CA1200400A - Particle analyzing and sorting apparatus - Google Patents
Particle analyzing and sorting apparatusInfo
- Publication number
- CA1200400A CA1200400A CA000424339A CA424339A CA1200400A CA 1200400 A CA1200400 A CA 1200400A CA 000424339 A CA000424339 A CA 000424339A CA 424339 A CA424339 A CA 424339A CA 1200400 A CA1200400 A CA 1200400A
- Authority
- CA
- Canada
- Prior art keywords
- particles
- particle
- stream
- liquid
- exit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 239000002245 particle Substances 0.000 title claims abstract description 72
- 239000007788 liquid Substances 0.000 claims abstract description 31
- 239000012530 fluid Substances 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 238000005286 illumination Methods 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 230000005855 radiation Effects 0.000 claims 2
- 238000005259 measurement Methods 0.000 abstract description 7
- 230000003287 optical effect Effects 0.000 description 14
- 238000004458 analytical method Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000005684 electric field Effects 0.000 description 2
- 238000004513 sizing Methods 0.000 description 2
- 241000272470 Circus Species 0.000 description 1
- 101100536883 Legionella pneumophila subsp. pneumophila (strain Philadelphia 1 / ATCC 33152 / DSM 7513) thi5 gene Proteins 0.000 description 1
- 101100240664 Schizosaccharomyces pombe (strain 972 / ATCC 24843) nmt1 gene Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000004020 conductor Substances 0.000 description 1
- LOWWYYZBZNSPDT-ZBEGNZNMSA-N delgocitinib Chemical compound C[C@H]1CN(C(=O)CC#N)[C@@]11CN(C=2C=3C=CNC=3N=CN=2)CC1 LOWWYYZBZNSPDT-ZBEGNZNMSA-N 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000009652 hydrodynamic focusing Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 238000012883 sequential measurement Methods 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
Classifications
-
- G01N2015/1028—
Abstract
ABSTRACT
A particle analyzing apparatus, for measurements of particles in a stream, comprising: a flow cell having a pair of channels connected by an interposed particle sensing aperture through which the particles pass, a nozzle mounted proximate to the downstream end of the downstream one of the pair of channels, and means for introducing sheath liquid into the downstream end of the downstream channel to sheath and hydrodynamically focus the particle stream as it proceeds through the downstream channel from the sensing aperture to the nozzle.
A particle analyzing apparatus, for measurements of particles in a stream, comprising: a flow cell having a pair of channels connected by an interposed particle sensing aperture through which the particles pass, a nozzle mounted proximate to the downstream end of the downstream one of the pair of channels, and means for introducing sheath liquid into the downstream end of the downstream channel to sheath and hydrodynamically focus the particle stream as it proceeds through the downstream channel from the sensing aperture to the nozzle.
Description
12~ 0~
The invention relat~s gen~ralLy to particle analyzing and sorting apparatus ancl more partictllarly is concerned with apparatuses in which studie.s may be made of particulate systems using the impedance sensing principle ancl optical measurements.
Since its conc~ption in the early 1950's, the principle of particl2 coun~ing and sizing invented by Wallace ~. Coulter has resulted in numerous methods and flow-through apparatuses for the electronic counting, sizing and analysis of microscop;c particles~
which are scanned in a fluid suspension~ as shown by the pioneer U.5.
Patent Z,656,508 to CouLter. In this prior art arrangement~ a D.C.
electric current flo~ is established between two vessels by suspending electrodes in the res-pective bodies of the suspension f1uid. ~he only fluid connection between the two l>odies~is through an orifice; hence, an electric current flow and field are established in the orifice.
The orifice and the re3ultant electric Eield in and around it constitute a sellsing zone. As each particle passes through the sensing zone, for the duration of t:he passage, the impedance of the contents of the sensing zone will change, thereby modulating the current flow and electric field in the sensing zone, and causing the generation or a signal to be app]ied to a detector suitably arranged to respon~ to such change.
