AU2015224429B2 - Apparatuses, systems, methods, and computer readable media for acoustic flow cytometry - Google Patents

Abstract

A flow cytometer includes a capillary having a sample channel; at least one vibration producing transducer coupled to the capillary, the at least one vibration producing transducer being configured to produce an acoustic signal inducing acoustic radiation pressure within the sample channel to acoustically concentrate particles flowing within a fluid sample stream in the sample channel; and an interrogation source having a violet laser and a blue laser, the violet and blue lasers being configured to interact with at least some of the acoustically concentrated particles to produce an output signal.

Description

APPARATUSES, SYSTEMS, METHODS, AND COMPUTER READABLE MEDIA 2015224429 09 Sep 2015

FOR ACOUSTIC FLOW CYTOMETRY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a divisional application from Australian Patent Application No. 2010326180, the entire disclosure of which is incorporated herein by reference.

BACKGROUND

[0002] Field: [0003] This application generally relates to flow cytometry and, more specifically, to apparatuses, systems, methods, and computer readable media for detecting rare events using acoustic flow cytometry.

[0004] Background: [0005] In traditional flow cytometry, a sample fluid is focused to a small core diameter of around 10-50 μηι by flowing a sheath fluid around the sample fluid at a very high volumetric rate (about 100-1000 times the volumetric rate of the sample fluid). The particles in the sample fluid flow at very fast linear velocities (on the order of meters per second) and as a result spend only a very short time passing through an interrogation point (often only 1-10 μβ). This has significant disadvantages. First, the particles cannot be redirected to the interrogation point because flow cannot be reversed. Second, the particles cannot be held at the interrogation point because focusing is lost without the sheath fluid. Third, the short transit time limits sensitivity and resolution, which renders rare event detection difficult and time-consuming.

[0006] Previous attempts at addressing these disadvantages have been unsatisfactory. The concentration of the particles in the sample fluid may be increased to compensate for some of these disadvantages, but this may not always be possible and may be costly. Also, the photon flux at the interrogation point may be increased to extract more signal, but this may often photobleach (i.e., excite to non-radiative states) the fluorophores used to generate the signal and may increase background Rayleigh scatter, Raman scatter, and fluorescence. Thus, there is a need for new apparatuses, systems, methods, and computer readable media for flow cytometry that allow high-throughput analysis of particles and fast and efficient rare event detection while avoiding or minimizing one or more of these disadvantages.

[0006a] A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that the document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any the claims. 1

SUMMARY 2015224429 11 May 2017 [0007] In accordance with the principles embodied in this application, new apparatuses, systems, methods, and computer readable media for flow cytometry that allow high-throughput analysis of particles and fast and efficient rare event detection while avoiding or minimizing one or more of the above disadvantages are provided.

[0008] According to an aspect of the present invention, there is provided a flow cytometer, including: a capillary including a sample channel; at least one vibration producing transducer coupled to the capillary, the at least one vibration producing transducer being configured to produce an acoustic signal inducing acoustic radiation pressure within the sample channel to acoustically concentrate particles flowing within a fluid sample stream in the sample channel; an interrogation source configured to interact with at least some of the acoustically concentrated particles to produce an output signal; a first pump configured to flow a fluid sample comprising particles in the sample channel; and a second pump configured to flow a sheath fluid in the sheath flow channel; wherein the first and second pumps are configured to maintain a total input rate of sample fluid and sheath fluid flowing in the capillary constant and flow the sample fluid into the capillary at a sample flow rate of at least 25 microliters per minute to 1000 microliters per minute.

[0009] According to another aspect of the present invention, there is provided a flow cytometer, including: a capillary configured to allow a sample fluid including particles to flow therein; a first focusing mechanism configured to acoustically focus at least some of the particles in the sample fluid in a first region within the capillary; a second focusing mechanism configured to hydrodynamically focus the sample fluid including the at least some acoustically focused particles in a second region within the capillary downstream of the first region; an interrogation zone in or downstream of the capillary through which at least some of the acoustically and hydrodynamically focused particles can flow; at least one detector configured to detect at least one signal obtained at the interrogation zone regarding at least some of the acoustically and hydrodynamically focused particles; and a sample fluid pump configured to flow a sample fluid into the capillary; and a sheath fluid pump configured to flow a sheath fluid into the capillary, wherein the first and second pumps are configured to maintain a total input rate of sample fluid and sheath fluid flowing in the capillary constant and flow the sample fluid into the capillary at a sample flow rate of at least 25 microliters per minute to 1000 microliters per minute.

[0010] According to yet another aspect of the present invention, there is provided a method for detecting a rare event using a flow cytometer, including: flowing a sample fluid including particles into a channel; acoustically focusing at least some of the particles in the 2 sample fluid in a first region contained within the channel by applying acoustic radiation pressure to the first region; hydrodynamically focusing the sample fluid including the at least some acoustically focused particles by flowing a sheath fluid around the sample fluid in a second region downstream of the first region; adjusting a volumetric ratio of the sheath fluid to the sample fluid to maintain a substantially constant overall particle velocity in an interrogation zone in or downstream of the second region; analyzing at least some of the acoustically and hydrodynamically focused particles in the interrogation zone; and detecting one or more rare events based on at least one signal detected at the interrogation zone, the one or more rare events being selected from the group consisting of one or more rare fluorescence events, one or more rare cell types, and one or more dead cells, wherein the system is configured to flow the sample fluid at a flow rate of 25 microliters per minute to 1000 microliters per minute. 2015224429 11 May 2017 [0011] Additional details of these and other embodiments of the invention are set forth in the accompanying drawings and the following description, which are exemplary and explanatory only and are not in any way limiting of the present invention. Other embodiments, features, objects, and advantages of the present invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate one or more embodiments of the present invention and, together with the description, serve to explain the principles of various embodiments of the present invention. The drawings are exemplary and explanatory only and are not to be construed as limiting or restrictive of the present invention in any way.

[0013] FIG. 1 illustrates a comparison of planar and line-driven capillary focusing.

[0014] FIGS. 2A and 2B illustrate a line-driven acoustic focusing apparatus.

[0015] FIG. 3 illustrates acoustically focused particles flowing across laminar flow lines in a line-driven acoustic focusing apparatus.

[0016] FIGS. 4A and 4B illustrate acoustically reoriented laminar flow streams in an acoustic focusing apparatus.

[0017] FIGS. 5A-5C illustrate the separation of micron-sized polystyrene fluorescent orange/red particles from a background of nanometer-sized green particles in an acoustic focusing apparatus. FIG. 5D illustrates acoustically focused particles flowing across laminar flow lines in an acoustic focusing apparatus.

[0018] FIGS. 6A-6C illustrate acoustic separation of particles across laminar flow boundaries.

[0019] FIGS. 7A-7C illustrate several acoustic focusing apparatuses. 3 [0020] FIG. 8 illustrates a schematic of an acoustical focusing flow cell in combination with an acoustic flow cytometer. 2015224429 11 May 2017 [0021] FIG. 9 illustrates a flow diagram of an acoustic focusing system.

[0022] FIG. 10 illustrates the diagram in FIG. 9 modified to include in-line laminar washing.

[0023] FIG. 11 illustrates acoustic focusing of a laminar wash fluid.

[0024] FIG. 12 illustrates a schematic of a parallel fluid acoustic switching apparatus. 3a 2015224429 09 Sep 2015 [0025] FIGS. 13A and 13B illustrate schematics ofsw.itchingof unlyscd whole blood.

[0026] FIG. 14 illustrates a schematic of an acoustic stream switching particle counting device.

[0027] .......... FIG. 15 illustrates the separation of negative con trast carrier particles front a blood samplllcore, [0028] FIG. 16 illfotfofpfoultiplexed immunoassaying in an acousti<s:wa^i:^|fjiiih.

[0029] FIG. 17 ilihsflitei: a flow chart for high-throughput screening :d|ih||dcoustic focusing, |0030J FIG. 18 illustrates a......two-chamber eulturing/harvesting vessel using acoustic washing. |0031| FIGS. I9A-19C illustrate aptamcr.selection from a library. |0032| FIG. 20 illustrates a dual-stage acoustic valve sorter, 10033] FIGS. 21Λ and 21B illustrate the optical analysis of acoustically repositioned particles and a medium. |0034] PIG, 22 i llustrates a diagram of particle groupings with different parameters.

[0035] FIG. 23 illustrates acoustically repositioned particles imaged by an imager.

[0036] FIG. 24 illustrates acoustic fusion of particles.

[0037] FIG. 25 illustrates acoustic focusing and separation of particles. |0038] FIGS. 26A-26F illustrate comparative output ploK for fluorescent microspheres run on a non-acoustic flow cytometer and on an acoustic focusing cytometer using various lasers I003||llli||s. 2711:¾¾ 27B are histogram plots illustrating thd|effo|t hi ceil cycle atiii|sis§ oflfoillfoidiifoeteaseliiilffositJime associated with acoustic cytdfoelfjfl: [0040] IFIG. 28A|i:S:ffo hotp§raph of blood having cells acous!fofl|:|ohcSnh^ a rope-like structure flowing in an acoustic cytometer. FIG. 28B is a photograph of more diluted blood with cells acoustically concentrated in a single file line in an acoustic c|fome|:®7ll [0041] FIG. 29 is a spectral graph showing the excitation and emission spectra of the v|pe|plifod fluor||h||e||Sdild [0042] FIG. 30 illustrates the detection of a rare event population of 0.07% CD34 positive cells as a subpopulation of the live CD45 positive cells. |0043| FIGS. 31A and 31B respectively show plots of FSCvs. SSC for lysed whole blood in an acoust ic focusing system and in a solely hydrodynamic focusing system. 4 2015224429 09 Sep 2015 |ci Jurkat cdlf ^ilinldihllhg die [0047] FIG. ill

10044] FIGS. 32A and 32B show plots οΓ I SC vs. SSC lame paFIGS. 31A and 31B. i|0045]!!!!FlG| 33 iiluStflfoliilhlematic diagram ofan acopSlc fli^eytbhfbtfy sysiedilllllls [0046] FIG. 34 illustrates a schematic diagram of an acoustic focusing capillary in an :acoustiCif]ow;:cytometS|||||||||||| d||fit||iii||ortion of an optical collection block in an acoustic flow

Cvtometiri i|0048] 111FIG. 36 i 11atic diagram of an optical data collection block in an acoustic :flow cylomctcrlllllllllll [0049] IIFIG. 37 illdSiliidiiliSChematic diagram of a ftuidibSlsystem in aiilgoustfc flow Cytometer.

[0050| FIG. 38 illustrates a schematic diagram of a single transducer acoustic focusing capillary' with downstream: hydrodynamic focusing, ]0051 j FIG. 39 illustrates a schematic d iagram of a blocker bar apparatus that may adj ust a forward scatter apedia|ihihhiS|ih|hc flow cytometer7|||||||||||| (0052 j FIGS. 4UA-40F illustrate the detection of rare event populations of 0.050% and 0.045% CD34 positive cells as a subpopulation of five CD45 positive cells, [0053] FIGS. 41A-4 ID illustrate comparative output plots for cell detection run on a non-acoustic flow cytometer anti oti ait acoustic focusing cytometer.

[0054] FIG. 42 ill ustrates a schematic of components of an acoustic focusing cytometer. |0055I Like symbols in the drawings indicate like elements.

