and prior to the breakoff point, ~.he jet stream 16 is interrogated by a light source or radistion mean~ 20 (normally a laser beam) and the re~ponse of the minute particle in the sample to the illumination (normally light scatter and fluore~cence) i8 tetected by the sensor sy3tem 22 also at a poiat prior to and above the breakoff point.
The 10w chamber 12 is mounted to and supported by 3 single vibrating means or second means for controlling the position of the breakoff point, a piezo-electric crystal assemblage 24, positioned above the radiation means 20, which vibrates the chamber 12 at 8 high frequensy. The exact frequency at which the chamber 12 vibrates i3 dependent on the selected diameter of the exit orifice 14 which requency is normally 20-40 KC. These vibrations impart small di~turb2nces, normally undulations, on the surface of the jet 16 which ~2~!6S5~
grow, due to well known aurface tension effect~, and eventually pinch the jet off at a breakoff point 26 into well defined droplets 28. The breakup of a regularly disturbed ~luid jet stream is ~hown in Fig. 9 and i~ included solely to illustrate ~uch effects. The exact distance from the nozzle containing the orifice 14 to the breakoff point 26 is inversely proportional to the amplitude of the initial disturbance or undulation. The si~e of the disturbance is propor~ional to the amplitude of the si~nal voltage applied to the crystal 24, if the mechanical coupling coefficients of the sy3tem hold constant.
Unfortunately, there are ~everal factors which can cause changes in the mechanical couplin~ coefficients and the~e factors are difficult to eliminate. The~e include air bubbles entering the flow chamber 12 with the 3ample or with the saline soiution and partial plug8 of the exit orifice 14 due to debri~, such a~ bro~en-up cell3 or fat.
The piezo-electric crystal i~ driven by a power ~mplifier 30 which normally derives its ~ignal from a frequency generator 32 through a variable potentiometer 34 which i8 usPd to vary the amplitude of the signal appliet to crystal 24 ant therefore vary the nominal breakoff point 26. Potentio~eter 34 serve~ as a coarse correction source to the drive of power amplifier 30 driving the crystal 24. Line 36 designates the normal path from the potentiometer 34 to the power amplifier in the ab~ence of the co~ponent~ of the present invention. The system of the presene invention ha~
incorporated 2 switch 59 ~hich is not present in the prior art sys~em3, for a purpose described hereinafter.
Connected to the sen30r 22 i8 the 30rt decision logic 38 in which the signal~ obtained from the deeectors (not shown but for~ing part oE
the sensor system 22) are applied to a set of criteria to decide )6SSi~9 whether or not it is desired to capture the particle originating those signals. If capture is desired, that decision must be delayed, as by sort delay 40, while the particle travels from the sense point to the breakoff point. A sort pulse is then formed by sort pulse forming circuit 41 and amplified and applied, through power amplifier 43, to the jet stream as a voltage just as the droplets which will contain the desired particle breaks off from the jet. Because of this impres~ed voltage the droplets break off with a net charge. The jet of droplets passes through an intense constant electric field which accelerates the charged droplets in the horizontal plane as they travel downwards. Thus charged droplets travel in a different path from the path of uncharged droplets and fall into different capture vessels, thereby effecting a phy~ical sorting of the particle~. A
typical rate of sorting for this process is 4,000 particle~ per second. Reference i5 made to U.S. patent 3,380,584 which discloses a way of impressing a voltage on a downstream portion of the Jet stream containing particle3 to be charged for subsequent collection and to an article by ~ulett, Bonner, Sweet and Her~enberg, CLI~ICAL CHEMIST~Y, Vol. 19, No. 8, 1973, which discloses impressing a voltage on an upstream portion of the jet ~tream for the same purpose.
The foregoing system is known in the art and no claim is made herein to this prior art method of analyzing and sorting minute particles. Such apparatus i8 disclosed in the aforementioned U.S.
patents and the references cited therein.
The present invention makes use of the relationship between the amplitude of undulation on the surface of the jet stream at any fixed point and the position of the droplet breakoff point to ascertain that ~2~)~55~
these has been a change in the droplet breakoEf point. As stated hereinabove, the amplitude of the undulation~ increases as the breakoff point is approached. An increase in amplitude of undulation at any given point along the jet is an indication that the breakoff point is closer while a decrease in the amplitude is an indication that the breakoff point i8 further away. The apparatu3 of the present invention monitors the position of the breakoff point to effect any one of the following three results: (1) to provide an indication, either visually, as by mean~ of a meter, or sudibly, by mean~ of an alarm, of a change in the breakoff point, (2) to di~able the 30rting proces3 and sound an alar~ Lf there i8 a fast change in the breakoff point, or (3) to automatically ant promptly restore the breakoff point, as by means of an automatic gain control loop, for ~mall and slow changes in the breakoff point.
The system components to the right of the dot-dash line 10 of the block diagram of Fig. 1 comprise parts of the present invention which couple to the known particle analyzer and sorter appearing at the left of dot-dash line 10, and include a photodiode or [sen~ing] sense ~eans 44 which detect~ the light ~cstteret by the jet3tream 16 a~ 8 result of the i~pact thereon by th~ concentrated beam of inten~e light 20.
An enlarged view of the non-electronic portions of the system co~ponen~s to the left of the dot-dash line 10 of the block diagra~ of Fi8. 1 aad the sense means 44 are shown in Fig. 10. This scattered light haa two components: a D.C. component which i~ proportional to the size of the jet stream 16 and the power of the li~ht beam 2~ ~the la~er, for exa~ple); and an ~.C. component which i8 proportional to the undulations on the jet ~tream 16 at the sen~e point 21, the point on the jet ~tream 16 at which the interrogating beam of light strikes it or i~pinges thereon, and the power o the intense interrogating light beam 20 ~the laser). The diode 44 is operated in the current mode, that i~ to say it is ter~insted in low impedance, thereby producing a linear optical power-to-current relationship and reducing the effect3 of diode capacitance for maximum 6~5S9 electronic bandwidth. Pho~o diode 44 is coupled to a breakpoint control means which includes an operational amplifier 46 which i8 operated as a transimpedance amplifier. Amplifier 46 converts the current from the diode 44 to a voltage linearly. The output from the tran~impedance amplifier 46 splits into an A.C. path including A.C.
amplifier 48 and a D.C path including D.C. amplifier 50.
The A C. path contains the basic information to be used in the practice of the invention, viz, the amplitude of the undulations on the jet stream Because the amplitude of the undulation signals generated at the photo diode detector 44 are low, this signal is given considerable gain by the A.C. amplifier 48, of the order of 103. In order to improve the signal-to-noise ratio in this A.C. path the bandwidth of the A.C. amplifier3 i8 limited to the range of frequencies of o~cillation applied to the pie20-electric crystal 24.