For many applications oE automated, ~1Ow-through particle analyzers, it is not possible to use just a small number of particle descriptors for identification of each type of cell present in a het2rodisperse cell population of a smaple. At present, many f1OW
systeins measure fluorescence, light scattering and electronic cell vol~une (impedance sensillg3. Additiona]ly, there have evolvecl flow~throllgh partic]e analy~ers wherein the particles are posil:ioned inside of l-iquid droplctY and the droplets are sorted u?on the abov2 I ~ZCI)13~
described measurern2nts. Such sorting particle analyzers are sho~n in U.S. Patent 3,710,933 to F~lwyler et al. and in an articl~ entitled "A
Volume-Activated Cell Sorter", The Journal of Histochemistry and . . .
Cytochemistry, by E. ~(enke et al., Vvl. 25, pp. 796-803, 1977.
__ _ _ __ ~ajcr design problems are brought about by the use of both optical measurements and ialpedance measurements in the sorting particle analyzers. T'ne above described sorting particle analyzers of the prior art perEorm electronic cell. volume measurements prior to the optical measurements, making it necessary to correlate the two types of measureMents. This correlation problem is not significant at very lo~ particle flow rates; howe~er, at high particle flow rates, it is possible for the rletected signals to be scrambled by such artifacts a~s aggregates of .ells which pull. apart after they traverse a volume-sensinv orifice, so as to move separately to the optical sensing ~one, the presence of nonfluorescing particl&s; and the possibility of two neighboring cells exchanging position in the Elow stream~ Addi.tionally, thi3 requires the use of special circuitry ~or C~mpeQ~atirlg fur the time delay between the optical and electronic signals for a given particle.
Where so~ting is not used, there has been developed a combined ele.-ero-optical partic].e analyzer in whi.ch all measure~ellts are made si~ultaneo(lsly, thereby eliminating the complexity and uncertaillty of correlating datfl obtained from sequentiaL measurements.
This electro-optical particle analyzer is descri'bed in an article entitled "Combined Optical and Electronic Analysis of Cells with A~C
Transducers", by Thomas et al.~, published in The Journal of Histochemistry and Gytochemistry, Vol. 25, No. 7, (1977), pp- 827--835.
This multiparameter particle analy2er uses a square sensing orifice wherein all parametels are measured simu'Ltatleously. The square ~z~v~
~tl, or;fice is enclo.sed in.side a cube Eormed by adhering Eo-lr pyramids together.
The present invention is directed toward a combined electro-optical partic]e analyzing and sorting apparatus, wherein both optical and elPctrical impedallce (electronic volume) measureillents are simultaneously made on a stream of particles passing through a particle sensing aperture. The flow cell has a pair of channels~ an upstrea~ channel and a downstrealn channel, clefining openings ~t opposed ends thereof, with the partic]e sensing aperture fluidly connecting the two channels. The improvement in the apparatlls comprises mounti~g a no~z'le~ containing an exit orifice, at the end of ~he downstream channel, so as to deEine a liquid~filled f~ow cha~he~.
A sheath liquid is provided at the 'lower end oE the flow chamber to provide a sheath for the particle stream over the entire lencth of the small diameter flow chamber, thereby leaving room in the flow cell, adjacent to the upper end oE the downs~tream channel, for illuminating the particles and collecting~ light therefrom. By virtue o this desigrl, the stream of particles are hydrodynamically focused as they proceed to the exit orifice and thereaEter become part of a liquid jet. '~le liquid jet, in a conventional manner, is broken into a plurality oE droplet3, which are chargecl and 30rted$ based upon the above-described signals.
Heretofore, droplet sorting had never been inclucled in an electro-optica]. apparatus wherein electrical impedance and optical ~5 measurenl2nts are inade simultalleously. Moreover, the applicants found that despite the small volume of the flo~ chamber, hydrodyna~nic focu3ing of the stream of particles by a liquid sheath could be acconiplis'necl by introducing the sheath liqu;d in the bottom of the ~2~
downstream orifice and thereby not interfering with the optical assembly.
By way oE example only illu~trat;ve embodiments oE the invention now will be described with reference to the accompanying drai~ings in ~hich:
FIGURE 1 illustrates a part cross-sectioned v;ew and part block diagram of a particle analyzing and sorting apparatus according to the invention; and FIGURE 2 is an enlarged cross-sectional view of the sensing aperture region of the f]o~ cell of FIGURE 1.