EXEMPLARY EMBODIMENTS

[0056] As used herein, "acoustic contrast" means the relative .difference in material properties of two objects with regard to the ability’ to manipulate their positions with acoustic radiation pressure, and may include, for example, differences in density and compressibility; "assaying" means a method for interrogating one or more particles or one or more fluids;

Incidding, for exhPIfeliifihssay kit.rJa|§ii^ "flow cell" means a channel, chamber, or capillary having an interior shape selected from rectangular, circular, round, s and trigonal; and "channel" means a course, pathway, or conduit with at least an inlet and cahldbntain an fluid ih||j|||:an in.terio|i|||]j|:::sel:eeted' from rectangular, square, elliptical, oblate circular, round, octagonal, heptagonal. hexagonal, pentagonal, and trigonal. 5 2015224429 09 Sep 2015 ί|)57] "acoustically focused", "acoustically

IfoCyses", ” mean|)h|:|ct of positioning particles witliin a flow cell by jhleans of.exampllbf acoustic focusing of particles is the alignment of particles ald^^liSiifiiiiSiiili iS . The spatial extent of the focal region where particles are :|be|]:ized the flqyypelfpeomelry, acoustic field, and acoustic contrast. ,plarisiiipit^ili^ilpw cell, the shape of an : observed focal region sh|||:)(ej|,, point,.....line, arc, e|ip|i)|pte:,) or it may be arbitrary. The primary force used to position the objects is acoustic radiation pressure. |!Ι$Ι$|ίίίίίίίΙΙίΙ$Ι$Ι$ίΙ$ί$?Ι$13 I he act of repositioning the location of miscible, partially miscible, or immiscible laminar flow streams of fluid or medium within a device with acoustic radiation pressure. This teehnicfue utilizes differences in the mechanical properties (acoustic contrast) of separate laminar streams in a flow1 channel. When two fluids are brought into contact, a large concentration gradient can exist due to differences in their molecular make-ups, resulting in an interfacial density and/or compressibility gradient (acoustic contrast between streams). Under Ihe action of an acoustic field, the streams may be reoriented within a flow ceil based upon their [¢1059] As used herein, "particle” means a small unit of matter, including, for example, biologica l cells, such as. euloiryotic and prokaryotic cells, archaea, bacteria, mold, plant cells, yeast, protozoa, ameba, profists, animal cells; cell organelles; organic/inorganic elements or molecu les; rnicrospheres; anil droplets of immiscible fluid such as oil in water.

[0U60] As used herein, "analyte” means a substance or material to be analyzed; "probe" means a substance that is labeled or otherwise marked and used to delect or identify another substance in a fluid or sample; "target" means a binding portion of a probe; and "reagent" 'means a substance known to react in a specific way. |00611 As used herein, "nucrosphere" or "bead" means a particle having acoustic contrast that can be symmetric as in a sphere, asymmetric as in liiuifibbell shape or a;thaeramol^iule having no symmetry. Examples of microspheres or beads include, for example, silica, glass and hollow glass, latex, silicone, rubbers, ||plymers such as ll|blpt|||ji|| polymethylmethacrylate, polymetbylenemelamine, polyacrylonitrile, polymethyfsicildpfo polyfvinilidene chloride-eo-aerylonifrile), and polylactid|i| [0062] As used .herein, "label" means an. identifiable substaiGPj such as ai dye or a radioactive isotope that is introduced in a system, such as a systefo/liiiii^feiihli^i ifbllowed: through the course of a flow cell or chaimelypf6vidipg|ih|pi|ihtio!i on thipartfcleS: 6 2015224429 09 Sep 2015 :or targets' in thefifiblliicell or an identifiable· substance, such as a dye or a radioactive isotope that is introduced in a system, such as a hiolpgical.system||h|i|u be us§iil|i;|ip|ffi lllllllllllllllllllll.

[0063] FIG. I illustrates a comparison of planar and line-driven capillary focusing According to. exemjjjii^ii&amp;mbodi^ inyeniiiiiiifti the particles 102, which may include one or more rare event particles, may be focused as a ilwo-dimensional {see i|i|||folF:::arrows}ih direction. in line-ii:^ifthriihSf f{39, the pafhd:i|Sli02 may be focused to the center axis of the e4pf||f$iiid^^ afob§;tle flow. The capillary may have a round, oi>l i ρχβίΤφ.^ί:ί:ί;ΙΐίίΡ tern at i vely, the particles 102 may also be focused to another axis within the capillary, or along the walls of the capillary, [0064| FIGS. 2Λ and 2B illustrate side and axial views of a line-driven acoustic focusing apparatus according to an exemplary embodiment of the present invention. The particles 203, which may include one or more rare event particles, may be acoustically focused using a transducer 205 to a pressure minimum in the center 209 of a tube 201, which may be cylindrical, for example. The particles 203 may form a single line trajectory 21 i, which may allow uniform residence time for particles with similar size and acoustic contrast, which may in turn allow high-throughput serial analysis of particles' without compromising sensitivity and resolution.

[0.100] FIG. 3 illustrates acoustically focused panicles flowing across laminar flow lines in a line-driven acoustic focusing apparatus according to an exemplary embodiment of the present invention. The particles 305, which may include one or more rare event particles, may be acousricalij^iiiiiFocxii^i^iiiiiiiijii^ 303 from sari)|j|§:rieam 3|||to thh^ephjililO'? of a flowing fluid/'xiv'aSli.:iiritheririhi{3'2jir a line-driven capillif||||l. Th||particles 305 may move across laminar flow lines and may then move as a single file line and be analyzed at analysis points 11,1|||||||||||||||||||| [0101] FIGS, 4A and 4B illustrate acoustically reoriented laminar flow streams in an acoustic focusing a]||8ijifi^ an exemplary emh||il|ient of the p||8|h|::i||||tion.

In FIG. 4A, there tFtc laminar flow streams 4Qli::i:|iind. to one another in capillary 401. In FlftfilB, an:||Ous||i:|btd is applied, by llie··transducer· the streams 403 an||||)7 ardiitouslfoally reorientel; based upon their acdhi?{i|ldhh|fhStillifhe stream with greate:r::acousti|;ieon||ist 403b may be reoriented' to the cerileJirfiCVifiiitlrri{ri401, while the strehth withribwcbacoustie contrast: 407b may be reoriehfoidlridar tlhlyiSlsiOf the capillary 401 |||f the ah|ustj|i field is acj;il|je|l 7 2015224429 09 Sep 2015 in a dipole the stream.. 403b: moves cJWti;iii: IiiiilJtiisl;i;0-f the eapil|i|T:4Ql, ';pli;ihl||||splacing the stream||07|| as tmmjscihiilpaiihliy-mjscjble, or miscible,.......A large eohe|h|rati|i::''gradiC«|ma|;b^!:St:' between the stfoifo|i:fiU|/to their different mbteetiihr make-ups. For purposes of/ifipstic. pressure, tire concentration gradient may besiyieweef as a density and/or compressibility gradient, and ll6/sli;PriSl may be viewed asliSola|ed||nt|ties with different densities and Idhtpressibi li li elpClisfibcontrasi) that can .be;f Ctedf 10102| FIGS, 5A-5C illustrate the separation of micron-sized polystyrene fluorescent orange,'red particles from a background of nanometer-sized green particles in art acoustic focusing apparatus according to an exemplary embodiment of Ihe present invention. FTG. 5D illustrates acoustically focused particles flowing across laminar flow lines in tin acoustic focusing apparatus according to an exemplary embodiment of the present invention. Separation may be based bollt on size and acoustic contrast because the time-averaged acoustic force scales with the volume of a particle. If a center wash stream has higher specific gravity and/or lower compressibility than an outer sample stream, the particles initially in the outer sample stream with greater acoustic contrast than the central wash stream will continue to focus to the capillary axis while the particles of lesser contrast will be excluded. FTG. 5A shows red 5.7 pin particles mixed with green 200 nm particles flowing through a capillary under epi-fluorescent illumination when the acoustic field is off. FIG. 511 shows that ihe 5.7 urn particles (which fluoresce yellow under blue illumination) are acoustically focused io a central line when the acoustic field is on, while the 200 nm particles remain in their original stream. FIG, 5C shows that the 5.7 pm particles fluoresce red under green illumination, with a red band-pass filter, while the 200 urn panicles are not excited. FIG, 5D illustrates a clean core stream 507 introduced alongside a coaxial stream 505 containing a fluorescent background fluid flowing in capillary 501. As a transducer 503 produces an acoustic standing wave (not shown), the particles 509, which may include one or. more rare event particles, are acoustically focused and move from the coaxial stream 505 to ihe core stream 507, where ’they flow in single file toward analysis point 511. |0103| FIGS, 6A-6C illustrate acoustic separation of particles across laminar flow boundaries according to an exemplary' embodiment of the present invention, A medium or fluid may be acoustically reoriented at the same time as particles in the medium or fluid, which may include One or more rare event particles, may be acoustically manipulated or focused. FIG. 6A. shows a fluorescence image of an optical ceil coupled to the end of a 250 pm acoustic focusing cell 609 when the acoustic field is off’. White lines 601 and 611 iiiiiiii 2015224429 09 Sep 2015 indicate the edges of the flow ceil A mixture of 10% whole blood in PBS buffer xpiked w it h :25//ft§/ml of flows· throu|| the;: bottom half 605 of the flow cell (the white blood cell ΌΝΑ is stained with SYTOX® Green); at the sop half 603 is 6% iodixanol in PBS buffer (dark). PIG. 6B show's that when the acoustic field is on, the 6% iodixanol in PBS buffer is acoustically reoriented to the center 613. the blood/PBS buffer/R-Phycoerythrin mixture is acoustically reoriented toward the sides|||:i|l;|l|f (top Ihd bottonFiffhe figure), and the white |ii|||Cbtls leave their original mddidtnlildihre acoustically focused to the center w-h|rl: they afpdfas: a green line (the: reds bidb|(Cell are similarly acoustically focused but arc not visibi$i;$|::the fluorescent imagfe)!: Iliiiifillustrates a ΜΑΙ-LAB plot 617 of the approximate acoustic force potential fo| iplibles that are more dfise/less compressible than the background. More dense, less iCbmpressible paitides/ili§|j^ iodixanol/PBS buffer) are acoustically, fbcused/acoustically (dark blue region, potential minimum), whereas less dense and/or more compressible media fe.g,, blood/PBS buffer/R-Phycoerythrin mixture) are acoustically fbcused/acoustically reoriented toward the left and right sides (dark red regions, potential maxima). If a sample stream of lower density (and/or higher compressibility) is flowed along the axial center of a substantially cylindrical capillary and a stream of higher density (and/or lower compressibility) is flowed adjacent to it, the streams will be acoustically reoriented to comply with the potential shown in FIG. 6C. a feature that has not been demonstrated or reported in planar systems.

[0104] !iii|.l|lill' illustrate aeddSl|lfob|sing appamtiik|Sli|||fdiiig tolbkefoplary embodiments of the present invention. FIG. 7A shows a flow cytometry system 700a in ilill|||iigll|:l||linciuding particies/7|:||/\|hich may includd|bl||b|' more |ii|e Cp:nt particles, and a wash buffer 713a are introduced in a capillary 703. A tine drive 701 te.g., a PZT'drive,.of prodt|bin||£m acoustic standing wave) introduces an acoustic standib|;fyave (nilllbl|ti|:|t a user-d§:§|ed mode (e.g., a dipole mode). Assa result,; the sample 715a and wash||ilie|I:i:|lniay be ae|(is|ically reoriented (a$ 715b and 713b) anti the. particles 712 may bd;|cbhStjbaIl|ii|51used (as/fll) based upon their acoustic coii||as| Al: ilidhiiatipn. source· 709ί|§ί§|^Α:::ΙΙ§11Ι1/ of lasers, or any "suitable illuminati<||:sbuie§|

|h||:ili|i|ht emittingsdiode) illuminhiessthe particles) ? 17 at an interrogation point 116,: f|e illumination source mdpbe a violet laSllle.g., a 405:nm laser), a blue laser (e.g., as488:nm lasi^|ili;|di|S^r(e.g|iil 640 nni laser), p/|:combi:hi|bl;thereQf An optical signal 719 lom the be .detectediiiiiii^iiiiii dctecldfifilrray of delectom 705 (e.g| A FjVlT array, a photo-multiplier tube, avalanche photodiodes (APDs). a multi-pixel APD device, 11111 2015224429 09 Sep 2015 silicon PMTs, etc.). FIG. 7B shows a flow cytometry system 700b where the clean stream 713a may be flowed independently through the optics cell. The particles 702, which may include one or mote rare event particles, may be acoustically focused to -flow as line 717. the sample buffer 715b may be discarded to waste 721, and line 717 may transit to a second acoustic wayiinducing shows a llow cytonietfy system 700e where the sample 7'Sligiytl^^piiiOU^ of the cehle|iiaiid floyflhext to capillary wall 703 while buffe||7l3a flows a|misf|lll(|i{)isite wal||IT|c trans.|hl§r 701 may acoustically reorient s^ilpli 715a (as 7T|||(|t||(h|biiically fo§h||:articles: 7|||which may include one..... or more raf|i||fent partkles|||s::|||(lll)||ftd may |Cb|s#cally reorient buffer 713a.