Since the size of the jet 16 i3 held constant during a test run of ssmple particles and i5 rarely changed, the D.C. component is proportional to the size of the jet 16 and the power of the laser 20, then a change in the D.C. level in the path including D.C. amplifier 50 can be considered to be a change in la~er power. Th~ ~ignal through the D.C. path controls the gain of a voltage controlled gain amplifier 52 in ~he output of the A.C. path, thereby enabling the A.C.
path 3ignal ~o be normalized as to laser power, as a result of which recalibration of the appara~us is eliminated each time ehe power of the laser 20 i8 changed. Stated another way, the D.C. path removes the effect of a change in laser power from the measurement of the distance from the sensing point 21 to the breakoff poin~ 26.
~Z~6~i5i9 The A.C. signal from the voltage controlled amplifier 52 is rectified by rectifier 54 (preferably a full-wave rectifier to obtain a smoother D.C. signal output therefrom) to provide a D.C. signal on lead 56 which is proportional ~o the amplitude of the undulations on the jet stream 16, and proportional to the distance f~om the sense point 21 to the breakoff point 26. The details of one suitable full wave rectifier which can be used is 3hown in Fig. 8.
The invention disclose3 three ways for utilizing the D.C. voltage on lead 56. The simplest is to drive a percentage modulation meter 58 which provides a vlsual indication as to the position of the droplet breakoff point 26 on the jet stream 16. Properly calibrated, such a ~eter 58 can be uYed to set the breakoff point 26 by ~anually adjusting a reference means or potentiometer 34 and using the normal path 36 in the known analyzer and sorter apparatus to bypass the AGC
system hereinafter described. The meter 58 is connected 80 as to be operable at all times.
The rectified voltage on lead 56 (the output from rectifier 54) can also be 3ed 28 a control voltage in an automatic gain control (AGC~ loop which will provide fine, a9 distinguished from co~rse, corrections to the voltage drive or power amplifier 30 which feeds the piezo-electric crystal trsnsducer 24 over path 60, and therefore fine corrections in the drift in ~he breakoff point 26, assuming, of course, that con~rol of the normal pa~h 36 is transferred by switching means 59 when the AGC feature of the invention is utili~ed. This AGC
loop includes an AGC amplifier sy3tem or control means 64 having an output, input and control terminals 65, 69 and 71, respectively, which is fed from the voltage o~ lead 56 over lead 66, and is 3imilar in design to the circuitry of voltage controlled gain amplifier 52 as illustrated in Fig. 7. A
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suitable double pole-single throw 3witch 59 i8 connected acros~ leads 36 and 37. switch 59 serve~ to effectively deactivate the AGC loop from the system when it is de~ired to initially set up the analyzer and sorter for a specific droplet delay. After the proper time delay has been ~et, the AGC system 64 i~ armed or activated to maintain the desired breakoff point. Prior to activation of the APC, the voltage output terminal VO (lead 37) i8 adjusted to the same amplieude a3 that on the normal path 36, by means oE potentiometer 67 a3 is indicated by the null detection meter 68, after which switch 59 can be thrown to transfer control of the power amplifier 30 to the AGC
system. The breakpoint control means also include~ amplifiers 46, 48, 50 and 52, rectifier 54 and said control means 64 and reference means 34. The sensin8 means includes such breakpoint control mean3 and the sense means 44.
lS Another way of monitoring the breakoff point 26 on the jet 16 is by mean3 of a ~ystem including a fa~t change detector 70 to which the rectified voltage on lead 56 is fed. The output 80 of fast change detector 70 is zero if a D.C. signal is being applied twhich i~ the ca~e when the breakoff point 26 is con~tant) and changes from zero if a "fast" change in droplet breakoff point i9 encountered. The polarity of the change in the output 80 of the fast change detector 70 indicates whether the rectified ~ignal level from rectifier 54 has increased or decrea3ed. The output from detector 70 can drive an alarm circuit 72 to alert the operator of a change in the breakoff point. This same output can al~o drive a relay system compo~ed of a driver latching mechanism 76, which, ;n turn, servss to automatically disable the ~orting system, via leads 78 in ~he particle analy~er and sorter. Operstor reset 74 serves to manually reset the driver latching mechanism 76. The alarm 72 can be made to indicate the type ~2~`6~
of change in the breakoff point based on the polarity of the output from the detector 70.
Fig. 2 diagramatically illustrates one form which the electrical circuitry of the fast change detector 70 may take, including the relay driving mechanism and the operator reset and the alarm. Fig. 2 sho~7s both the audible alarm 72 and a visual alarm 71. The change of the signal on lead 56 is coupled to the amplifier 70 by capacitor Cl.
This A.C. signal is amplified and applied to lead 80. If the signal on lead 56 is a constant D.C. value lead 80 will be near ~ero potential. Lead 80 is connected to two comparatorY 77 and 79 which are configured to sense variations in the signal on lead 80 in either polarity away from a zero potential. A slight guard band is used to allow for amplifier off~et. When the signal on lead 80 changes, the appropriate comparator senses the change and switches. The switching is sensed and latched by the latch 81. The Q output of the latch 81 switches, disabling ~AND gates in 78, thus interrupting the sort signals. The switching of the latch also activates both a visual alarm 71 and an audible alarm 72. The values of capacitor Cl and resistor Rl are selected for speed of change while Rl and R2 are selected for sensitivity to change.
Fig. 4 illustrates the circuitry of the transimpedance pre-amplifier 46 of Figs. 2 and 3. Fig. 5 illustrates the circuitry of the A.C. æmplifier 48 of Figs. 2 and 3. Fig. 6 illustra~es the circuitry of the D.C. amplifier 50 of Fig3. 2 and 3. Fig. 7 illustrates the circuitry of the voltage controlled gain amplifier 52 of Figs. 2 and 3. Fig. 8 illu~trate3 the circuitry of the full wave rectifier 53 of Figs. 2 and 3.
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l6 Referring now to FIGS. 10-11 and 12-14 wherein the photodiode sense means 44, which is positioned in the scattering plane, and located "generally" in the Y-Z plane and more particularly essentially radially of the jet stream's axis 104 as well as being perpendicular to the bundle of scattered rays, only one of which is shown, scattered ray 106, which ray 106 is located in the scattering plane 108, the X-Y
plane. As particularly shown in FIG. 12 the laser's one over e squared beam 20 is on the order of twice the width of the jet stream 16 and its intensity profile is gaussian shaped. Furthermore, the laser beam 20 itself is generally elliptically shaped having a major axis and a minor axis and is focused so that its major axis lies in the scattering plane 108 and is perpendicular to the jet stream's axis 104 and its minor axis is parallel to the axis 104 of the ~et stream 16. Referring now specifically to FIG. 12 the laser beam's longitudinal or central axis 110 is shown intersecting the longitudinal axis 104 of the jet stream 16. A portion of the interrogating laser beam 20 impinges on or upon the jet stream 16 at the sense point 21; another portion of the interrogating laser beam 20 ~ goes directly by the jet stream 16 without impinging on it and is referenced to as the "direct" or non-impinging radiation. The impinging radiation produces scattered radiation having a lobe pattern and the mechanism of such radiation scatter includes reflection, diffraction and refraction, all of which emanate from the jet stream 16, and it is essentially the refracted radiation which i5 detected by the photodiode 44. A more detailed description of light scattering used in flow systems for cell sizing can be found in Chapter 5 of Flow Cytometry and Sorting, Edited by Melamed, Mullaney and Mendelsohn, published by John Wiley and Sons, 1979. Referring now particularly to FIGS. 13 and 14, since the sense point 21 is located upstream of the 6S~
droplet breakoff point 26 (above the breakpoint region) where the amplitude of su.face undulation on the jet stream 16 are essentially cylindrically or barrel shaped when viewed sideways or in the vertical or Z axis, the droplets within the jet stream 16 act as weak positive and negative lens; that is their radius of curvature is relatively large. The result is that the radiation which is incident upon the jet stream's surface and which is scattered by the jet stream 16 is converged and diverged only slightly along the vertical Z axis; that is such scattered radiation is scanned in the vertical direction as shown. This effect is illustrated in said FIGS. 13 and 14 only for the refracted portion of the scattered radiationbut its effect on tlle reflected and diffracted radiation is the same.