FIGURE 1 illustrates a flow through, particle analyzirlg and sorting apparatus 10 having a sample introduction tube 12, a sheath tube 14 positioned in surro-lnding, coaxial relationship to the tube 12, and an optically transparent Elow cell l6 positioned at the end of , the tube 12. The flo~J cell 16 has formed therein a pair of opposed bores or channels 18 and 20 and a microscopic sensing apertu~e Z~, which forms a fluid passageway between the ends o the channels. The aperture 22 defines a particle sensing ~one to be described hereinafter. A liquid stream of individuslly suspended particles;
originally from a pressurized reservoir 23A, proceeds through the tube 1~. A saline lami~ar liquid sheath, originally from another pressurized reservoir 23B, proceeds through the tube 14 so as to surround tlle stream of particles. ~.s the liquid stream of particles exits from the tube 12, and enters t:he first channel 18, hydrodynamic pressures reduce the diameter of the stream of particles as the stream obtains the velocity of the liquid sheath. ~e liquid sheath also acts to center the stream of particles so tnat particles pass through t'he orifice 22. After leaving the orifice 22, the particles enter the second channel 20, of the flow cell 1~.
Various system coTnponents are supported by a cylindrical frame 25. ~ 1loz21e 24, with an exit orifice 26 Eo~med thereill, i.s mounted to the end of the flo~ cell 16, so that the noz~le 24 and second channel 20 define a liquid--filled flow chamber 28. A tu~e 29 i9 coupled to the Erame 25 by a conduit fitting 30. A second sheat'n l;q~id is fed via the tube 29 to a liquid cavity ~ hich is in fluid comm~n;catic)n wi.th ~hree inlet orifices 32 formed in the wall of the flow cell 16 ~ue to the pressure drop associated with the aperture 22, it is necessary to introduce the second sheath liquid into the flow chamber 28 to create a second shea-h haviQg æufficient hydrodynamic pressure.s to pass the particles through the flow charnber 28 and out ,he exit orifice 26.
Contrary to the prior art de.signs, the second liquid sheath is i.ntroduced at the lower port1on of the flow chamber 28, resultlng in advantageæ ;n opticaI illumination and collection, which w;li be described hereinafter. More specificaliy, the æheath liquid enters the second channel 20 through the plurality o~ inlet orifices 32 ~.
~: poæitioned at locations a considerable distatlce below and downstre&m of the sensing aperture 22. Moreover, the second sheath liquid is introducecl in a non concentric manner relative to the particle stream exitin~, from the sensing aperture 22 and ;s in;ected into a relatively small inter;or vollune of the second channel 20. Deæpite the small volume of the second channel 20 and the non-concentric introductic)n of ,he second liquid sheath at the bottom of the seconcl channel 20, it has been found that a portion of the second æheath liqu;d travels "uphill" to capture the particle stream ex;ting from the sensing ~zc~o~
aperture 22, while a portion of the second sheath liquid goes i~nediately to the exit orifice 26 and all points in-between. In th;s manner, good hydrodynamic focusing of the particle stream throug'n the flow chamber 28 is accomplished, tnereby allo-~ing the stream to e~it from the exit orifice 26. In the pre~erred embodiment, three inlet orifices 32 are shown. However, it should be understood that the number of orifices 32 are a mere matter of design choice, and one wil suffice, although, depending upon their diameter, it is convenient to have nlore ~han olle, so as to allow Eor cleaning and flushing of the flow chamber 280 The system components shown schematically in block form ase those which exist normally in conventiona'l particle analyzer and sorting systems, somet;mes referred to as Elow cyto~etric sorting syste~s. Only those components of the particle analyæer and sort~r 10 have been shown which are necessary to exp'lain the operation of the present invention.
In a conventional manner, vibratory energy is applied to the liquid jet 34, exiting from the exit orifice 26, by vibratory means 36. As one possibility, the vibratory means 36 can comprise a piezo~electric crystal. The 10w cell ]6 is mounted to and supported by a piezo-electric crystal which vibr~tes the flow cell 16 at a high frequency. The e~act frequency at w~ich the cell 16 vibrates is dependent on the selected diameter of the exit oriEice 26. These vibrations impart small disturbances, normally undulations, on the surface of the jet 34 which grow, due to well known surface tension effects, and eventually pinch the jet ofE at a breakofE point 38 into well defined droplets 40. The diameter of the exit orifice 26, the velocity of the liqui& jet 34 and the dilution of the particle ~2~
sus?ension are all predetermined so that normally there is no m~re than one cell in a given droplet 40.