[0105] FIG. 8 illustrates a schematic of an acoustical focusing flow cell in combination with an ifobiStic flow cyfpSiStplj®!! particles and flow streams·;; aceordingillllii exemplary 801 including..... particles |||||07, and 809_(iirivlaii:Gi3·i;ιΐΐcilfliidfei^:Ciflu^:i:<bi|iUu<ore:rare: .#Ch||||ticles, is introduced to a flow ceil 810 cQntaining/$:iiiiii;|ih^^ 803^ΙΙθ||| and. 809 .a|!prtieles '815 th$|?!|if y|§§!!^ A wash or other reagent 805 from wash container 802 is introduced in flow cell 810 as background stream |||||whi.eh|bxits laterally, is introdueedlfrOnfipash .container 817 into a/flow.cellvIfOliifrllgibcus cytometer 850 havi:h|;hilpbUs(i||fleld genejffor 822, which acoustically focuses particles 823 as particles 825/827 before entry at 829 into another flow cell 851. A transducer 831 further acoustically focuses the particles 825/827 as particles 832 before iiiterix>||dtie)rT £»t it)terrogation poinf|8||/b|/ihfofrb|h|i A signal 854 from the· 'interrpgd(p|:p^fl|l0s. is .sent' to de|e|fo||||||i|b||h collection of the..... iTiierFogale^l^S^iiji^Siiiii^ ec tioti point :81111111111111111111111111111111111 [0106] FIG. 9 illustrates a flow diagram of an acoustic focusing system according to an cotemplaryi/g^lj^lii^ti^ the present -ρίί^ϊίίΜίΐ^,ί^ iuding particles is collected aiil/liijltddstlia controllable ;|B||iji|t!!:fi flow pump! pumps lhe|Slii!pti;ih|6/hh acoustic focxjsfr'ig:;/CtiS^iciS!!TtA:i;:9/iiiatjicttistic· focusing device! focuses at!i|ist!sbih|!bi'the parficles!;#hteh mayiiSslfdl! event particles!! into a 1ί^!Ρ!!|||||;!:!||| the partideiy/lrf/ihen dinf§f|i(| zone for optical...... excitation.lhli^lctdliidnii Τη. step 90T^hiSi:!ji^ja.st :Som^;i;i^i|ii|feibiii^e^iii^^!i^ excited and..... at least soi||!:|ipiiiifo:the exeits|/fl|fiie1es is les are then cither directed to further analysis or to waste or some other processing. In step 909, the particles may be further analyzed by a longer transil lime data collection and analysis section. In step 911, the particles may be extracted as waste or subjected to additional processing by a waste 10 2015224429 09 Sep 2015 or additional processing section. The controllable pump may be adjustediTiSi!:ι<5ii flow rate for a desired/liifear velocity of the particles, "wiiiGli .aaiay' m/s to IQ nvs, of about 0 m/s to about' 0,3. trt/s, example. The excitation/detection may be p'ulsed: suitable excitation/defection methods knowh!:ii!(!ii$||i and/or:ibpihS:|: including using· a Rayleigh scatter detector. Jllllllllllllllllllllllllllllllllllil' |0107| The control of particle velocity has many advantages. First, it may improve the signal by increasing the number of photons given off by a fluorescent/hmnrieseent label, as tiro label may be illuminated for a longer time period. At a linear velocity of 0.3 m/s, the number of photons may increase by about 10-fold and about 3,000 particles per second may then be analyzed when using acoustic focusing (assuming an average distance between particle centers of TOO microns). And at a linear velocity of 0.03 m/s, that number may increase by about 100-fold and 300 particles per second may be analyzed. Second, markers that are not typically used because of the fast transit times in traditional flow cytometry (e.g,, lanthanides, lanthanide chelates, nanoparticles using europium, semiconductor nanocrystals (e.g., quantum dots), absorptive dyes such as cylologicai stains and Trypan Blue, etc,) may become usable. Third, other markers (e.g., fluorophorc.s or luminophores that have long lifetimes : and/or: : low quantum yields/extinction coefficients; most chcmi-bioluminesccnt species; labels with life limes greater than about 10 ns, between about 1() us to. about 1 ps, between about 1 us to about 10 ps, between about 10 ps to about 100 ps, and between about 100 ps to about 1 ms) may benefit from lower laser power that reduces phoiobieaching and from the longer transit limes made possible by the control of linear velocity. Pulsing at a rate o|i|lil|i|d times pcl|iillblllii?t|||i may, example, allow 10 cycles of excitation and luminescence collection in which virtually all of pulse rate without the benefit of longer transit times made possible by the control of linear

Ye)pih|||f|pfded by)|ifi|p||hients of )1||::§»:[§|§||Τηγεη1ΐ:ΟΙ1 might pass withoui e ver being interrogated. If the pulse rate were increased to 100 kHz with a 1 ps pulse, there may still be nearly 9 ps in which to monitor a lanthanide luminescence (as md|||il||i!i||^ores hav;|1 -2. ns lifetimes .and|l|bsfaiitofl|(fbstence decays williiflffjiiS)!!| |0I08| |i||p. 10 illustrates the diagram ::|||flG. 9||i|difi.ed to include in:-hl||i||iihar wailiilpbbprdtng to an exemplary; emboditi|nt of the present invention. In st|()l|0|l, a san|ple|;mei|ding particles is collblfed and directed tp||i|ontrollable sample flow Ihhfp. in step 1005, the controllable sample flow pump pumps the sample into the laminar washing; 11 2015224429 09 Sep 2015 devidevice 1Q19 {which maysbe based' ohliebustic reoriei||idIilI^|ill|{tes andsfiuids), Meanwhile, ilf||ep 1003, awash difecilldlalChiidiabk: wash fluid pump, in stej||§07,. the cotiihcjiiabie was||iii||p|il|:i pum|Sd|dl^ishi®lid into the laminar washing desled; In step 1009, the laihthh|5dilSihih|; device washes the sample; particles in-line. In step 1011, clean fluid is collected, and the 'w^-hiSidiiiiiiaSiEfc^ which may include, one or more rarb^bfent partieles|ldiitl;i|iehi diri&amp;i^ zone for optical excitation and detection. In step didlliiliiehf! some of the particles are optically excited and at least some signal from the excited particles i|i||fde|e||:itnd the particleslaje then either dirceteliidi&amp;rther analysis or to other processing. In step: 10:|§i|.the particles may|be;|Itl|her analyzed:by a ionge{|rli|8t lime idaihicpltlddon and analysis seeiion. In step 10l||4he:j)arti:c1es extmcteiiiSiiij^iiiiii^ia Op iii^ideilddo additional processing by a waste or additional proces|ii|i^ction, :||||||| 10109] 1 FIG, 11 ilhfstrateslidoustic:: fbcustn^l::6flh|:iaxninar wash......fluid according to an iejteihliiilry embodiment of t^i;;;j^ii«ser^:i;iiliyentiQ!ti^ saxnpjjisiiifeid®iji^ pfSjiiles 1109, swhidifiiriay include one or ihb(|:Tar|::§yent p^dll^lldSlinti'Qdue||lihld::planar ejebUStic flow ielilOl along with a .lamih|i|^al|ilfliiid' 1:ί<$ί|ίϊ!||:||||ρ^^ wave 1105 that acoustically focuses the particles Π09 to a trajectory passing through node Itll based on acoustic contrast. The planar acoustic flow cell 1101 may also have an acoustic node located externally, in which case particles 1109 may be acoustically focused to the top of the flow cell.

[0110] FIG. 12 illustrates a schematic of a parallel fluid acoustic switching apparatus according to an exemplary embodiment of the present invention. There, a first, outermost sample medium 1205 containing first and second part icles 1202 and 1204/. which may include one or more rare event particles, is introduced in capillary 1201. A second, intermediate medium 1213 along with a third, innermost medium 1209 arc introduced in capillary 1201. A line drive 1203 may acoustically reorient the first, second, and third media and may acoustically focus the first and second particles based on their acoustic contrasts. The particles may then flow out of the capillary. Upon switching, some of the particles may be acoustically focused from the first, medium to the third medium, passing through the second medium (which may be a reagent stream), or they may be acoustically focused from the second medium to the third medium.

[0111) FIGS. 13A and 1313 illustrate switching of unlysed whole blood according to an -exemplary embodiment of the present invention. The .blood sample 1309 and wash buffer 1307 are introduced at different locations in the capillary 1302, Upon activation of the !!!!!!!!!!!!!!!!!!!ll;:!!!!!!!!!!!!! 2015224429 09 Sep 2015 transducer 1304, the red blood cells 1303 and white blood cells 1305, which may include one or more rare event red/white blood cells, are acoustically focused, and the sample 1309 and i cally reoriented ldpiiitunlei||l^tte:i in. the rope-lihd ·§|ίίϊ^ 11111111111111111: [0112] FIG. 14 iliuslrates a sehe matic of·£$ίϊίϊί§^ embodimj$i$iiiiL&amp; :;iiidldd|iddlll00 of a sample 1mayiiieladdldielbr more rare event particles, flowing along with an unknown or unusable conductivity buffer 1403. The panicles 1409 may be acoustically focused to the buffer 1403 using transducer 1407 while the sample mediuni may be discarded at waste outlets 1411. The particles may be analyzed and counted by any suitable electronic detector 1417 detecting signals at electrodes 1415 as the particles move past the second transducer 1413 to the detection point 1419 with pore size 1419b, lllil [0113] F IG. 15 illustrates the separation of negative contrast carrier particles from a blood sample core according to an exemplary embodiment of the present invention. There, a transducer 1507 may acoustically focus negative contrast carrier particles 1505, which may include one or more rare event particles, from a blood core sample 15.11 initially including them and blood cells 1503 to cross the interface 1502 between the blood core sample 1511 and a clean buffer 1513, moving toward the capillary walls 1501. In other acoustic modes, the blood cells 1503 may be driven to the walls while the negative contrast carrier particles 1505 may be driven, to the central axis.

[0114] FIG, 16 illustrates multiplexed immunoassaying ill an acoustic wash system according to an exemplary embodiment of the present invention. In an acoustic wash -system 1600, competitive immunoassaying may be performed quickly by flowing analytes 1609/1611/1613 in a center stream 1607 and pushing beads 1603/1605/1621 pre-bound with fluorescent antigen, from outer .stream 1601 info the center stream 1607. Specific chemistry may placed on each of populations that are mixed i n a single reaction vessel and processed in flow. The populations 1615 exiting the :vessel, which may include one ormore rare event populations, may be distinguished by size and or fluorescence color and/or fluorescence at analysis point 1617.