Although, as previously indicated, the photodiode 44 and its associated light source 20 can be moved to a point closer to the droplet breakoff point 26 where the undulations are larger, it cannot be moved to a point any closer to the breakoff point 26 which would result in the basic modulation information, the amplitude of the undulation of the jet stream 16, being lost; that is any point on the jet stream 16 beyond which would result in the output signal from the diode 44 no longer being proportlonal to the amplitude of the undulations on the jet stream 16. The portion of the jet stream 16 wherein such amplitude information is lost is referred to as the breakpoint region and all that portion of the jet stream 16 prior to or upstream to the actual breakoff point 26 is referred to as the uninterrupted portion of the jet stream 16.
Apparatus of the foregoing kind may be referred to as flow cytometric sorting systems and are used in the medical research and diagnostic field for the rapid analysis of blood cells and other biological cells. Systems for cell separation and soreing are described in U.S. patents 4,038,556; 3,963,606; 3,710,933 and 3,380,584, in SCIENCE, Vol. 198, pages 149-157, published October 14, 1977, and in the references cited therein.
U.S. patent 4,038,S56 discloses a me~hod of and apparatus for the simultaneous optical mea~urement of several characteristics of each particle of a group of small particles while the particles are suspended in a liquid.
U.S. patent 3,963,606 discloses a particle separator for separating particle~ in a liquid according to certain characteristics including a device for adjusting an electrical delay to be equal to the time between the emergence of a particle from a jet forming aperture to the breakoff point.
U.S. patent 3,710,933 disclos2s an apparatus for automatically analyzing and sorting minute particle~ suspended in a liquid on the basis of certain preselected characteri~tics.
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U.S. patent 3,380,584 discloses a particle separator in which electrical pulses cause an acoustic coupler driver to vibrate the fluid which contains the particles.
The SCIENCE article, Vol. 198, pages 149-157 discloses a flow cytometer. Fluorescence from biological cells within a fluid stream is measured at the intersection with a laser beam. Droplets containing cells of interest are sorted out of the fluid tream. To the extent that it might be necessary to understand fully the techniques involved and ~he teachings of the invention herein, the above patents are incorporated herein for reference.
A major problem in u~ing the cell sorter systems wherein a jet stream subjected to vibrations breaks off into droplets îs ascereaining if a change has occurred in the point at which the jet stream i8 breaking into droplets. If the precise instant of breakoff with relation to the sense point change3, then the instrument cea3es to be a cell sorter and become~ a water or saline solution sorter of unknown cell content. An even wor~e undesired result is that the unwanted particles are qorted when there i~ such a change in the breakoff point. Such a change i3 often due to changes in the mechanical coupling coefficients, such as air bubbles entering the flow ~hamber and partial plug5 of the jet stream exit orifice.
Optical sensitivities, such a~ the presence of undesired light, render impractical the use of an illuminating strobe source to observe the breakoff point on the jet stream while the system is taking data or sorting the cells. It is at this time of 80rting that a monitor i8 most needed.
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An object of the present invention is to sense in a droplet generation sy~tem a change in the amplitude of the undulations at a fixed point on the surface of a liquid suspension jet stream prior to the droplet breakcff point, and to indicate that such a change has occurred.
Another object is to automatically disable the ~orting function in a particle analy~er and sorting system when there is a significant change in the droplet breakoff point.
~ A further object is to utilize the information derived from a change in the amplitude of undulation at a fixed point on the surface of a jet stream to restore the level of the amplitude of undulation at that fixed point to its original state.
Other objects will appear from a reading of the detailed description herein.
The liquid jet stream containing the minute particle3 in suspen~ion is driven at the frequency at which it is desired to generate droplet~, nor~ally 20 to 40 KC depending on the diameter of the jet exit orifice; for example, 32 KC frequency for a 76 micron jet exit orifice diameter and 40 KC frequency for a 50-60 micron jet exit orifice diameter. The jet is vibrated by introduciag a small disturbance, as from a driven piezo-electric crystal, at the exit orifice of the jet stream. This disturbance is in the form of an undulation or a ~tanding wave on the surface of the jet stream and this undulation grow~ as the jet advances and causes the breakoff of droplets downstream. The amplitude of the disturbance or undulation at any point along the jet stream is a function of the distance of that point from the point of droplet breakoff and this a~pect is ~2~J6~
described in the report oF Richard G. Sweet, SEL-64-004, ~arch 1964, of the StanEord Laboratories of the Stanford University. As here described, the amplitude of the undulation at a fixed point is monitored. A change in this amplitude at that fixed point indicates a change in the breakoEf point. In a prefer~ed embodiment herein, a laser beam, used as the illumination source in the optical cell sorting systems described in the foregoing U.S. patents, is utilized as a concentrated source of light rays in the monitoring scheme. The interrogating 1aser-beam strikes the jet stream at a fixed point nnd then the laser beam scatters to provide a lobe pattern of scattered light that i9 in the same plane as the laser beam and is perpendicular to the jet stream.
As is well known, thi~ scattered laser light is modulated by the surface undulations on ehe jet. Heretofore, this modulation has been considered to be an undesirable phenomenon when cell light scatter is being measured and has usually been blocked out in the in~trument.
The present apparatus detects such modulation by positioning a photo diode in the plane of laser light scatter, hereinafter also referred to a~ the scattering plane. The output of the photo diode is converted to a voltage which may be used (1) to alert the operator either visually or audibly that a change has occurred in the point at which the jet stream is breaking up into droplets, ~) to automatically control the intensity of the vibrations applied to the jet for restoring the amplitude of undulation at the point on the jet at which it is being monitored, or (3) to automatically disable the apparatus. Any one or any combination of the foregoing happenings can be utilized itl the method or apparatus here described.
If desired, a secondary source o concentrated light rays other than the laser beam can be used to impinge on or interrogate the jet ~65iS~
stream at a point closer to the droplet breakoff point where the undulations are larger and, therefore, where the signal obtained therefrom i~ larger. In thi~ case, the photo diode would be judiciously positioned to receive the light scattered or reflected from the jet.