By means of a conventionaJ. sorting arrangement 60, the .selected droplets 40 are charged by, for example, a charging collar having ~ voltage applied thereto. Other droplets are not charged.
The sorting arrar.gel~ent 60 also inclaldes a pair of deflector plates having an electrical potential difference applied therebetween. As thQ droplets pass between the plates, the charged droplets are deflected in the electric field, thereby a].lowing the charged droplets i:o be separated from the uncharged droplets. The decision to charge a given dro~1.et is based upon the previously described optical ar~d inpedance measllrements for the particle contained ~ithin that drcplet.
Ihe above description o~ the droplet fo.ming and droplet sorti.ng is only briefly given, since this portion of the apparatus 10 is well kllO~Jn in the art.
In the flow cell lfi the particle suspension is itlurqinated iTI a conventional manner, while passing through the sensing aperture 22~ by a light beam provided by an illumination source ~2, for example a laG~r. The respQnse of the particle in the sample suspension to the ~0 i.llumir.ation, typically light scatter, fluorescence, or absorbance, is detected by an optical detector system 44. ~s îs-well known in the art, there are numerous illumination and light collection arrangement.s ~hich can be used with the flo~ cell 16. Ho~evers by positioning the inlet orifice 32 substantially downstream of the aperture 22 according "5 to the in~Qntion~ the orifices 32 do not inter~ere w:ith light i3.1umination and collection; hence, greater solid angles of light illl~Qinat;on and collection are possib].e.
The .serlsing aper~ure 22 not cnly serves as an optical sensing æone afi descr;bed above, but also serves as an electronic .~
volume sens;ng zone, according to the principle of l~allace Cou1te., as w;ll be described below. .~n upstream electrode 46 is preferabl~
mounted interior'Ly to the sheath tube 14. A downstream electrode 48 preferably is mounted in a remote chamber 50, which is in fluid cor~unication with the flow chamber 28 ~hro~lgh the tube 29. A lo-~
frequency current, including D.C., or high frequency cur~ent source 52 or both is electricaLly coupled to the electrodes 46 and 48 by way of electrical conductors 54 and 56 respectively. As the particles pass through the aper~ure 22, they modulate the electrical cur.ent so as to produce particle pul~ses detected by conventional ~etector circuitry 58. Illustrative current source 52 and detection circu;try 58 are shown in U.S. Patents 3,710,933; 3,502,974 and 3,502,973.
Preferably, but not necessarily, the channels 18 and 20 have a circular cross section of .05 inches with the overall Length of the flow cell 16 being .25 inches. The flow cell 16 is formed from a monolithic piece of quartz, which allows for the flow cell 16 to be quite small. The srnaller the si~e of the flow cell 16, the better its - optical ch~racteristics, in that the flow cell approaches a point source for the optical signals. The cross section of the partîcle sensing aperture 22 preEerably approximates a square. ~s seen in the further enlargement of ~IGURE 2, the ends of the channels 18 and 20 are formed with spherical surfaces 62 and 64, which are each interrupted by the aperture 22. By ~roviding rounded ends for the bores, the aperture 22 does not have to be precisely located. The outside surfaces are flat and parallel to the walls of the aperture 22. Typically, the aperture 22 has walls with lengths of 50 to 100 ~icro~eters. Preferably, the aperture 22, the exit orifice 26 and the channels 18 and 20 are coaxially aligned. The above-described 1(1 dimensions and configurat;ons described itl this paragraph are merely illustrative and can assume other shapes and sizes, respectivel;~.
Although the apparatus is used primarily for study-ng cells, it is equally applicable to other kinds of particles.
Although particular embodiments of the invention have been sho.~n and described herein, there is no intention thereby to limit the invention to the details of such embodiments. On the contrary, the intention is to cover all modifications, alternatives, embodi~ents, usages and equivalents of the subject invention as fall ~ithin the spirit and scope of the invention, specification and the appended clai~s.