[011-51 FIG. 17 illustrates a flof6|||i/|or 'high-throughput seedling usil||leb§sflS|flmIi according to an exemplary' embodiment of the present invention. In step 170.1, cell/bead-type :|hriidi|l|Which may include one or more rare even$$|i§icles, are;;pi|ered ii^i/tffriirriict'i fli!;f!Sii§h|step. .1703, they are inCUbatid/^itte dra|/dShdidatds!bfs! 111111111111111113 11111111111 2015224429 09 Sep 2015 to· an a(foiis:ije;;;;fo||^^ Meanwhile, in· step 1:||9|ipther· drug(’s) slii^Sliiadditional 'folcithtpj;;;^^ to the· acoustic fbcuser/sir|ldl|swilcher. in ;;:s(®p;;;;4i;05, the acoiifilliS 'switcher focuses and/or switch||||it|i|Ics and/or streams, and may separate the particle’s from excess drug/ligand. In step 1707, clean fluid is i^dllddiid immediately idilpfldr additional switching using the acou^iiiiliii^iiiyigi^jr^ iiSMtCMdi In step 1711, lire particles may be identified and/or sorted.

Idhy/dhled particles may be sent to waste, and, in step 1715, selected parti.cl:0:$i::i/t^iiiJ/i1S/1/il/0ii iidddilidhiai analysts or processing. In step 1717, additional processing;.........including:: tisfoi^iiltion of drug bound, scintillation counting, viability/apoptosis detdiftiiialdhlih^:: :i|did:|||fossion analysis, may be performed.

[0116] !§1G, 18 illustrates a two-chamber eulturing/harvesting vessel using acdustic i;:||hihid|;d|dording to an exemplary embodiment of the present invention. There, cells, which Sfoayllihdiddd: one or more rare event cells, are cultured in chamber 1801 and may he:; :;:|dfi:ddi:dMi§:; sent tpsbdacoustieally foeised in the channel 1805 where they may be examined for cell density/growth by titc optical detector 18! 7. When growth goals are met and the growth medium in chamber 1801 is spent, valves may be activated io allow fresh media from the reservoir 1803 to flow along channel 1805 and spent media to be harvested in chamber 1811. The ceils may then be acoustically focused into the fresh medium and transferred to the second culture chamber 1809. The same process may then be repeated in reverse such that cells are -cultured in the chamber 1809 and transferred into fresh media in the chamber 1801. |«117j TIGS. 19A-19C illustrate aptarner selection from a library according to ah .exemplary embodiment of the present invention. TIG. 19A shows multiplexed beads/edls 1903 with target molecules incubated with aplamer library 1901. TIG, 1913 illustrates the use Of in-line acoustic medium switching to separate beads/cclls 1903 and 1907 from unbound apiamers 1904. Salt and/or pH of the wash core (center circle) may be adjusted to select for higher affinity apiamers, and serial washes may be performed, to increase purify, FIG. 19C illustrates sorted beads 1911. 10118] TIG. 20 illustrates a dual-stage acoustic valve saner enabling in-line non-dilutive high speed sorting of rare cell populations according lo an exemplary embodiment of the present invention. There, a sample including: particles 2007 'isintroduced into part 2001 of an acoustic valve sorter 2000. A first transducer 2002 induces an acoustic wave in channel 2004, and an interrogation source 2013 interrogates the particles 2007 at an interrogation point 2006. Unwanted particles detected at sorting point 2009 may be directed past waste II!;! 2015224429 09 Sep 2015 valve 2010a, whereas selected particles may be directed to downstream processing 2011 along the channel 2004 for further focusing by a second transducer 2002, interrogation by light source 2015, and appropriate sorting toward either waste valve 2010b for unwanted particles or an exit from the channel 2004 for selected particles 2019. For rare cells, this provides high speed initial valve sorting that captures cells of interest, thus enriching: the ratio of desired cells in the sorted fraction, which may then be run again at a slower rate tor enhancing purity. If, for example, cells are analyzed at a rate of 30,000. cells per second and the valve sorting were capable of sorting at 300 cells per second, each initial sort decision should contain an average of about 100 cells. If these 100 cells are then transferred to a second sorter (or the same sorter after the initial sort) at a slower .flow rate, the cells of interest may be purified considerably, |0119| FIGS. 21A and 21B illustrate the optical analysis of acoustically repositioned particles and a medium according to an exemplary embodiment of the present invention. There, particles 2102, which may include one or more rare event particles, in a sample 2103 are acoustically focused (as particles 211.5) based upon acoustic contrast by a line drive 2105. The particles-2115 enter an optics cell 2117 and an interrogation source 2111 interrogates them. An array of detectors 2107 then collects an optical signal 2113 front each particle and, if the -signal meets certain user-determined criteria, the corresponding particle (or group of: particles) is illuminated by light source.......... (e,g,, a flash LED (wideband or Li’Vfrsand ii'lllliMfry an imager, 2109. In FlQ|2!^|fid7i:mage is acquired and the flow rale 2|:||||f

In7||iG:,lllB, however, the'.flow riifr|l27 of pd§i|le;8 illllifrdeeillid appropriateliibfllhe. required, imaging resohtfcm to acquire an [0120) FIG, 22 illustrates a diagram of particle groupings with different parameters such as may be analyzed in a system as shown in FIGS. 21A and 21B. Each particle within a group of particles 2202d!Stliilar as to slhllmgter 1 and Parameter 2 (.each of which ma|!|dv for example, forward scalier, side scatter, or fluorescence». The user-defined threshold 2201 identifies particles that meet the threshold for imaging based on values for Parameter 1 and for Parameter 2, If die particle meets the user-defined threshold, then flow may be reduced to an appropriate rate for the imager to capture an in-focus image of the particle. Other detection thresholds 2205, 2209, and 2215 may also be· established. Of course, not every particle need be imaged. Rather, a sampling matrix of particles from gated subpopulations may be constructed to define a set of particle images to be captured based upon their scalier and fluorescence signatures, which may allow high particle analysis rates (in excess of 2000 111 2015224429 09 Sep 2015 . Images may ea'ptiij*feiiii^liiiijU o;iogy» orientation|;and internal ijt$iii$ and number of nucli^l?ib||ii|ylbe obtained using any suitable jagitjgiii;iiaowu in the art, inciuding||psi|bile||!||© panning technology. Imaging iiiSiilii^iiliii^^ -<jup to .300 xells/sec|^lbit!s|b#illfiow may allow long Integration iitlliS&amp;iflsb high· and allow (up to 0.5 microns), [¢121] |||ir||231 illustrates acoustically ii;|§§&amp;^ by .an imager; embodiment of :;§|(;sl|lws a photograph of from an aeousticil^iepph|dd(| the stream ipl the optics cell .2307 is slowed for in-focus image capture of blood cells 2303. To create the of mrver an optica] cell (which may be, for example, a borosilicate glass cube with an interior circular cylindrical channel having the same diameter as the inner diameter of the line-driven :ΰ§Ρίίί®$|ί of excifatiOhililij&amp;iliiiji^ consumption of the acoustic device is 125 milliwatts. Line-driven capillaries may yield fine focusing of 5 pm latex particles and blood cells at volumetric flow rates exceeding 5 ml/min. The line-driven capillary may be attached 1o a square cross-section quartz optics cell. The inner cavity of the optical cell may be circular in cross-section, and it may have the same inner diameter as the line-driven capillary to extend the resonance condition of the fluid column and thereby extend the acoustic focusing force into the optical cell.

[0122] FIG. 24 illustrates acoustic fusion of particles according to an exemplary embodiment of the present invention. A first, sample 2401 containing a first particle type is pumped through a first acoustic focuser 2402 driven by a PZT transducer 2404 and the particles arc acoustically focused into a line 2408. Λ second sample 2403 containing a second particle type is similarly pumped'and focused into a line 2409 in the second acoustic focuser 2405 driven by a PZT transducer 2407.. The samples are flowed into a third acoustic focuser 2410 driven by a PZT transducer 2411 such that the tines of particles are focused to form a single line where the particles can interact, Downstream, the particles pass through an electric field produced using electrodes 2413 that fuse particles 2412, potentially forming one or more rare event particles.

[0123] FIG, 25 illustrates acoustic focusing and separation of particles according to an exemplary embodiment of the present invention. The particles 2503, which may include one or more rare event particles, are moved to first acoustic focuser 2505, which focuses them in single file fine 2509 with first transducer 2507. The line 2509 may subsequently be fed into acoustic separator 2513 equipped with second transducer 2512 and multiple exit bins 251:9a, 16 2015224429 09 Sep 2015 2519b, and θ:ϊΐ;:;:^η:ίϊ;:;β;ίΰ:Ι!§^ up on ,o:ne or more of size and ab0|sl:ic properties. The position of line 2509 may be adjusted upon entry in the acoustic separator 2513 by drawing fluid away or otherwise removing fluid through, for example, side channel 2511, [0124] According Jo exemplary embodiments of the present invention, the amount of assaying in clinical immunuphenolyping panel assaying on a single patient’s blood may be reduced by performing such assaying using an acoustic flow cytometer capable of controlling particle velocity and allowing long transit times as described herein, which increases the number of markers that may be assayed at once. Larger compensation free panels of, e.g., 4, 6 or more antibodies at once may be performed. For example, in a panel of anti-CD45, CD4, and CD8 antibodies used for CD4 positive enumeration of T-cells in AIDS progression monitoring, for example, CD3 may be added or substituted to aid identity T-eells. The assaying may be done using a blue (e.g., 488 nm) anti red (e.g., 635 ivm) laser cytometer with each antibody haring a different fluorochrome (e.g., F'lTC, PL, PL~€y5 and APC). Many four-antibody assaying combinations for leukemiaTyraphoma classification may be used, for example, including (1) Cl.)3, CD 14, HLADr, and CIM5; (2)-01)7, CD13, CD2, and CD19: (3) CDS, l.ambda. CD19, and Kappa; (4) CD20, CD lie. CD22, ami CD25; (5) C’D5, CD19, CD10. and CD34; and (6) CD 15, CD56, CD 19. and CD34, for example. Further, protocols described in Sutherland et al, "Enumeration of CD341 Hematopoietic Stem and Progenitor Cells,” Current Protocols in Cytometry, 6.4.1-6.4.23 (2003), which is incorporated herein by be used \Vlhtp||tpf niOflpfilre exemplary ethlbdeiM d escribed' herein. |0l25j combinations, for:;i;KijrirriylymjhlhiSclassification mafjbildiSblhSddfr examples shown in Tablclll|ffil|iefl. column indicates the nssh|ih|jhiihbCr and th|ib|jbolumn indicates the fluoraclfpiricjlsed iolpheh antibody; the ;speciicri|lh|':each anttlodpriS listed left to right underji|Pt|;it| respective :fluorochrome label|lil|leplacing ftuofdChfomesiwtth a long-lifetime rCS|piisihnd narfpw band reagents, i|hinitii|::Criihpensation .|i$l|i|y· piilels are possible. A |§i$illd!e examples of labels that may accomplish compensation minimized results that do not require compensation controls are shown in Table 2. The assaying may use 405 nm and 635 nm pulsed diode lasers, for example.

TABLE I 1. Kappa Lambda CD5 CD 10 <71 >34 CDI 9 2. <4)38 CD 1.1c CD22 CD 19 CD23 CD20 .1. CD?" CD? 6 C 1.)33 CDS C.D161 CD3 4. CD! lb CD 13 CD33 HLADi CD34 CD45 5. CD7 j CD.32 CDi 1 a CD! 6 CD64 CD45 TAW.E 2 Odol©.545:ss illhdoliloo EuroDiumDi- \ f 11 PerCP APC iAiekSsifbbrti: 405' 1 CD7 CD4 CD2 CDS CD 3 CD45 1. Kappa Lambda CD? CD 10 CD34 CD 19 2. CD38 CD lie CD22 CD 19 CD23 CD20 3. CD?7 CD56 CD33 CDS CD 161 CD3 4. CD 11b CD 13 CD33 HLADr CD34 CD45 ?, CD7 i CD32 CD41a CD 16 CD64 CD45 2015224429 09 Sep 2015 |0126J Immunophenotyping in blood may be performed witli red cell lysis by incorporating u .rapid red cell lysis reagent mlo the central wash stream to lyse red cells inline in a flowing separator. After lysis, the unlyscd white cells may be quickly transferred to a quenching buffer in a subsequent separator. This may be performed in seconds, minimizing damage or loss of while cells, and may also be used to exclude debris including lysed red cell ‘ghosts'5 that have decreased acoustic contrast resulting from the lysis process. Staining of while blood cells for immunophenolyping may be done in a small volume of blood prior to lysis, or it may be done after lysis (while carefully controlling the sample volume and number of white cells to ensure the proper immune-reaction). An acoustic wash system as described herein may be used to concentrate target cells or particles to a small volume for proper immunostaining. which is useful for samples with a low concentration of target cells. For example, such a system may be used lo decrease the cost of assaying in CD4 positive T cell counting for AIDS progression monitoring.