A feature of this disclosure is the sorting disabling circuitry which includes a fast change detector responsive to a rectified output whose D.C. level i9 proportional to th~ percentage ~odulstion of the beam of concentrated light ray~ (laser beam, for e~ample) as affected by the undulations on the surface of the jet stream. This detector controls a relay driver latchin~ mechanism which acts to disable the sorting function of the particle analyzer whenever there i8 a significant change in the amplitude of undulation on the jet stream.
This circuitry i9 provided with an operator reset device and an alar~
which, if desired, can indicate the type of change ba~ed on the polarity of the output from the fast change detector.
Another feature i9 an automatic gain control (AGC) loop ~ystem for varying the intensity of the vibrations applied by the pie~o-electric crystal to the jet stream, for 310w changes in the amplitude of modula~ion. The aforementioned rectified output serves a~ a control voltage for the AGC loop. Thi3 loop system varies the power which is applied to drive the piezo-electric crystal. As a result, fine, a~ distinguished from coarse, amplitude corrections on the surface of the jet stream i9 obtainable. An arm-disable control for the AGC syste~ enable ;.nitial ~etting of the droplet breakoff point.
~Q6~9 More particularly in accordance with one aspect of the invention there is provided, a method for use in a droplet ~eneration system wherein a liquid jet steam is produced and vibrations applied thereto in order to produce undul~tions on the surface of said stream and subseguent breakup of the jet stream into droplets which are collected downstream, said stream having a breakpoint region, and sensing the undulation on the ~urface of said jet stream at a location on said stream prior to the breakoff point of said stream into droplets, said method comprising the step of: controlling said vibrations as a function of the amplitude of the undulation at said location.
In accordance with the second aspect of the inventlon there is provided, in an apparatu~ for analyzing and sorting particles suspended in a liquid9 said apparatus including first means for producin~ a jet stram from said liquid suspension, second means for controlling the position of the breakoff point, vibrating mesns for vibrating said jet stream to produce an undulation on the surface of said stream and subsequent breakup of said stream into droplets for collection downstream, said stream havin~ a breakpoint region, radiation means for providin~ a beam of radiation to interrogate a portion of said jet stream at a location thereon prior to the brea~point re~ion by impingin~ said beam thereon, the improvement comprising: sensing means, coupled to said second means, responsive to radiation from said radiation means which interrotages said portion of said stream at said location, for providing an output which is a function of the amplitude of undulation at said location on said portion of said jet stream.
In accordance with a third aspect of the invention there is provided, in an apparatus for analyzin~ and sorting particles suspended in a liquid, said apparatus including first means for producing a jet stream from ~aid liquid suspension, second means for controlling the position of the breakoff point, vibratinæ means for vibratin~ said jet stream to produce an undulation on the surface of said stream and subsequent breakup of said stream into droplets for collection downstream, ssid stream having a breakpoint region, radiation means for providin~ a beam of radiation to interrogate a portion of said jet stream by impinging said beam thereon, the improvement comprising: sensing means, coupled to said second means, responsive to radiation from said radiation means which interrogates said - 6a -~2~5~i~
portion of said stream, for providing an output to said second means whichis proportional to the amplitude Oe undulation at said portion of said jet stream.
In accordance with a fourth aspect of the invention there is provided, in an apparatus for analyzing and sorting particles suspended in a liquid, said apparatus including first means for producing a jet stream from said liquid suspension, second means for controlling the position of the breakoff point, vibrating means for vibrating said jet stream to produce an undulation on the surface of said stream and subsequent breakup oE said stream into droplets at a breakoff point for collecting downstream, said stream having a breakpoint region, radiation means for impinging a beam of radiation upon a portion of said ~et stream to thereby scatter said radiation impingine thereon, the improvement comprising: sensing means, coupled to said second means, responsive to radiation scattered by said stream, for providing an output to said second means which is a function of the magnitude of the sensed scattered radiation.
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_, By way of example only illustrative embodiments of the invention now will be described with reference to the accompanying drawings in which:
Fi8. 1 ia a block diagra~ showing how the electrical elements of the invention are coupled to a known type of particle analyzer and sorter;
Fig. 2 6hows the electrical circuitry of the fa~t change detector, the relay driver latching mechani~m and the alarm circuit3 of the diagram of Fig. l;
Pig. 3 ~hows diagra~matically the electrical circuitry for converting the signal obtained from the photo diode to a receified D.C. which is proportional to the modulation of the light rays by the surface undulations on the jet stream; and Figs. 4 to 8 illustrate in more detail the electrical circuitry of the blocks shown in Fig. 3.
Fig, 9 illustrates in side elevation, to scale, the breakup of a regularly disturbed fluid jet stream;
Fig. 10 ia an enlarged side elevation view o~ the non-electronic portions of the system components to the left of the dot-d~sh line 10 of the block diagram of Fi8. 1 and the aense means 44;
Fig. 11 schematically illustrates the laser light being scattered fro~ the surface of the jet ~tream 16 and bein8 detected by the sense means 44 of the system shown in Figs. 1 and 10;
Fig. 12 is an enlarged, simpliEied, view of the laser beam impinging upon the sense point of the iet stream, in the X-Y
scattering plane of Fig. ll, with the jet streamis ~inimum diameter shown in dot-dash, of the syate~ shown in Figs. 1 and 10;
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Fig. 13 is an enlarged, simplified, view of the laser beam impingin~ upon the sense point of the jet stream viewed in the X-Z
plane of Fig. ll, when the jet stream is at its maximum diameter at the sense point, of the system shown in Figs. l and l0; and Fig. 14 is an enlarged, simplified, view of the laser beam impinging upon the sense point of the jet stream viewed in the X-Z
plane of Fig. ll, when the jet stream is at its minimum diameter at the sense point of the system shown in Figs. l and l0.
Throughout the figures of the drawings the same parts are designated by the same reference numerals.
The block diagram of Fig. l is divided into two parts by a dot-dash line l0. System COmpOQentS to the left of the dot-dash line are those which normally exist in a known type particle analyzer and sorting system, sometimes referred to as a flow cytometric sorting system. One such known sorting system is found in the TPS and EPICS
series of instruments manufactured and sold by Coulter Electronics, Inc. of Hialeah, Florida 33010. Only those componenes of the particle analyzer and sorter have been shown which are necessary to explain the operation of the present invention. System components eO the right of the dot-dash line l0 comprise parts of the present invention which have been added and couple to the known particle analyzer and sorter for achieving the objects of the invention. It should be noted that the automatic gain control (AGC) feature and the disabling feature involving a relay driver latching mechanism of the invention are inserted at two locations into the normal signal paths of the particle analyzer and sorting systems a~ will appear in more detail hereinafter.