The invention relat~s gen~ralLy to particle analyzing and sorting apparatus ancl more partictllarly is concerned with apparatuses in which studie.s may be made of particulate systems using the impedance sensing principle ancl optical measurements.
Since its conc~ption in the early 1950's, the principle of particl2 coun~ing and sizing invented by Wallace ~. Coulter has resulted in numerous methods and flow-through apparatuses for the electronic counting, sizing and analysis of microscop;c particles~
which are scanned in a fluid suspension~ as shown by the pioneer U.5.
Patent Z,656,508 to CouLter. In this prior art arrangement~ a D.C.
electric current flo~ is established between two vessels by suspending electrodes in the res-pective bodies of the suspension f1uid. ~he only fluid connection between the two l>odies~is through an orifice; hence, an electric current flow and field are established in the orifice.
The orifice and the re3ultant electric Eield in and around it constitute a sellsing zone. As each particle passes through the sensing zone, for the duration of t:he passage, the impedance of the contents of the sensing zone will change, thereby modulating the current flow and electric field in the sensing zone, and causing the generation or a signal to be app]ied to a detector suitably arranged to respon~ to such change.
For many applications oE automated, ~1Ow-through particle analyzers, it is not possible to use just a small number of particle descriptors for identification of each type of cell present in a het2rodisperse cell population of a smaple. At present, many f1OW
systeins measure fluorescence, light scattering and electronic cell vol~une (impedance sensillg3. Additiona]ly, there have evolvecl flow~throllgh partic]e analy~ers wherein the particles are posil:ioned inside of l-iquid droplctY and the droplets are sorted u?on the abov2 I ~ZCI)13~
described measurern2nts. Such sorting particle analyzers are sho~n in U.S. Patent 3,710,933 to F~lwyler et al. and in an articl~ entitled "A
Volume-Activated Cell Sorter", The Journal of Histochemistry and . . .
Cytochemistry, by E. ~(enke et al., Vvl. 25, pp. 796-803, 1977.
__ _ _ __ ~ajcr design problems are brought about by the use of both optical measurements and ialpedance measurements in the sorting particle analyzers. T'ne above described sorting particle analyzers of the prior art perEorm electronic cell. volume measurements prior to the optical measurements, making it necessary to correlate the two types of measureMents. This correlation problem is not significant at very lo~ particle flow rates; howe~er, at high particle flow rates, it is possible for the rletected signals to be scrambled by such artifacts a~s aggregates of .ells which pull. apart after they traverse a volume-sensinv orifice, so as to move separately to the optical sensing ~one, the presence of nonfluorescing particl&s; and the possibility of two neighboring cells exchanging position in the Elow stream~ Addi.tionally, thi3 requires the use of special circuitry ~or C~mpeQ~atirlg fur the time delay between the optical and electronic signals for a given particle.
Where so~ting is not used, there has been developed a combined ele.-ero-optical partic].e analyzer in whi.ch all measure~ellts are made si~ultaneo(lsly, thereby eliminating the complexity and uncertaillty of correlating datfl obtained from sequentiaL measurements.
This electro-optical particle analyzer is descri'bed in an article entitled "Combined Optical and Electronic Analysis of Cells with A~C
Transducers", by Thomas et al.~, published in The Journal of Histochemistry and Gytochemistry, Vol. 25, No. 7, (1977), pp- 827--835.
This multiparameter particle analy2er uses a square sensing orifice wherein all parametels are measured simu'Ltatleously. The square ~z~v~
~tl, or;fice is enclo.sed in.side a cube Eormed by adhering Eo-lr pyramids together.
The present invention is directed toward a combined electro-optical partic]e analyzing and sorting apparatus, wherein both optical and elPctrical impedallce (electronic volume) measureillents are simultaneously made on a stream of particles passing through a particle sensing aperture. The flow cell has a pair of channels~ an upstrea~ channel and a downstrealn channel, clefining openings ~t opposed ends thereof, with the partic]e sensing aperture fluidly connecting the two channels. The improvement in the apparatlls comprises mounti~g a no~z'le~ containing an exit orifice, at the end of ~he downstream channel, so as to deEine a liquid~filled f~ow cha~he~.