[0127] Immunophenotyping in blood may also he performed without red cell lysis by triggering cl election sighhililttther than scatter signai$|lSi;^^ then be stained with airtib0jjii^;;iiBtiid fed into a cylometertiyitftbui: lysis, in 80¾¾ cases with virtually hb|:|il|ri|i||| Acous|| (Cytometers according t:o|||||b|iments of the: liiflepl: jpyeption unay|pf||ri|(|||| type blSpaying on approxim.ately|||||l:i)0 μ! of whole i|i3|i:p|pit|ie sincel||:biriid||ells can iCConcentrated into a centrafbc||;§vith very'little to|||||!i||!||||!:As thlliliiiciibirild cells iifilbrmal patients usually malii;|i|i|ess'than 1% of the total number of cells in whole blood, coincidence of white blood cells in the dense blood core is rare, yfliblsole usfe||f:iiJilfodynamic ‘focusing does not appear 10:(1¾¾ such a solid core, which limits the number.....of cells passitig through a given cross iSbiilibnal area. An 18 2015224429 09 Sep 2015 acoustic.....wash step that transfers th|;/ilbbd ipelii;|\|h|;/irom free· antiboicite:Siii;iartCi;i;ttittiiiiiicfeaii: biif&amp;i/foijy/hlso be performed, vyh(c|/foa| redtiebs/ffdorescent bacfogJr^U:ft^:!ia^B:!tn;GteaSS:i sensiti^lllP' [0128] FIGS. 26A-26F illustrate comparative output plots for fluorescent microspheres run on a nou-aeousiie ild^fo|fofo|fo;ifh|l?bn air:iac(|ptfoiifo||sing cytometeil§i^ and sensitivity increa|ed|tf|til|||ltine that'may acoustic cytom||||||||p|th|l|d' an exdiiipj#|liliii|dime:ni of iJti;::ii;ii$y:rif|:ti:£ϊϊι;y Fluorescent microspheres (available from Spherolech, Liberlyville, IL, under the trade ::dcstgiiation.i)-5A., 3,2 pnip;jvqfo(i|jfo:bi/h:non-acou^ only hydrodynamic focusing (FIGS. 26C and 26F) and on an acoustic focusing cytometer using upstream acouslic focusing followed by downstream hydrodynamic focusing (FIGS, 26A, 26B, 26D, anti 26E) using a 488 run blue laser (top row, FIGS, 26A-26C) and a 405 run violet laser (bottom row, FIGS. 26D-26F). The non-acoustic How cytometer was run at its highest sensitivity setting with a sample input rate of 15 μΐ/'min (right column, FIGS. 26C and 26F). The acoustic focusing cytometer was run both at its standard sensitivity setting with a 100 μΙ/min sample input rate (middle column, FIGS, 26B and 26E) and at its highest sensitivity selling with a 100 μΐ/min sample input rate with an approximately 4-fold increase in time the particle spends illuminated by the laser (left column, FIGS. 26A and 26D). Two overall flow rates (2.4 ml iron and 0.6 ml/min) were considered. The sheath and sample input rates were adjusted relative to each other to allow sample input rates of 25 μΙ/min to 1000 ul/min for the 2.4 ml/min overall rale, and 25 μΙ/min to 200 μΐ/ininibr the 0,6 ml/min Overall rale. FIGS, 26A-26F shows that the 8-peak fluorescent rainbow microspheres (which consisted of 8 populations of different fluorescent intensity levels) are more clearly resolved by tiie acoustic focusing cytometer, as can be seen by the greater and clearer separation between peaks in the 8-peak bead set, especially in FIGS. 26A and 260. which benefit from the 4-fold increase in lime the panicle spends illuminated by the laser (e.g., from about 10 ps to aboul 40 fcs). This demonstrates the better resolution of fluorescent populations that results from slowing flow and increasing transit times. 101291 FIGS. 27A and 27B are histogram plots illustrating the effect, in cell cycle analysis of an approximately 8-fold increase in transit time associated with acoustic cytometry according to exemplary embodiments of the present invention. FIG, 27A shows data obtained upon miming ST486 B lymphocytes labeled with a violet stain (available from Life Technologies Coip,, Carlsbad. CA under the trade designation FxCye!ciNI) through a non-acoustic (hydrodynamic only) flow cytometer using a violet laser and a low sample rale iiiiiiiiiiiiiiiin 2015224429 09 Sep 2015 setting (transit time was. about 5 j*s). HQ. 27B shows the same type of data but obtained using tin acoustic focusing cytometer with upstream acoustic focusing followed by downstream hydrodynamic focusing using a violet laser and a; 25 μί/inin sample input rate (transit time was about: 40'ps). Approximately 15,000 total events were acquired in both eases. The data analysis, performed using curve Oiling software available from Verity Software House under the trade designation ModFit LT v. 3.2.1 yielded the underlying cell cycle phase distributions and the percent Coefficient of Variation (%OV) of the software defined GqGj peak and G2/G1 ratio. The %CV is a measurement of the precision of the cells falling in the GfjGj peak (the lower the %CV, the more precise the measurement). FIG. 27A shows more distinct populations and a lower %CV (2.81% vs. 5,84%) when using acoustic ibchil|(!l (0130] Other similar cell cycle analysis experiments have shown that although data quality and %CV may diminish as sample rates increase using only hydrodynamic -focusing, the data quality and %CV may suffer little or no changes as sample rates increase when using acoustic focusing. Specifically, for hydrodynamic focusing only at a concentration of i x 10* cells/ml, %CV values for sample rates of 12 μΙ/min, 35 μΐ/min. and 60 μί/ιπίη were» respectively, 4,83%, 6,12%. and 7,76%, and S-Phuse data changed from 37,83% for the low 12 μί/inin rate to 26.17% lor the high 60 ulinin rate. But for downstream hydrodynamic focusing on an already acoustically focused sample, %C V values for sample rates of 25 μιΐ/min, 100 μΐ/min, 200 μΐ/min, 500 μΐ/min, and 1000 μΐ/min were, respectively, 3.22%, 3,16%, 3.17%, 4,16%,. and 4.2170, and S-Phase data only changed from 40,29% for the low 25 μΐ/min rate to 38,55% for the high 1000 ul/rnin rate, Thus, even at. sample rates tar exceeding those of non-slidiisiieifoc using, systems, acouPefS||foths.:ii|ipn||fo^ [0131J According to exemplary7 embodiments of the present invention, acoustic cytometers fmhjfeiiflbw one to aecjiire statistjfei$!|$;^^ e9^|tS|it|drastically shorter i|6ffolslof lime because such c|foiiie|§l|| ihpt)|||tp· that are nearly an· fofopibf magnitude|ligher. 17c)riieiJeSiijElf)!fft:e:ifTIow ey|othdters: usually have a. fSgiillelinput rate 150 - e sttmated: mm· time to run a 2 ml fsiilpillat a conceηΐϊϊίϊίon.oT-S'.tsililij5:iiiiii;^liij^ 13 mimitCs, whereas an acoustic itpClfSmg'cytometerhildy have aiSdfoplelihpiiti^ μ1/βύη,: which may lead to an iiSiliSled run time liljhin a 2 ι|ίί&amp;!!ί^ of 5. x 10 cells/ml of about 2 iilifip. Table 3 sht§iv$ the ' attairted. ::ibr various combination fof/shtiple concentration and. safopieiisflitfig course possible to increase, the· 20 2015224429 09 Sep 2015 number of events by increasing the concentration. Rut by using high volumetric sample input rates possible with acoustic focusing, one may attain the same number at a lower concentration, i.e,, high sample input rates allow lor high data rates without the need to increase sample concentrations associated with non-acoustic systems.

Table 3

Sample Flow Rate (pL'min) .Sample Concentration (part/ml) lllillll ||30 60 100 120 150 mm 500 1000 1.001* 04 WM&amp;i ins 10 ::111:7 20 25:11 S3 1116? $.0011-04 8 112$ 50 S3 100 Ϊ25 111:6711 417 833 i .00F · 05 ffffllll mm 100 167 200 250 1133311 833 1,667 5.Θ0Κ+05 llislfff 1250 500 ililii 1,000 1,250 1,667 4,167 22331 1.00R-06 500 1,000 1,66? 2,000 2,500 3,333 8,333 i.0tBff)l!!!!!!!l: ill 2,500 mm 5.000 10.000 8,333 10,000 [0132] The wide range of sample input rates afforded by acoustic focusing cytometers enables high volumetric sample throughput combined with either low sheath or no sheath, or high volumetric sheath if desired. For low concentration samples, the high volumetric throughput translates to much faster particle analysis rates, which in turn translates to shorter assay times, particularly for rare event analysts in which the volumes that must be processed to achieve a statistically significant result are on the order of a milliliter or greater. This volumetric throughput can also translate to ultra high particle rates for moderately high concentration samples. If, for example, an acoustic cytometer were to use a sample concentration, of 6 million eells/ml and the sample input rate were 1000 μΐ/min, the cells would be pushed through the instrument lasers at a rate of 100,000 eeils/s. At the overall fltiffiralif of 2,4: m'i/min, this.οοτιοόϊΐϊ'ί^ϊΐ.<3ί^!:ΐ!ν^ί<3 in a:very:lngh rate of coinei§$n$: evfttillbut the instrument could use hilllllsflSier overall flow: rate such as 24 ml/iti:i|lfol exatf |16 Such performance is consid^fibl^fbetter than in conventional cytomefefliWh^fC tratisifdimes through an interrogation laser arc usually only about 1-6 ps. With:lifa^bf£i|e event rale of 0.1 per unit time, 10 (|||;|btxesponds;dfo|i|i!i|:|^a1ysis raldlpf abtiif!t0,000 pariieles/s. For acceptable coi:ncidencel|nd an eyeh|lf|t||pf: 1000 p|ff[bl|si^|l|nlPoustic system of the present invention .may adcommodai|i|fSlsifiitiines pfifllliplidlliEge' that:; 2015224429 09 Sep 2015 greatly improves photon statistics and opens the field of application lor the longer acting photo-probes. The rate of particle analysis in aeouslie focusing cytometers may be up to 70,000 panicles/s, and may reach more than 300,000 edis/min when periodically adjusting the velocity of the focused stream. (0133] For a 300 pm diameter aeouslie focusing capillary, a 10 ps transit lime through the ;inteixogaiiOii laser, and a particle rate of 10,.000 particles/W:a; eoncenlration of abplllllii^llp' ii|||||tti|or less .meaai. event rate offosiihafvone in ten tifflxi;;i:::