The known particle analy~er and sorting system shown to the left of the dot-dash line 10 will now be briefly described. It includes a flow chamber 12 into which a saline solution (normally 13 p.s.i.g) is introduced unde~ pressure and exits through a small orifice 14 (diameter ranges from 50 u to 200 u, depending upon the application of the system) to form 8 liquid jet stream 16 all of which i~ referred to as a first mean3 for producing a jet stream. The sample ta suspension of minute particles, ~uch as blood cells or biological cells) i8 introduced into the flow chamber 12 through a tube 18. Below the exit orifice 14 and above and prior to the breakoff point, ~.he jet stream 16 is interrogated by a light source or radistion mean~ 20 (normally a laser beam) and the re~ponse of the minute particle in the sample to the illumination (normally light scatter and fluore~cence) i8 tetected by the sensor sy3tem 22 also at a poiat prior to and above the breakoff point.
The 10w chamber 12 is mounted to and supported by 3 single vibrating means or second means for controlling the position of the breakoff point, a piezo-electric crystal assemblage 24, positioned above the radiation means 20, which vibrates the chamber 12 at 8 high frequensy. The exact frequency at which the chamber 12 vibrates i3 dependent on the selected diameter of the exit orifice 14 which requency is normally 20-40 KC. These vibrations impart small di~turb2nces, normally undulations, on the surface of the jet 16 which ~2~!6S5~
grow, due to well known aurface tension effect~, and eventually pinch the jet off at a breakoff point 26 into well defined droplets 28. The breakup of a regularly disturbed ~luid jet stream is ~hown in Fig. 9 and i~ included solely to illustrate ~uch effects. The exact distance from the nozzle containing the orifice 14 to the breakoff point 26 is inversely proportional to the amplitude of the initial disturbance or undulation. The si~e of the disturbance is propor~ional to the amplitude of the si~nal voltage applied to the crystal 24, if the mechanical coupling coefficients of the sy3tem hold constant.
Unfortunately, there are ~everal factors which can cause changes in the mechanical couplin~ coefficients and the~e factors are difficult to eliminate. The~e include air bubbles entering the flow chamber 12 with the 3ample or with the saline soiution and partial plug8 of the exit orifice 14 due to debri~, such a~ bro~en-up cell3 or fat.
The piezo-electric crystal i~ driven by a power ~mplifier 30 which normally derives its ~ignal from a frequency generator 32 through a variable potentiometer 34 which i8 usPd to vary the amplitude of the signal appliet to crystal 24 ant therefore vary the nominal breakoff point 26. Potentio~eter 34 serve~ as a coarse correction source to the drive of power amplifier 30 driving the crystal 24. Line 36 designates the normal path from the potentiometer 34 to the power amplifier in the ab~ence of the co~ponent~ of the present invention. The system of the presene invention ha~
incorporated 2 switch 59 ~hich is not present in the prior art sys~em3, for a purpose described hereinafter.
Connected to the sen30r 22 i8 the 30rt decision logic 38 in which the signal~ obtained from the deeectors (not shown but for~ing part oE
the sensor system 22) are applied to a set of criteria to decide )6SSi~9 whether or not it is desired to capture the particle originating those signals. If capture is desired, that decision must be delayed, as by sort delay 40, while the particle travels from the sense point to the breakoff point. A sort pulse is then formed by sort pulse forming circuit 41 and amplified and applied, through power amplifier 43, to the jet stream as a voltage just as the droplets which will contain the desired particle breaks off from the jet. Because of this impres~ed voltage the droplets break off with a net charge. The jet of droplets passes through an intense constant electric field which accelerates the charged droplets in the horizontal plane as they travel downwards. Thus charged droplets travel in a different path from the path of uncharged droplets and fall into different capture vessels, thereby effecting a phy~ical sorting of the particle~. A
typical rate of sorting for this process is 4,000 particle~ per second. Reference i5 made to U.S. patent 3,380,584 which discloses a way of impressing a voltage on a downstream portion of the Jet stream containing particle3 to be charged for subsequent collection and to an article by ~ulett, Bonner, Sweet and Her~enberg, CLI~ICAL CHEMIST~Y, Vol. 19, No. 8, 1973, which discloses impressing a voltage on an upstream portion of the jet ~tream for the same purpose.
The foregoing system is known in the art and no claim is made herein to this prior art method of analyzing and sorting minute particles. Such apparatus i8 disclosed in the aforementioned U.S.
patents and the references cited therein.
The present invention makes use of the relationship between the amplitude of undulation on the surface of the jet stream at any fixed point and the position of the droplet breakoff point to ascertain that ~2~)~55~
these has been a change in the droplet breakoEf point. As stated hereinabove, the amplitude of the undulation~ increases as the breakoff point is approached. An increase in amplitude of undulation at any given point along the jet is an indication that the breakoff point is closer while a decrease in the amplitude is an indication that the breakoff point i8 further away. The apparatu3 of the present invention monitors the position of the breakoff point to effect any one of the following three results: (1) to provide an indication, either visually, as by mean~ of a meter, or sudibly, by mean~ of an alarm, of a change in the breakoff point, (2) to di~able the 30rting proces3 and sound an alar~ Lf there i8 a fast change in the breakoff point, or (3) to automatically ant promptly restore the breakoff point, as by means of an automatic gain control loop, for ~mall and slow changes in the breakoff point.
The system components to the right of the dot-dash line 10 of the block diagram of Fig. 1 comprise parts of the present invention which couple to the known particle analyzer and sorter appearing at the left of dot-dash line 10, and include a photodiode or [sen~ing] sense ~eans 44 which detect~ the light ~cstteret by the jet3tream 16 a~ 8 result of the i~pact thereon by th~ concentrated beam of inten~e light 20.
An enlarged view of the non-electronic portions of the system co~ponen~s to the left of the dot-dash line 10 of the block diagra~ of Fi8. 1 aad the sense means 44 are shown in Fig. 10. This scattered light haa two components: a D.C. component which i~ proportional to the size of the jet stream 16 and the power of the li~ht beam 2~ ~the la~er, for exa~ple); and an ~.C. component which i8 proportional to the undulations on the jet ~tream 16 at the sen~e point 21, the point on the jet ~tream 16 at which the interrogating beam of light strikes it or i~pinges thereon, and the power o the intense interrogating light beam 20 ~the laser). The diode 44 is operated in the current mode, that i~ to say it is ter~insted in low impedance, thereby producing a linear optical power-to-current relationship and reducing the effect3 of diode capacitance for maximum 6~5S9 electronic bandwidth. Pho~o diode 44 is coupled to a breakpoint control means which includes an operational amplifier 46 which i8 operated as a transimpedance amplifier. Amplifier 46 converts the current from the diode 44 to a voltage linearly. The output from the tran~impedance amplifier 46 splits into an A.C. path including A.C.
amplifier 48 and a D.C path including D.C. amplifier 50.
The A C. path contains the basic information to be used in the practice of the invention, viz, the amplitude of the undulations on the jet stream Because the amplitude of the undulation signals generated at the photo diode detector 44 are low, this signal is given considerable gain by the A.C. amplifier 48, of the order of 103. In order to improve the signal-to-noise ratio in this A.C. path the bandwidth of the A.C. amplifier3 i8 limited to the range of frequencies of o~cillation applied to the pie20-electric crystal 24.