A sheath liquid is provided at the 'lower end oE the flow chamber to provide a sheath for the particle stream over the entire lencth of the small diameter flow chamber, thereby leaving room in the flow cell, adjacent to the upper end oE the downs~tream channel, for illuminating the particles and collecting~ light therefrom. By virtue o this desigrl, the stream of particles are hydrodynamically focused as they proceed to the exit orifice and thereaEter become part of a liquid jet. '~le liquid jet, in a conventional manner, is broken into a plurality oE droplet3, which are chargecl and 30rted$ based upon the above-described signals.
Heretofore, droplet sorting had never been inclucled in an electro-optica]. apparatus wherein electrical impedance and optical ~5 measurenl2nts are inade simultalleously. Moreover, the applicants found that despite the small volume of the flo~ chamber, hydrodyna~nic focu3ing of the stream of particles by a liquid sheath could be acconiplis'necl by introducing the sheath liqu;d in the bottom of the ~2~
downstream orifice and thereby not interfering with the optical assembly.
By way oE example only illu~trat;ve embodiments oE the invention now will be described with reference to the accompanying drai~ings in ~hich:
FIGURE 1 illustrates a part cross-sectioned v;ew and part block diagram of a particle analyzing and sorting apparatus according to the invention; and FIGURE 2 is an enlarged cross-sectional view of the sensing aperture region of the f]o~ cell of FIGURE 1.
FIGURE 1 illustrates a flow through, particle analyzirlg and sorting apparatus 10 having a sample introduction tube 12, a sheath tube 14 positioned in surro-lnding, coaxial relationship to the tube 12, and an optically transparent Elow cell l6 positioned at the end of , the tube 12. The flo~J cell 16 has formed therein a pair of opposed bores or channels 18 and 20 and a microscopic sensing apertu~e Z~, which forms a fluid passageway between the ends o the channels. The aperture 22 defines a particle sensing ~one to be described hereinafter. A liquid stream of individuslly suspended particles;
originally from a pressurized reservoir 23A, proceeds through the tube 1~. A saline lami~ar liquid sheath, originally from another pressurized reservoir 23B, proceeds through the tube 14 so as to surround tlle stream of particles. ~.s the liquid stream of particles exits from the tube 12, and enters t:he first channel 18, hydrodynamic pressures reduce the diameter of the stream of particles as the stream obtains the velocity of the liquid sheath. ~e liquid sheath also acts to center the stream of particles so tnat particles pass through t'he orifice 22. After leaving the orifice 22, the particles enter the second channel 20, of the flow cell 1~.
Various system coTnponents are supported by a cylindrical frame 25. ~ 1loz21e 24, with an exit orifice 26 Eo~med thereill, i.s mounted to the end of the flo~ cell 16, so that the noz~le 24 and second channel 20 define a liquid--filled flow chamber 28. A tu~e 29 i9 coupled to the Erame 25 by a conduit fitting 30. A second sheat'n l;q~id is fed via the tube 29 to a liquid cavity ~ hich is in fluid comm~n;catic)n wi.th ~hree inlet orifices 32 formed in the wall of the flow cell 16 ~ue to the pressure drop associated with the aperture 22, it is necessary to introduce the second sheath liquid into the flow chamber 28 to create a second shea-h haviQg æufficient hydrodynamic pressure.s to pass the particles through the flow charnber 28 and out ,he exit orifice 26.
Contrary to the prior art de.signs, the second liquid sheath is i.ntroduced at the lower port1on of the flow chamber 28, resultlng in advantageæ ;n opticaI illumination and collection, which w;li be described hereinafter. More specificaliy, the æheath liquid enters the second channel 20 through the plurality o~ inlet orifices 32 ~.