According to Poisson statistics, this corresponds to a probability of about 1% that a time will contain morcithip|pnl!|vent, meaning abohilllloi of events will be coincident.;; iilidlivbhimeiric flow rate'T|:i|tlfdilfbr this 10,000 piillcid^s rate example is||bbi|lli|| ilpbtbili For such a 300 pm 'dpi^i^iicapillafy, a conc|lhh|ipb:of about 2.8 x 10? Ρρΐ|^η||1|ίί optimal for maximum. tlmougbjJblBil about^. For larger particles or; lafpllaser beams, or if fcwe|iii$iiinii^ one may reduce coincident·; evfnt|:by decreasing eoneentfati$|i|ii.^ cytometer with a. flow;:ratdfo|i 2. j|||j/min may 'be diluted up tp:i§$||$i^ needed, to process the samples titan for a non-acoustic :cylomet#§djiiii§:ip;| 10 td min. Thus, with simplpi up-front dilutions, air acoustic tHrougliput than a non-acoustic cytometer for concentrations up to about 6 x 10 eclls/tnl. The 6 x 10° cells/ml concentration sample can be conventionally processed at a maximum rate of 1000 cclls/s. An input rate of approximately 30 μΐ/min is typically diluted about 20-fokl to reach the optimum concentration for an acoustic cytometer. By running al 2 ml/min, particles may be analyzed at nearly 10 times the rate of a non-acoustic cytometer using an acoustic focusing cytometer, in some embodiments, the particles may be analyzed at a rate of at. least 2 times, at least 4 times, at least 5 times, at least 8 times, or at least 15 times the rate of a non-acoustic cytometer. If a user prefers to take advantage of longer transit times through the laser, a sample could be slowed to 0.2 ml/min where it would have similar particle analysis rates to. the non-acoustic cytometer, but with longer transit times that opens the field of application for the longer acting photo-probes. (0134] Diluting samples stained with excess antibody reduces the concentration of free antibody in solution, therefore reducing background signal and increasing sensitivity, it can therefore be possible to perform sensitive assays without a centrifugation wash, while still maintaining a relatively high analysis rate, if the dilution factor is high enough. Alternati vely, one can increase the amount of staining antibody in order to drive the staining reaction faster and can then quickly dilute to reduce non-specific binding. This can result in a 22 2015224429 09 Sep 2015 much faster overall work flow. A sample that normally requires a 15 minute incubation and a 15 minute centrifugation can potentially be done in just 2 minutes, if for example a 2 μΐ sample is stained with overall antibody Staining concentration 10-fold greater lhan used for a 15 min incubation, the staining could be done over a very short period of just 2 minutes, after which it is diluted 500 fold Ιο I ml and an antibody concentration of 50 fold less than the normal staining concentration. A 1 ml sample can be analyzed in just 1 minute at a 1000 μΙ/min sample input rate, [0135] FIG. 28A is a photograph of blood having cells acoustically concentrated to form a rope-fike structure flowing in an acoustic cytometer according to an exemplary embodiment of the present invention. FIG. 28B is a photograph of more diluted blood with cells acoustically concentrated in a single file line in an acoustic cytometer. The cells in FIG. 281) are concentrated enough to result in many coincident scatter events but scatter height data using violet excitation of similar samples may still be capable of resolving different while blood cell populations from each other, if ceil concentration is reduced such that the rope-like sftucture becomes a dense line, it is possible to continue to use scatter to distinguish white ceil populations from the red cells using scattering measurements. The spacing of cells in this line may be much closer than what is normally acceptable for coincident events if a fluorescent- marker that Stains only the desired population, (e.g., a fluorescent GD4S antibody or DNA dyes that used, [0136J FIG. 29 is a spectral graph showing the excitation and emission spectra of the violet excited Pacific Blue™ fluorophore. It shows the 405 ran violet laser excitation and a ifiitd||p!|||||||{iaj!cd|ld|:l|||sed. for autoflirorescence correction in idfobjhhtpit wij||. thiiittrti^:|iirtj:Grptikkii^rutoflu-orescertce may be collectedsmlh: tight, band of color near the peak emission of the autofluorescence (peak emission near 430 nrrt) but in a region of relatively low Pacific Blue™ fluorescence (peak emission near 455 nra); [0137] FIG, 30 illustrates the detection of a rare event population of 0,07% CD34 positive cells as a subpopulation of the live CD45 positive cells according to an exemplary embodiment of the present invention. Approximately eight hundred CD34 positive KG~la cells were spiked into 100 μΐ whole blood collected from a normal donor. The sample was labeled with a CD45 Pacific Blue™ conjugate and a C.D34 phyeoerythrin conjugate. After incubation. High Yield Lyse solution was added for red blood cell lysis, and SYTOXS) AADvanced™ Dead Cell Slain was added lor labeling. The ceils wore analyzed on an acoustic focusing cytometer with upstream acoustic focusing followed by downstream 2015224429 09 Sep 2015 hydrodynamic focusing at a high throughput rate setting (200 μΐ/min) and about 200,000 total events were collected. Dead cells-were eliminated from the analysis by gating on SYTOX® AAPvanccd™ negative ceils and then·looking at CD45 vs. CD34 events.

[0138] FIGS. 31A and 3 IB respectively show plots of Forward Scatter (FSC) vs. Side iSeafter (SSC|liiipi|4|Sed whbfoi*bll<l)d .in .ih|acoustlelid|d|ihg sysfofo**hid inlSii|plely· hydrodynamic focusing system. FIG. 31A shows a plot of FSC vs. SSC for lysed whole glided at 405 niilexeiistion inijib apdSStic fcMj'USiA;g;(ii:with a 1ΟΟΙ^1|6ίίί1 sanipilginput rate. FIG. 31B shows the same at 488 rtnt excitation in a hydrodynamic focusing system with a 15 μΙ/min sample input rate. Both FSC and SSC were collected using a 405 nm laser (violet) as the primary laser line. FIG. 31A shows a greater separation-between populations relative to FIG. 3 IB. as well as the possible creation of an additional population of cells that appears to be consistent with dead ceils.

[0139] FIGS. 32A and 32B show plots of FSC vs, SSC for Jurkai cells obtained using the same systems and parameters described above for FIGS..3.1 A and 31B, respectively. Again. FIG. 32A shows a greater separation between populations relative to FIG. 32B, showing the increased performance of the acoustic focusing system.

[0140] FIG. 33 illustrates a schematic diagram of an acoustic flow cytometry system according to an exemplary embodiment of the present invention. The system 3000 lias a sample tubel3i(|llsdit(t|ning. a saii|il|3t|04'may iiMuSe· b%::pX:: more rare event particles, pumped through a capillary 3006. A piezoelectric element 3008, arranged adjacent to tire capillary 3006, may be operated by control circuitry 3009 under the fodnirol of a''|fo|ds|6f||;022, and may apply .aeousiiljeiergy 'Ibjlfoustkailyjfocus and/or ifraetionate th|||i||:iy|^|based m||t|ej|i:|fo|erties, ifljiiding, c.g.. size, and ||jisily. I tie Alouslically fodp6dlp;iicles maf|thliflljel an interrogation zone 3010 wlfofo they pass lifoughthe .betb$:i||iii$li$^ (e.g., a highly focused laser beam or libre laser beahlsj/|i||§ll or safo|;6|:||lii3i:||tsbe collected a waste site 3014. i|^|e sen tiered slight resulting Iroiiiihli iljthffoiation source 3012 with the particles mayi|g*; collected by a collection lens and optical collection block 3016. and may be analyzed u uh an *|iiy of photom|lti|Ml|ubes |||8 imerconiClfob with a data acquisition modile 3020 and* jhliproeessor 302iillll!l [01411 FIG. 34|ill|is|fates aisdhematic dia|i|li|of air acoustic focusing: capllary msam: acoustic flow cytometersaccording to an exemplary embodiment of the present inventiomtoi shbibThe effect of:|i|lpiezoelec|riC element on thdipartieles, The particles 300|ifand 3001 b i illlli sample 3004.....travel in lllackground or Cltrielsfluid 3003 in the acoustic focusing* 24 1111111111111 2015224429 09 Sep 2015