Since the size of the jet 16 i3 held constant during a test run of ssmple particles and i5 rarely changed, the D.C. component is proportional to the size of the jet 16 and the power of the laser 20, then a change in the D.C. level in the path including D.C. amplifier 50 can be considered to be a change in la~er power. Th~ ~ignal through the D.C. path controls the gain of a voltage controlled gain amplifier 52 in ~he output of the A.C. path, thereby enabling the A.C.
path 3ignal ~o be normalized as to laser power, as a result of which recalibration of the appara~us is eliminated each time ehe power of the laser 20 i8 changed. Stated another way, the D.C. path removes the effect of a change in laser power from the measurement of the distance from the sensing point 21 to the breakoff poin~ 26.
~Z~6~i5i9 The A.C. signal from the voltage controlled amplifier 52 is rectified by rectifier 54 (preferably a full-wave rectifier to obtain a smoother D.C. signal output therefrom) to provide a D.C. signal on lead 56 which is proportional ~o the amplitude of the undulations on the jet stream 16, and proportional to the distance f~om the sense point 21 to the breakoff point 26. The details of one suitable full wave rectifier which can be used is 3hown in Fig. 8.
The invention disclose3 three ways for utilizing the D.C. voltage on lead 56. The simplest is to drive a percentage modulation meter 58 which provides a vlsual indication as to the position of the droplet breakoff point 26 on the jet stream 16. Properly calibrated, such a ~eter 58 can be uYed to set the breakoff point 26 by ~anually adjusting a reference means or potentiometer 34 and using the normal path 36 in the known analyzer and sorter apparatus to bypass the AGC
system hereinafter described. The meter 58 is connected 80 as to be operable at all times.
The rectified voltage on lead 56 (the output from rectifier 54) can also be 3ed 28 a control voltage in an automatic gain control (AGC~ loop which will provide fine, a9 distinguished from co~rse, corrections to the voltage drive or power amplifier 30 which feeds the piezo-electric crystal trsnsducer 24 over path 60, and therefore fine corrections in the drift in ~he breakoff point 26, assuming, of course, that con~rol of the normal pa~h 36 is transferred by switching means 59 when the AGC feature of the invention is utili~ed. This AGC
loop includes an AGC amplifier sy3tem or control means 64 having an output, input and control terminals 65, 69 and 71, respectively, which is fed from the voltage o~ lead 56 over lead 66, and is 3imilar in design to the circuitry of voltage controlled gain amplifier 52 as illustrated in Fig. 7. A
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suitable double pole-single throw 3witch 59 i8 connected acros~ leads 36 and 37. switch 59 serve~ to effectively deactivate the AGC loop from the system when it is de~ired to initially set up the analyzer and sorter for a specific droplet delay. After the proper time delay has been ~et, the AGC system 64 i~ armed or activated to maintain the desired breakoff point. Prior to activation of the APC, the voltage output terminal VO (lead 37) i8 adjusted to the same amplieude a3 that on the normal path 36, by means oE potentiometer 67 a3 is indicated by the null detection meter 68, after which switch 59 can be thrown to transfer control of the power amplifier 30 to the AGC
system. The breakpoint control means also include~ amplifiers 46, 48, 50 and 52, rectifier 54 and said control means 64 and reference means 34. The sensin8 means includes such breakpoint control mean3 and the sense means 44.
lS Another way of monitoring the breakoff point 26 on the jet 16 is by mean3 of a ~ystem including a fa~t change detector 70 to which the rectified voltage on lead 56 is fed. The output 80 of fast change detector 70 is zero if a D.C. signal is being applied twhich i~ the ca~e when the breakoff point 26 is con~tant) and changes from zero if a "fast" change in droplet breakoff point i9 encountered. The polarity of the change in the output 80 of the fast change detector 70 indicates whether the rectified ~ignal level from rectifier 54 has increased or decrea3ed. The output from detector 70 can drive an alarm circuit 72 to alert the operator of a change in the breakoff point. This same output can al~o drive a relay system compo~ed of a driver latching mechanism 76, which, ;n turn, servss to automatically disable the ~orting system, via leads 78 in ~he particle analy~er and sorter. Operstor reset 74 serves to manually reset the driver latching mechanism 76. The alarm 72 can be made to indicate the type ~2~`6~
of change in the breakoff point based on the polarity of the output from the detector 70.
Fig. 2 diagramatically illustrates one form which the electrical circuitry of the fast change detector 70 may take, including the relay driving mechanism and the operator reset and the alarm. Fig. 2 sho~7s both the audible alarm 72 and a visual alarm 71. The change of the signal on lead 56 is coupled to the amplifier 70 by capacitor Cl.
This A.C. signal is amplified and applied to lead 80. If the signal on lead 56 is a constant D.C. value lead 80 will be near ~ero potential. Lead 80 is connected to two comparatorY 77 and 79 which are configured to sense variations in the signal on lead 80 in either polarity away from a zero potential. A slight guard band is used to allow for amplifier off~et. When the signal on lead 80 changes, the appropriate comparator senses the change and switches. The switching is sensed and latched by the latch 81. The Q output of the latch 81 switches, disabling ~AND gates in 78, thus interrupting the sort signals. The switching of the latch also activates both a visual alarm 71 and an audible alarm 72. The values of capacitor Cl and resistor Rl are selected for speed of change while Rl and R2 are selected for sensitivity to change.
Fig. 4 illustrates the circuitry of the transimpedance pre-amplifier 46 of Figs. 2 and 3. Fig. 5 illustrates the circuitry of the A.C. æmplifier 48 of Figs. 2 and 3. Fig. 6 illustra~es the circuitry of the D.C. amplifier 50 of Fig3. 2 and 3. Fig. 7 illustrates the circuitry of the voltage controlled gain amplifier 52 of Figs. 2 and 3. Fig. 8 illu~trate3 the circuitry of the full wave rectifier 53 of Figs. 2 and 3.