~: poæitioned at locations a considerable distatlce below and downstre&m of the sensing aperture 22. Moreover, the second sheath liquid is introducecl in a non concentric manner relative to the particle stream exitin~, from the sensing aperture 22 and ;s in;ected into a relatively small inter;or vollune of the second channel 20. Deæpite the small volume of the second channel 20 and the non-concentric introductic)n of ,he second liquid sheath at the bottom of the seconcl channel 20, it has been found that a portion of the second æheath liqu;d travels "uphill" to capture the particle stream ex;ting from the sensing ~zc~o~
aperture 22, while a portion of the second sheath liquid goes i~nediately to the exit orifice 26 and all points in-between. In th;s manner, good hydrodynamic focusing of the particle stream throug'n the flow chamber 28 is accomplished, tnereby allo-~ing the stream to e~it from the exit orifice 26. In the pre~erred embodiment, three inlet orifices 32 are shown. However, it should be understood that the number of orifices 32 are a mere matter of design choice, and one wil suffice, although, depending upon their diameter, it is convenient to have nlore ~han olle, so as to allow Eor cleaning and flushing of the flow chamber 280 The system components shown schematically in block form ase those which exist normally in conventiona'l particle analyzer and sorting systems, somet;mes referred to as Elow cyto~etric sorting syste~s. Only those components of the particle analyæer and sort~r 10 have been shown which are necessary to exp'lain the operation of the present invention.
In a conventional manner, vibratory energy is applied to the liquid jet 34, exiting from the exit orifice 26, by vibratory means 36. As one possibility, the vibratory means 36 can comprise a piezo~electric crystal. The 10w cell ]6 is mounted to and supported by a piezo-electric crystal which vibr~tes the flow cell 16 at a high frequency. The e~act frequency at w~ich the cell 16 vibrates is dependent on the selected diameter of the exit oriEice 26. These vibrations impart small disturbances, normally undulations, on the surface of the jet 34 which grow, due to well known surface tension effects, and eventually pinch the jet ofE at a breakofE point 38 into well defined droplets 40. The diameter of the exit orifice 26, the velocity of the liqui& jet 34 and the dilution of the particle ~2~
sus?ension are all predetermined so that normally there is no m~re than one cell in a given droplet 40.
By means of a conventionaJ. sorting arrangement 60, the .selected droplets 40 are charged by, for example, a charging collar having ~ voltage applied thereto. Other droplets are not charged.
The sorting arrar.gel~ent 60 also inclaldes a pair of deflector plates having an electrical potential difference applied therebetween. As thQ droplets pass between the plates, the charged droplets are deflected in the electric field, thereby a].lowing the charged droplets i:o be separated from the uncharged droplets. The decision to charge a given dro~1.et is based upon the previously described optical ar~d inpedance measllrements for the particle contained ~ithin that drcplet.
Ihe above description o~ the droplet fo.ming and droplet sorti.ng is only briefly given, since this portion of the apparatus 10 is well kllO~Jn in the art.
In the flow cell lfi the particle suspension is itlurqinated iTI a conventional manner, while passing through the sensing aperture 22~ by a light beam provided by an illumination source ~2, for example a laG~r. The respQnse of the particle in the sample suspension to the ~0 i.llumir.ation, typically light scatter, fluorescence, or absorbance, is detected by an optical detector system 44. ~s îs-well known in the art, there are numerous illumination and light collection arrangement.s ~hich can be used with the flo~ cell 16. Ho~evers by positioning the inlet orifice 32 substantially downstream of the aperture 22 according "5 to the in~Qntion~ the orifices 32 do not inter~ere w:ith light i3.1umination and collection; hence, greater solid angles of light illl~Qinat;on and collection are possib].e.
The .serlsing aper~ure 22 not cnly serves as an optical sensing æone afi descr;bed above, but also serves as an electronic .~
volume sens;ng zone, according to the principle of l~allace Cou1te., as w;ll be described below. .~n upstream electrode 46 is preferabl~
mounted interior'Ly to the sheath tube 14. A downstream electrode 48 preferably is mounted in a remote chamber 50, which is in fluid cor~unication with the flow chamber 28 ~hro~lgh the tube 29. A lo-~
frequency current, including D.C., or high frequency cur~ent source 52 or both is electricaLly coupled to the electrodes 46 and 48 by way of electrical conductors 54 and 56 respectively. As the particles pass through the aper~ure 22, they modulate the electrical cur.ent so as to produce particle pul~ses detected by conventional ~etector circuitry 58. Illustrative current source 52 and detection circu;try 58 are shown in U.S. Patents 3,710,933; 3,502,974 and 3,502,973.