The piezoelectric bel|||irezoceramicl||gp|nt| acoustically focuses the particles 3001a into an inner coaxial stream 3030 and the particles . obiter coax.t a1 stream 3032111111111111111111111111¾ [0142] ΙΪΙρ. 35 illustrates a portio&amp;Siiil&amp;illii^ btedliiift. an acoustic· flow cytometer according to an exemplary embodiment of the present invention. The optical hbiiiieiphlblock 3016 includes an interhdigstiti0ili:·i|1); 1:0¾ilaaving.'iilla^Sier'3040 (e.g||a ;|0|l|tii:ytplet laser) and a second 1 ascr ·ΐΐ^Ιglί 1 1 lbJ·υ« lh^ef||!fie|beams. emitted by the lasers 3040 and 3042 enter an arrangement of beam shaping optics 3044, which tightly fotlhseiiilhem on the acoustically focused ci.es 3001a (FIG. 34). As the particles 3001a pass through the laser beams, the scattered light is collected by a collector lens 3046 and enters an optical collection block 3048 (FIG. 36)before passing Jo the detector array 3018 (FIG. 33). The 405 nm wavelength may be very useful with or without pulses when coupled with long transit times, ami is especially useful for excitation of quantum dots useful for many-colored assaying. Other wavelengths may also be used, including 640 lira, for example. The same particle may be analyzed by two different lasers. A stronger laser may be used to analyze dimmer particles, white a different, weaker laser may be used for brighter particles. A single weaker laser may also be used with increased transit time with signal integration, and sucli a laser may also be used in a pulsed system by administering stronger and weaker pulses at different times, [0143] The use of two lasers is useful to improve auto-fluorescence and background variance concerns and increase signal-to-noise ratio by reducing the variance of both signal and background. For example, the first laser may excite aulo-fiuorescence above the wavelength of the excitation laser, and the signal detected above that wavelength may used to estimate the auto-fluorescence contribution expected for the primary detection laser. This may be done with a system having a violet laser and a blue laser, or only a violet laser, or a violet laser exciting more than one color if there is a. separate color band to monitor the auto-fluorescence. Only the blue fluorescence: channel may be monitored, and expected contribution in other channels may then be subtracted. A red laser may also be used. For pulsed or modulated systems with long lifetime probes, the short lived contribution of the auto-fiuorescencc combined with the initial output of the long lifetime probe may be measured. Fluorescence of the long lifetime probe after the auto-fluorescence has decayed may also be measured and back calculated to determine the auto-fluorescence contribution in all channels. 25 2015224429 09 Sep 2015 [0144] According lo exemplary embodiments oi'lhe present invention, four-color assaying with only auto-fluorescence compensation may be performed using Qdot® 525, 585. 655, and 800 and a single violet diode laser. If a second laser, such its, c.g., a 650 ran or 780 rim laser diode is added, other combinations that are virtually compensation free can be added with even more colors. For example, Qdot® 525, 565, 605, 705 and Alexa Fluor® 750, which is excited very efficiently at 780 ran, may be added. Other dye combinations may also be used, as may other lasers or diodes, including a 473 am UPSS blue laser, a 488. nm wavelength laser, and a green DPSS module. If, for example, the rest period is 1 ps and four different lasers are used, with 10 ns pulses, each laser is triggered every microsecond, with a pulse of a different wavelength hitting the target about every' 250 ns. A second low power pulse for each laser may be used lo extend dynamic range (the brightest signals may be quantified from the low power pulse, dimmest from the high power pulse). Using lasers at 405 nm, 532 nm, 650 nm, and 780 tun, four colors and autofluorcsecnec may be monitored with virtually no compensation using: 405 nm-autofluorescence and Pacific Orange™, 532 nm-ΡΕ or Cy3, 635 mn-Alexa Fluor® 647, and 780 nm-Alexa Fluor® 790, although because there is some excitation of PE at 405 nm and some excitation of Alexa Fluor® 790 at 635 ntn, :i;|f might |||ife|iif gdlllllll: [0145] FIG. 36 illustrates a schematic diagram of an optical data collection block in an aeoaisticsflowscytomcteriiaceording to an exemplary embodiment of the present invention. The: scattered ilight: 3050 from the lasers 3040 and 3042 is collected by the eolleetarfons: 304||eh|efslfii6lCbl1ection block 3048 in tire direction of arrow A, and proceeds to a pair of spalaf IMltg pinholes 3052, one of which being backed by a mirror (not shown), fihtehi:: sepiUtes the beam 3050 into a primary beam 3054 and a secondary beam, 3056. The primary: beam 3054 enters a collimating lens 3060 and traverses beam splitters BS1, BS2, and BS3 ai|dsassociated focusing lensesfo enter fluorescence channels FL4, FL5, and FL6, as wellas* s|$i scatter channel 3062 in )|||detector array 301.8 (FIG. 33). The secondary beam :3056:: • enters a ppllimating lens SQTliplifraverses mirror 3072 and beam splitters BS5 and BS4 and ihllocihjldiiditising lenses pliiliiiluorescence channels FL1, FL2, and FL3 in the detector: (play .Ili|i!!!!!!!!|, [0146] |||i|i|I:||iljmtraffe|||i|S of a fluidics system., in an acQusticlfldwi' cytometer according to an exemplary embodiment of the present invention. In system 31.00, a sample jSfppillbllpI sample flyi||||p4 (FIG. 34) ppr sample tube 3705; at(||fo|g||i:aphragm pump 3106 after passing by a bubble sensor 3008 used to detect the presence of air in the sample 2015224429 09 Sep 2015 line. As a' result, there mayed voluirf|bi^:|i^|s||i^ws5 and samples may be drawn to .of ail w|||Sii.||tli|lend'Ofiii sample during the sample. 'dr|§i||l||ap|| 3008 rl&amp;y modalities, including ultrasonic, impedance, capacitive, optical, or any other type of sensor modality that .can· determine fluid sample. d.ίιΐ!ί manifold 3104 may then be 110 in aiiilp' '3703 for entry into the acouSfi|:jfbd||^ A iyieatl|||ii||l|ip||3.124 pumps the sheath fluid 3205 (FIG. 38), which is maintained in a sheath reservoir 3120 in reservoir section 3701, into intoiiii^ipi xipper-::fii^i^;Sii(i^ 30' in upper manifold section 3704 at the capillary' lube 3006. Water and wash fluid may be maintained In a water reservoir 3140 and wash fluid reservoir 3150, and pumped into the system as necessary, while waste is collected in a waste reservoir 3160. |0147] FIG. 38 ilhisJrai.es a schematic diagram of a single transducer acoustic focusing capillary with upstream acoustic focusing followed by downstream hydrodynamic focusing according to an exemplary embodiment of the present invention. The sample fluid including particles 3204, which may include one or more rare event particles, flows through the capillary 3206. A single transducer 3208 may then acoustically focus particles 3204 (as 3201): in a first region along a substantially central axis of the capillary 3206. This may be done prior to any hydrodynamic focusing. A. sheath fluid 3205 may then be used to flow around the acoustically focused sample fluid and particles and further focus, hydrodynamically, the fluid and particles 3201 in a second region downstream of the first region. It would be possible to use more than one .transducer; for the upstream acoustic focusing, but a single transducer is preferred. Also, if would be possible to use a first, upstream acoustic focusing phase followed by a second, downstream dual focusing phase that would both acoustically and hydrodynamically focus the particles 3201. Preferably, however, the particles are first acoustically focused and then are hydrodynamically focused at a second location downstream of the acoustic focusing location, without further acoustic :foduiih||las it tf offers particularly impressive rai'e -event iddfolidilbilitiesi! be used alone.ihilieu of'hydrodynamic focusing; that certain configurations jointly using acoustic i|:chSih|;|;;and h.ydare particular||||ijseful. For example, joint stabilize ihglhfisoiuie location of a particle sff|ilj||ainst extemaijlpills^ tighten the fodusjbf the focused particle stream 27 2015224429 09 Sep 2015 usfeflil wh^ng;:itJliiS:i:$^Ιΐΐijis;:iicliI0:r where “st{Ci|liiei!i|:;iilS't be kept al lower concentration to prevent aggregation); and may help ensure that the sample does not contact the walls (which may be important in some applications). Finally, it turns Pit,· \ιη.εχρ^ίί!^ΐϊί^ϊί!ί^ stingjSi;;tiiSfe of ilplslic fbCipl|ji|stream, folkJwedtb^liidtiiiiiStream pe of'hyd.-n^^iiiiS^iiiiiiiiiiife thdihme chatihililield excellent propertiiShlbtytng the detection of 'ige^ij^ii^iiii^^ relatively short periodipf time, as describedihipme of the above exempih(j|dbl§l|ime^ [0149) According to exemplary embodiments of the present invention, (he sample pump 5102 and ihc sheath fluid pump 3124 may be controlled by a processor to adjust the volumetric ratio of sheath fluid to sample fluid in the capillary tube 3006 to maintain a substantially constant overall particle velocity in Ihc interrogation zone. For example, the volumetric ratio of sheath fluid to sample fluid may be maintained from about 1:10 to about 100:1. I hc ability to adjust sample input, rates while maintaining a light focused particle stream enables adjustment of velocity through (and thus time spent in) an interrogating laser. Longer inlcrrogalion times allow higher sensitivity measurements bv allowing the collection of more photons over time. When a particle analysis system may only control sample flow, the adjustable How rate limits the ability to increase or decrease particle analysis rales for a given sample concentration as increasing or decreasing the sample input rate necessarily increases or decreases the transit time. By including a sheath flow that is adjusted in response to sample input such that the overall fluid flow is kept constant, it is possible to allow a wide range of sample input rales without changing the overall fluid velocity. Then, by changing overall fluid velocity, it is possible to lake advantage of the benefits of longer

By not accelerating th|S|>§^^ the coaxial sheath flow, particle the laser i;nterrogation|f||ph|df;in acoustic flow cytometer may be than in conventiohh|h||fo||'hami focusing systems. Preferably, i|§||j||||))|:|||||||:|θ [IS, at least 25 ps, at J|ii§:l||lji;||il least 35 [is, at least 40 ps, at least ibOsipSf; or at least 100 pslll|ihislhi|y allow higher sensitivity Optical similar particle ihhi|kiS:liitis.

[0150] |:|j^23lsi||tiarates a schematic diagr||j||||lliii)Cker bar apparatus that may adjust a in .an 'acoustic fldiy||cj)idiiieler according to· an exemplary . The bl|ckd|!|ll 3300 .may be used to change the apeil|hlbllli!fbpii| scattered lighlibetbre tb|j|h|ihf| 'Scattered lightsCnters the· collector 1ρηι|ί$($^ 3041 eil|i|d| by th|l|ase:fsl:3040 |h!|||3|l||intC]'aet with die acoo|$i:i&amp;|$^ of particlssstflowingiphldflie plahl:6|4lOij?hper in the fi.gure)f 2311111111111 2015224429 09 Sep 2015

The blocker bar 3300 may be mounted on the underside and olT-axis of a cylindrical peg 3302 so that spinning the peg 3302 changes its location relative to the propagation of the scattered laser beam 3050. This allows art operator to align the blocker bar 3300 with the lasers 3040 and 3042 once the lasers are aligned with the particle stream. The aperture .for the forward scatter 3050 may be changed by changing the shape of the blocker bar 3300, which may be done by adding a. collar onto the bar. The peg 3302 may be rotated to position the blocker bar 3300 lo provide ari aperture a of the forward scatter 3050 of between about 15° and about 23°, or between about 17" and 21", or about 19". |015l| FIGS. 40A-40F illustrate the detection of a rare event populations of 0.050% and ():045% CD34 positive cells as a subpopnlation of the live GD45 positive cells according to an exemplary embodiment of the present invention Thao, peripheral blood was stained and run using an acoustic focusing cytometer with upstream acoustic focusing followed by downstream hydrodynamic focusing at flow Tates of 500 μΐ/min (FIGS. 40A-40C) and 1000 iil/min (FIGS. 40D-40F') with a stop gate set at 500,000 total cells. FIGS. 40A and 40D shows the total cells stained with SYTOX® AADvanced™ Dead Cell Stain and show the live cell gate. FIGS. 40B and 40E· show cells gated on live cells. FIGS. 40C and 40F show cells gated on live 0)45 positive cells. At the 500 μΙ/min flow Tate, the acquisition time was about 6 minutes, 26 seconds: 0.050% CD34 positive cells of leukocytes were detected; and direct measurement of CD34 positive cells was 0.07 eelFpl. At the 1000 μΐ/min flow rate, the acquisition time was about 4 minutes, 28 seconds: 0.045% CD34 positive cells of leukocytes were detected; and direct measurement of CD34 positive cells was 0.05 cell/μΙ.

[0152) FIGS. 41A-41D illustrate comparative output plots for cell detection run on a non-eUrr cyidii|fb|.ls:.Peripheral Jibpd|ifom' a normal donor was spiked with CD34 positive cells and 50 μΐ of' Count Bright™ Absolute Counting Beads and used to calculate CD34 counts, FIGS. 41A and 41B show data gated on live.cells (FigillAlifthd CF$|li|(§|itive cells (Fig. 4fB):|ifh onabydildlhalnd focusing flow cytometeilllPMs. 41C||h|||lD show data pteilon. live eells7|ltp54lCi:i:|i|d. CD4:5; positive cells (Fi||;||:D) Eti||dilgn acodsltdifochsihf. cyt|ffefer with' upstrl|i||||dQusiie focusing foilowedll^l dowhltfoSin hyd;rbiipiamt§ fbcu§iii|l|edprding to an exemplary jCmlddiment of thelipresentllinvihtion, ||IISmg:liithe| h^dfol^h|fhtc locusing onlyfiflpw acquisition tjf|iii§i$i$ 13 miiit|s,. lOvlecOiids; th||li|ilj||i|iiv^||i?n ctlill ileltYllihsmi,heads fpl .8.(|;[||el§/pl; art|iii$i$i^i$c|iim|puremenf th|i isfObiiltle flo\fl|yiorii:|(|i||hc acqui|iii:pn itin# #4$' 1 minute,ii||iSecond$^ 29 2015224429 09 Sep 2015 ipbklOve cell co#llifef|^|i|tisi:ng beads was|||i:f||Slls/gl; and difobi Measurement d|::p§)||l;: positive cells [01531 FIG·. 4llil^S|(‘a||sia schematic o|;|bti|baents of a||lc:bliStic. focusing cytometer;: according to an c|Stf|>iaf|le:fnbodimeni ofjhiiptfitelt mventiOji(:i|?he exemplary cytometer IlOludes a first ΑηιΡ:ΡΙ0ί:42θ5 for sheath ΑηίΡΐΙΡΟίΟΡίηβ a sfibaihifluid 4208, a sheatifilluidi: ifiler 4207, 'and a ^1ί||:1|ί||Ι|}ΐ|ί reservoir 422G;6iii|tl|a|^e:colid.ffl:Uiiif|paIII 4209 for samplf;||hi|[:: meludmg a samplclfiimBllli^, a btibBIc4211, and a capillary.; assembly 4210. The sheath and sample fluids may flow in a flow cell 4204 and first and lecond lasers 42021|iilll||||may intepjp|ii&amp;S$ be in the sample ;:fluid. iFinally, some sheathihid:^Pp:e;fluid. mhyijl&amp;iiiilbl^ 4201.

[0154] ErnbodimehlsiOllilb^tdsent ipyepijpp!^^ events faster; run more;; dells in less time, -of antigens in cells;;;and;: resolve cell poputatiohSliSibfd|distinctljr|wi:ihlieSSldihbi;pii:fyillThey may provide powerful;; control over sample concentration, flow rate, the number of photons delectable, experiment length, and sample· thi$j§|^ may.reshape the way many current cellular assays|i^||ij|p|ii^ opportunities for creating new cellular assays. It may2' MHz, for example, io position cells into a single focused line along the central axis of a flow channel without high-velocity or high-volume sheath fluid, and may concentrate cells regardless of volume. Acoustic focusing may exploit the physical differences between cells or particles relative io Ihe background medium, allowing cells Io remain lightly focused. The acoustic focusing may concentrate cells in the center of the fluid with sound energy, which creates considerable flexibility in the sample concentration analyzed. More importantly, acoustic focusing may separate the alignment of cells from the particle flow rate, so the flow rate of the cells may be increased or decreased without.disrupting the focus of cells in the capillary. The precision of this adj ustable flow’ rate may help researchers to determine the number of cells analyzed and the amount of time the cells spend in the focused laser beam. Additional features and advantages of acoustic flow' cytometry may be found in Ward et al·., Fundamentals of Acoustic Cytometry, Current Protocols in Cytometry, Supplement 49, 1.11,1 1.22,12 (2009), the entire disclosure of which is incorporated herein by reference.