~l~0~
l6 Referring now to FIGS. 10-11 and 12-14 wherein the photodiode sense means 44, which is positioned in the scattering plane, and located "generally" in the Y-Z plane and more particularly essentially radially of the jet stream's axis 104 as well as being perpendicular to the bundle of scattered rays, only one of which is shown, scattered ray 106, which ray 106 is located in the scattering plane 108, the X-Y
plane. As particularly shown in FIG. 12 the laser's one over e squared beam 20 is on the order of twice the width of the jet stream 16 and its intensity profile is gaussian shaped. Furthermore, the laser beam 20 itself is generally elliptically shaped having a major axis and a minor axis and is focused so that its major axis lies in the scattering plane 108 and is perpendicular to the jet stream's axis 104 and its minor axis is parallel to the axis 104 of the ~et stream 16. Referring now specifically to FIG. 12 the laser beam's longitudinal or central axis 110 is shown intersecting the longitudinal axis 104 of the jet stream 16. A portion of the interrogating laser beam 20 impinges on or upon the jet stream 16 at the sense point 21; another portion of the interrogating laser beam 20 ~ goes directly by the jet stream 16 without impinging on it and is referenced to as the "direct" or non-impinging radiation. The impinging radiation produces scattered radiation having a lobe pattern and the mechanism of such radiation scatter includes reflection, diffraction and refraction, all of which emanate from the jet stream 16, and it is essentially the refracted radiation which i5 detected by the photodiode 44. A more detailed description of light scattering used in flow systems for cell sizing can be found in Chapter 5 of Flow Cytometry and Sorting, Edited by Melamed, Mullaney and Mendelsohn, published by John Wiley and Sons, 1979. Referring now particularly to FIGS. 13 and 14, since the sense point 21 is located upstream of the 6S~
droplet breakoff point 26 (above the breakpoint region) where the amplitude of su.face undulation on the jet stream 16 are essentially cylindrically or barrel shaped when viewed sideways or in the vertical or Z axis, the droplets within the jet stream 16 act as weak positive and negative lens; that is their radius of curvature is relatively large. The result is that the radiation which is incident upon the jet stream's surface and which is scattered by the jet stream 16 is converged and diverged only slightly along the vertical Z axis; that is such scattered radiation is scanned in the vertical direction as shown. This effect is illustrated in said FIGS. 13 and 14 only for the refracted portion of the scattered radiationbut its effect on tlle reflected and diffracted radiation is the same.
Although, as previously indicated, the photodiode 44 and its associated light source 20 can be moved to a point closer to the droplet breakoff point 26 where the undulations are larger, it cannot be moved to a point any closer to the breakoff point 26 which would result in the basic modulation information, the amplitude of the undulation of the jet stream 16, being lost; that is any point on the jet stream 16 beyond which would result in the output signal from the diode 44 no longer being proportlonal to the amplitude of the undulations on the jet stream 16. The portion of the jet stream 16 wherein such amplitude information is lost is referred to as the breakpoint region and all that portion of the jet stream 16 prior to or upstream to the actual breakoff point 26 is referred to as the uninterrupted portion of the jet stream 16.
Claims (20)
1. Method for use in a droplet generation system wherein a liquid jet stream is produced and vibrations applied thereto in order to produce undulations on the surface of said stream and subsequent breakup of the jet stream into droplets which are collected downstream, said stream having a breakpoint region, and sensing the undulation on the surface of said jet stream at a location on said stream prior to the breakoff point of said stream into droplets, said method comprising the step of: controlling said vibrations as a function of the amplitude of the undulation at said location.
2. Method according to claim 1, wherein said sensing is of the amplitude of undulation on the surface of an uninterrupted portion of said jet stream at said location on said stream which is prior to the breakpoint region.
3. Method according to claim 1, wherein said sensing is in proportion to the amplitude of the undulation at said location on the surface of the stream.
4. Method according to claims 1, wherein said sensing is of the amplitude of undulation on the surface of an uninterrupted portion of said jet stream and is by radiation which is scattered by said stream at said location on said stream which is prior to the breakoff point.
5. Method according to any one of claims 2 to 4, wherein said location is fixed.
6. Method according to any one of claims 2 to 4, which includes interrogating said stream by applying a concentrated beam of rays to said jet stream at said location on said stream and wherein said step of sensing includes deriving a D.C. signal from said interrogation which is proportional to the amplitude of the undulation at said location, and wherein said step of controlling said vibrations includes controlling the intensity of said vibrations by said D.C.
signal in response to a deviation of said amplitude from a predetermined level.
signal in response to a deviation of said amplitude from a predetermined level.
7. Method according to any one of claims 2 to 4, wherein said step of controlling said vibrations includes automatically controlling the intensity of the vibrations applied to said stream in response to a change in said amplitude of undulation at said location in such direction as to restore said amplitude of undulation at said location to its original state, to thereby prevent drift in said breakoff point.
8. In an apparatus for analyzing and sorting particles suspended in a liquid, said apparatus including first means for producing a jet stream from said liquid suspension, second means for controlling the position of the breakoff point, vibrating means for vibrating said jet stream to produce an undulation on the surface of said stream and subsequent breakup of said stream into droplets for collection downstream, said stream having a breakpoint region, radiation means for providing a beam of radiation to interrogate a portion of said jet stream at a location thereon prior to the breakpoint region by impinging said beam thereon, the improvement comprising: sensing means, coupled to said second means, responsive to radiation from said radiation means which interrogates said portion of said stream at said location, for providing an output which is a function of the amplitude of undulation at said location on said portion of said jet stream.
9. In an apparatus for analyzing and sorting particles suspended in a liquid, said apparatus including first means for producing a jet stream from said liquid suspension, second means for controlling the position of the breakoff point, vibrating means for vibrating said jet stream to produce an undulation on the surface of said stream and subsequent breakup of said stream into droplets for collection downstream, said stream having a breakpoint region, radiation means for providing a beam of radiation to interrogate a portion of said jet stream by impinging said beam thereon, the improvement comprising: sensing means, coupled to said second means, responsive to radiation from said radiation means which interrogates said portion of said stream, for providing an output to said second means which is proportional to the amplitude of undulation at said portion of said jet stream.
10. In an apparatus for analyzing and sorting particles suspended in a liquid, said apparatus including first means for producing a jet stream from said liquid suspension, second means for controlling the position of the breakoff point, vibrating means for vibrating said jet stream to produce an undulation on the surface of said stream and subsequent breakup of said stream into droplets at a breakoff point for collecting downstream, said stream having a breakpoint region, radiation means for impinging a beam of radiation upon a portion of said jet stream to thereby scatter said radiation impinging thereon, the improvement comprising: sensing means, coupled to said second means, responsive to radiation scattered by said stream, for providing an output to said second means which is a function of the magnitude of the sensed scattered radiation.
11. The apparatus according to claims 8 or 9, wherein said sensing means is responsive to radiation scattered by said stream.
12. The apparatus according to claims 8 or 10, wherein said output is proportional to the amplitude of undulation.
13. The apparatus according to claims 8 or 9, wherein said beam of radiation impinges said jet stream at said location thereon which is prior to the breakpoint region.
14. The apparatus according to claims 8, 9 or 10, wherein said second means includes said vibrating means.
15. The apparatus according to claims 8, 9 or 10, wherein said vibrating means is a single means.
16. The apparatus according to claims 8, 9 or 10, wherein said vibrating means is positioned above said radiation means.
17. The apparatus according to claims 8, 9 or 10, wherein said beam of radiation impinges said jet stream only upon an uninterrupted portion thereof.
18. The apparatus according to claims 8, 9 or 10, wherein said beam of radiation only impinges said jet stream at said location thereon which is prior to the breakpoint region.