Preferably, but not necessarily, the channels 18 and 20 have a circular cross section of .05 inches with the overall Length of the flow cell 16 being .25 inches. The flow cell 16 is formed from a monolithic piece of quartz, which allows for the flow cell 16 to be quite small. The srnaller the si~e of the flow cell 16, the better its - optical ch~racteristics, in that the flow cell approaches a point source for the optical signals. The cross section of the partîcle sensing aperture 22 preEerably approximates a square. ~s seen in the further enlargement of ~IGURE 2, the ends of the channels 18 and 20 are formed with spherical surfaces 62 and 64, which are each interrupted by the aperture 22. By ~roviding rounded ends for the bores, the aperture 22 does not have to be precisely located. The outside surfaces are flat and parallel to the walls of the aperture 22. Typically, the aperture 22 has walls with lengths of 50 to 100 ~icro~eters. Preferably, the aperture 22, the exit orifice 26 and the channels 18 and 20 are coaxially aligned. The above-described 1(1 dimensions and configurat;ons described itl this paragraph are merely illustrative and can assume other shapes and sizes, respectivel;~.
Although the apparatus is used primarily for study-ng cells, it is equally applicable to other kinds of particles.
Although particular embodiments of the invention have been sho.~n and described herein, there is no intention thereby to limit the invention to the details of such embodiments. On the contrary, the intention is to cover all modifications, alternatives, embodi~ents, usages and equivalents of the subject invention as fall ~ithin the spirit and scope of the invention, specification and the appended clai~s.
Claims (6)
1. A particle analyzing apparatus for studying particles in suspension, said apparatus including a flow cell having a particle sensing aperture through which a stream of particles in suspension is passed, said flow cell having an upstream channel and a downstream channel, with said particle sensing aperture being positioned therebetween and being the only fluid connection between said channels, exit means positioned proximate the downstream end of said downstream channel, and liquid introducing means constructed and positioned downstream of the downstream end of said downstream channel and operating for introducing liquid which flows both into said exit means as well as toward the upstream positioned particle sensing aperture for hydrodynamically focusing the stream of particles as it flows downstream from said particle sensing aperture into said exit means.
2. A particle analyzing apparatus according to claim 1 in which said liquid introducing means is constructed and arranged with respect to the stream of particles so as to introduce the liquid in a non-concentric manner.
3. A particle analyzing apparatus according to claim 1 in which said liquid introducing means is constructed and arranged with respect to the stream of particles such that the liquid enters said flow cell at approximately right angles to the stream of particles.
4. A particle analyzing apparatus according to any one of claims 1, 2 or 3 and further including: illumination means for providing radiation to illuminate particles in said particle sensing aperture, said illumination means and its radiation being oriented and positioned substantially remote from said liquid introducing means.
5. A particle analyzing apparatus according to any one of claims 1, 2 or 3 in which said liquid introducing means is positioned proximate to said exit means and said exit means includes a nozzle having an exit orifice for jetting the stream of particles as a liquid jet from said nozzle.
6. A particle analyzing apparatus according to any one of claims 1, 2 or 3 in which said liquid introducing means is positioned proximate to said exit means, said exit means includes a nozzle having an exit orifice for jetting the stream of particles as a liquid jet from said nozzle, and further including disturbing means for periodically disturbing the liquid jet to produce droplets containing particles and sorting means for sorting said droplets, said sorting means being controlled by signals generated when the particles pass through said particle sensing aperture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000424339A CA1200400A (en) | 1983-03-24 | 1983-03-24 | Particle analyzing and sorting apparatus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000424339A CA1200400A (en) | 1983-03-24 | 1983-03-24 | Particle analyzing and sorting apparatus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1200400A true CA1200400A (en) | 1986-02-11 |
Family
ID=4124857
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000424339A Expired CA1200400A (en) | 1983-03-24 | 1983-03-24 | Particle analyzing and sorting apparatus |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1200400A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108024669A (en) * | 2015-09-30 | 2018-05-11 | Toto株式会社 | water discharge device |
-
1983
- 1983-03-24 CA CA000424339A patent/CA1200400A/en not_active Expired
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108024669A (en) * | 2015-09-30 | 2018-05-11 | Toto株式会社 | water discharge device |
| CN108024669B (en) * | 2015-09-30 | 2020-10-13 | Toto株式会社 | Water discharge device |
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