19. The apparatus according to claims 8, 9 or 10, wherein said sending means includes a sense means, responsive to radiation from said radiation means which interrogates said portion of said stream, and breakpoint control means for maintaining the position of the breakpoint constant and having an output and an input, said input coupled to said sense means and said output coupled to said second means, and for providing an output to said second means which is proportional to the amplitude of undulation at said portion of said jet stream.
20. The apparatus according to claims 8, 9 or 10, wherein said sensing means includes a sense means, responsive to radiation from said radiation means which interrogates said portion of said stream>
reference means for providing a reference signal having an amplitude which is proportional to the nominal breakoff point position of said jet stream, and control means having output, input, and control terminals, said output terminal coupled to said second means, said input terminal coupled to said reference means, and said control terminal coupled to said sense means, and said control means providing an output signal which is proportional to the amplitude of undulation at said portion of said jet stream.
reference means for providing a reference signal having an amplitude which is proportional to the nominal breakoff point position of said jet stream, and control means having output, input, and control terminals, said output terminal coupled to said second means, said input terminal coupled to said reference means, and said control terminal coupled to said sense means, and said control means providing an output signal which is proportional to the amplitude of undulation at said portion of said jet stream.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19833307789 DE3307789A1 (en) | 1983-03-04 | 1983-03-04 | METHOD AND DEVICE FOR DISPLAYING A CHANGE IN THE DECOMPOSITION POINT IN A DROP PRODUCTION SYSTEM |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1206559A true CA1206559A (en) | 1986-06-24 |
Family
ID=6192580
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000421053A Expired CA1206559A (en) | 1983-03-04 | 1983-02-07 | Method of and apparatus for detecting change in the breakoff point of a droplet generation system |
Country Status (8)
| Country | Link |
|---|---|
| JP (1) | JPS59159046A (en) |
| AU (1) | AU563027B2 (en) |
| CA (1) | CA1206559A (en) |
| CH (1) | CH654925A5 (en) |
| DE (1) | DE3307789A1 (en) |
| FR (1) | FR2542110B1 (en) |
| GB (1) | GB2136561B (en) |
| NL (1) | NL8300783A (en) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3512090C1 (en) * | 1985-04-03 | 1986-10-02 | Eltro GmbH, Gesellschaft für Strahlungstechnik, 6900 Heidelberg | Circuit arrangement for the preparation of signal amplitudes of very different sizes which are to be processed at the same time |
| EP2264427B1 (en) | 1997-01-31 | 2017-05-03 | Xy, Llc | Optical apparatus with focussing reflector for converging radiation onto a flow of particles, and related method of analysis |
| US6149867A (en) | 1997-12-31 | 2000-11-21 | Xy, Inc. | Sheath fluids and collection systems for sex-specific cytometer sorting of sperm |
| US7208265B1 (en) | 1999-11-24 | 2007-04-24 | Xy, Inc. | Method of cryopreserving selected sperm cells |
| AU760689B2 (en) * | 2000-03-28 | 2003-05-22 | Matsushita Electric Works Ltd. | Particle sensor |
| BRPI0115792B1 (en) | 2000-11-29 | 2020-05-05 | Colorado State Univ | system for separating frozen / thawed sperm in populations with x-chromosome and y-chromosome |
| US7713687B2 (en) | 2000-11-29 | 2010-05-11 | Xy, Inc. | System to separate frozen-thawed spermatozoa into x-chromosome bearing and y-chromosome bearing populations |
| US8486618B2 (en) | 2002-08-01 | 2013-07-16 | Xy, Llc | Heterogeneous inseminate system |
| JP4595067B2 (en) | 2002-08-01 | 2010-12-08 | エックスワイ，エルエルシー | Low-pressure sperm cell separation system |
| CA2534394C (en) | 2002-08-15 | 2013-01-08 | Xy, Inc. | High resolution flow cytometer |
| US7169548B2 (en) | 2002-09-13 | 2007-01-30 | Xy, Inc. | Sperm cell processing and preservation systems |
| ES2918578T3 (en) | 2003-03-28 | 2022-07-19 | Inguran Llc | Apparatus and methods for providing sexed animal sperm |
| NZ544103A (en) | 2003-05-15 | 2010-10-29 | Xy Llc | Efficient haploid cell sorting for flow cytometer systems |
| CA2561519C (en) | 2004-03-29 | 2017-08-22 | Monsanto Technology Llc | Sperm dispersions for use in insemination |
| CA2574499C (en) | 2004-07-22 | 2016-11-29 | Monsanto Technology Llc | Process for enriching a population of sperm cells |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3380584A (en) * | 1965-06-04 | 1968-04-30 | Atomic Energy Commission Usa | Particle separator |
| BE793185A (en) * | 1971-12-23 | 1973-04-16 | Atomic Energy Commission | APPARATUS FOR QUICKLY ANALYZING AND SORTING PARTICLES SUCH AS BIOLOGICAL CELLS |
| US3963606A (en) * | 1974-06-03 | 1976-06-15 | Coulter Electronics, Inc. | Semi-automatic adjusting delay for an electronic particle separator |
| NL180704C (en) * | 1976-06-14 | Coulter Electronics | DEVICE FOR SIMULTANEOUS OPTICAL MEASUREMENT OF CHARACTERISTICS OF PARTICLES CONTAINING IN SUSPENSION. | |
| US4047183A (en) * | 1976-11-04 | 1977-09-06 | International Business Machines Corporation | Method and apparatus for controlling the formation and shape of droplets in an ink jet stream |
| US4318480A (en) * | 1979-08-20 | 1982-03-09 | Ortho Diagnostics, Inc. | Method and apparatus for positioning the point of droplet formation in the jetting fluid of an electrostatic sorting device |
-
1983
- 1983-02-07 CA CA000421053A patent/CA1206559A/en not_active Expired
- 1983-02-08 AU AU11244/83A patent/AU563027B2/en not_active Ceased
- 1983-02-08 GB GB08303440A patent/GB2136561B/en not_active Expired
- 1983-03-02 JP JP58033000A patent/JPS59159046A/en active Granted
- 1983-03-03 CH CH1159/83A patent/CH654925A5/en not_active IP Right Cessation
- 1983-03-03 NL NL8300783A patent/NL8300783A/en not_active Application Discontinuation
- 1983-03-03 FR FR8303501A patent/FR2542110B1/en not_active Expired
- 1983-03-04 DE DE19833307789 patent/DE3307789A1/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS59159046A (en) | 1984-09-08 |
| JPH0324626B2 (en) | 1991-04-03 |
| FR2542110B1 (en) | 1988-11-04 |
| FR2542110A1 (en) | 1984-09-07 |
| CH654925A5 (en) | 1986-03-14 |
| DE3307789A1 (en) | 1984-09-06 |
| GB2136561A (en) | 1984-09-19 |
| NL8300783A (en) | 1984-10-01 |
| AU1124483A (en) | 1984-08-16 |
| GB8303440D0 (en) | 1983-03-16 |
| GB2136561B (en) | 1986-06-25 |
| AU563027B2 (en) | 1987-06-25 |
| DE3307789C2 (en) | 1991-09-19 |
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