[0155] in systems using hydrodynamic focusing only, the sample core is ‘‘pinched” by the fast flowing sheath fluid, and the volume of sheath fluid is typically greater than 100 to 1000 times that of the sample flow. Such large ratios lead to low sample input rates, which usually hinders resolution. According to exemplary embodiments of the present invention, however, 30 2015224429 09 Sep 2015 a previously acoustically focused sample may be further focused, hydrodynamically, downstream of the acoustic focusing, the volumetric: ratio between the slveath fluid and the sample fluid may be reduced significantly. For example, that volumetric ratio may be reduced to about 50 to i, 40 to 1., 30 to 1, 20 to 1., 10 to .1, 9 to 1, 8 to 1., 7 to 1,6 to 1, 5 to i, 4 to 1, 3 to 1, or 2 to 1, for example. That volumetric ratio may also be about 1 to 1, I to 2, 1 to 3, J to 4, 1 to 5, 1 to 6, 1 to 7, I to 8, 1 to 9, and J to 10. These numbers are exemplary and other fractional ratios between them may also be used. Preferably, the volumetric ratio between sheath fluid and the sample fluid may be between about 10 to 1 and 1 to 10, or, between about 5 to 1 and 1 to 5. The System may flow' a fluid sample with particles in a sample channel in the capillary at a sample fluid input rate of about 200 μΐ/min to about 1000 μΐ/min and a slveath fluid in a sheath flow channel at a sheath fluid input rate of about 2200 ul/min to about 1400 μΐ/'min, while maintaining a total input rate of sample fluid and sheath fluid constant to ensure that an interrogation time of the particles through one or more interrogating lasers remains constant regardless of the sample fluid input rate. The system may also How the sample fluid at a sample flow rate between about 25 μΙ/min to about 1000 μΐ/min and the sheath fluid at a sheath flow rate between about 2375 (.tl/min to about 1400 μΐ/miii. The system may also How the sample fluid at a sample How rale of at least 200 plan in and the sheath fluid at a sheath fluid flow rate of at most 2200 μΐ/min, while adjusting the volumetric ratio of the sheath fluid to the sample fluid to a ratio between about 11 to 1 and about 1.4 to 1. The system may also flow the sample fluid at a sample flow’ rate of at least 500 μΐ/min and the sheath fluid at a sheath fluid flow rate of at most 1900 μΙ/min, while adjusting the volumetric ratio of the sheath fluid ip the sample fluid to a ratio between about 1.0156] According to an embodiment of the present invention, there is provided a flow cytometer, including (I) a capillary including a sample channel; (2) at least one vibration producing transducer coupled to the capillary, the at least one vibration producing transducer being configured to produce an acoustic signal inducing acoustic radiation pressure within the sample channel to acoustically concentrate particles flowing within a fluid sample stream in the sample channel; and (3) an interrogation source including a violet laser and a blue laser, the violet and blue livsers being configured to interact with at least some of the acoustically concentrated particles to produce an output signal. |0157] In such a flow cytometer, the at least one vibration producing transducer may include a piezoelectric device, the violet laser may have a wavelength of about 405 nanometers, and the blue laser may have a wavelength of about 488 nanometers. Farther, the 111111:31 2015224429 09 Sep 2015 |i|i;llary may.:ii£31::ii:i:iiil'ttS^tWi::::iiC'hiSJrtii'leIi:iCOnfigured, to flow aisSii)llili||SfOun4l the fluid sample stream downstream of the acoustic concentration of (he particles by the acoustic pM:;i;1 ft?IiiiIjp;i;;<2:<3(.Η.ο.οΐϊίΐθ3'ΐ6 the acou^t|||;|::i:||c|hirated::i particles wliihfhiiiilfe;:;;SihhfJ5iitrrhiijisii;i;i;i:iF’hi'tilib:ftitdbe, such a flow cyto|||yi;:||||||iii|thde al iilrst pump:;iiiSlg$ii§^ particles its the sati|i|:;4hl|i||l|hi the capillary at a sample fluid input rate of about 200 microliters per minute to about 1000 mi eroli flow a sheath flui|;i|hSll:l|||i|i|sfl'OW^ channel at a sheath fluid input rate of about 2200 microiiters per minute to about 1400 microliters per minute in the capillary, and the first and second pumps may be configured to maintain a total input rate of sample fluid and sheath fluid flowing in the capillary constant to ensure that an interrogation time of the at least some of the acoustically concentrated particles through the violet and blue lasers remains constant regardless of the sample fluid input rate.

[0158] Such a flow cytometer may also include an optical module to collect the output signal from the interrogation source; a detector module to detect an output signal of the optical module; and a data acquisition module to process an output of the detector module, and it may further include a processor configured to control at least one of the at least one vibration producing transducer, the detector module; and ihc data acquisition module. Further, such a flow cytometer may include a blocker bar between the capillary and the optical to ;a substantially cylindri|||pi 'slut is rotatable to . apcrliure of the :|htputi|i||ia| of the intemjgatiQfi sqnrcediiiptllliiil output signal, of tile inte|rb|iilton source may be: beiw&amp;e^ii:i^ii^|i!!i!|ii:iii:^i|iiigii^ 21 degrees. Furthermore, ifldlbptical module may includlililllitolitlhi! output signal from the inteffdpbion source, and an output of the collection lens may be split into two beams with a spatial tillering pinhole deviddl^idreiti a fimt::|d|hil|;|dt||l|rom the violet laser and a second beam is output from the iillllitser. And, thniay:|||)ude detectors to i|d|ect a forward scatter signalsand a side scatter signal from the: first beamoutput by the violet laser.

[0159] s According to another embodiment of the present invention, there is provided a flow cytometer, including (1) a a sample fluidlhcluding particles to fldwj|h|fein; (2) a first focu:sih|diddh!lhism ephfi|Ifed to acoustically; focus at least some of tilpi||ilicles in the sample fTtiii|liij::!:j^ the capiliaiyf;||:) a second focusing bfdbhlhism configured to hydiidfhhmicaliy i|pdhil|he sample fluidiilpluding the at least some acoustically focused particles in a· second region· within lire capillary downstream of the firsl region; (4) an interrogation zone in or downstream of the capillary through which at least 111112 2015224429 09 Sep 2015 :;;^^;^!:ii^^:::iS^:;::^0^^tica!!y|^^:::hydrodyTOam_seraIly focused particles can fiowiltifi (5) at least iieteci Sfiffeast one signal obtained at the ijifoifogatioii zone .aco'usticia.ily arid hydmdynamieally focused papitiles.

[0160] mlyllso include a sample fluid pump configured to flow a sample fluid inib||i|:dSpilaiy at alSSniple flow fate between about 25 mi#blifei|: per minute to about 1000 mforoljfors/per mibhfoiiand a sheath fluid pump configuredltplflb^w a sheath fluid into the capillary at a shllfhlfibw rate between about 2375 micrQlfd!!!!!!:minute to about|l|i0 microlilers per nlliiie|||iurther, the first focusing meehanis.fo:|iia|:s|0^ toflbclliilleast some of tlbiadOlltically focisdl? particles in the first fo|foiii|bl|ili:ngle file di|d;ibiifh|ifrOiri- the firS:fpppi:fo:the secotii;::rd|ion, and the saniple;;|:Ui|i|illshd|th fluid a total t^fe of sample fluid and :^hdaiilflhidlip^tng in that an :i:|ii||j|||ation time of thd||||i||||||||h0||f the jaiii^iii^iiiii^lfe^ 1 ly fbcusedi|dtif|lds through the inffi|b|atfofo fobhstant reg^die:S|foii;l|f' sample flow ::t^td|lli|:u;0h a flow oytofo;ei$i^^ i: sample fluid' pti|i|iid|fiiured to flow a||i|t|l0i:||lid into the capj!fi$i|| between about 200 microlilers per minute to about 1000 microlilers per minute and a sheath fluid pump configured to flow a sheath fluid into the capillary at a shealh flow rate between about 2200 microlilers per minute to about 1400 microliters per minute.

[0161] According···to another embodiment of the. present invention, there is provided a method for delecting a rare event using a flow cytometer, including: (1) flowing a sample flujd including particles into a channel; (2). acoustically focusing al least some of Ihe particles in the sample fluid in a first region, contained within the channel by applying acoustic radiation pressure to the first region; (3) hydrodynamically focusing the sample fluid including the at. least some acoustically focused particles by flowing a sheath fluid around the sample fluid in a second region downstream of Hie first region; (4) adjusti ng a volumetric ratio of the sheath fluid to the sample fluid lo maintain a substantially constant overall particle velocity in an interrogation zone in or downistream of the second region; (5) analyzing at least some of the acoustically and hydrodynarnically focused particles in the interrogation zone; and (6) detecting one or more rare events based on at least one signal detected at the interrogation zone, the one or more rare events being selected from the group consisting of one or more rare fluorescence events, one or more rare ceil types, and one or more dead cells.

[0162] Such a method may also include flowing the sample fluid at a sample flow rate of al least 200 microliters per minute and the sheath fluid at a shealh fluid flow rate of at most 311111111111111111: 2015224429 09 Sep 2015 2200 microliters per minute, and adjusting the volumetric ratio of the sheath fluid to flic sample fluid may include adjusting the volumetric ratio of the sheath fluid to the sample fluid to a ratio between about 1.1 to 1 and about 1.4 to 1. Furl tier, such a method may include flowing the sample fluid at a sample flow rate of at least 500 microliters per minute and the sheath fluid at a sheath fluid flow rale of at most 1900 microiilcrs per minute, and adjusting the volumetric ratio of the sheath fluid Id the sample fluid may include adjusting the volumetric ratio of the sheath fluid to the sample fluid to a ratio between about 3.8 to 1 and about 1.4 1o 1. Furthermore, the method may include ensuring that a transit lime of the acoustically and hydrodyimmtcally focused particles through the interrogation zone exceeds about 20 microseconds, or ensuring that a transit time of the acoustically and hydrodynamically focused particles through the interrogation zone exceeds about 40 microseconds.

[0163] According to another exemplary embodiment of the invention, there is provided a computer readable medium including computer readable instructions, which, when executed by a computer in or in communication with an acoustic flow cytometry apparatus, control the apparatus to: (1) flow a sample fluid including particles into a channel; (2) acoustically focus al least some of the particles in flic sample fluid in a first region contained within the channel by applying acoustic radiation pressure to the first region; i3.> hydrodynamically focus the focused particles bv·;i;i;i:έ»;fsht;e;:3:th::: fluid around the sample fluid in a second region downstream of die first region; (4) adjust a fodhilibtfiii the ti. to· maintain a s iilbiitailMiilfyr':fi ovefhl|pa|p:ie velocity or downstream of analyze at least some of focused particles.....in.....the

Interrogation zone; and. (6)idbfb§|i:0^ events based on Ifilbip bhb|si|hi]| detected at the interrogation. events being· selects^ consisting of one or more ra^iiiiijSiaeitr^ one or more rare ce||j||es, and one or more dead cells, [0164] S. rch gillhildter .also control· the· ap|afS<us to flow the paiftple fluid at|ii|fipbpow rat|ii$|::|||basf:$ per minuteilidlfhc sheath fluid illlisheath al mieroihers per minute, and to adjust the volumetric llffolblithi! the sampillfluid bpldjlsting the vo1um.etn|j||fio of the sheath ratio between, about 11 to 1. and about 1.4 tbs!!! Further, such a may also controlltHbli^pparalus to flow thb|sgmplg|fluid at a microliters peilfomlte and the sheath, i|hl|iat afsleath fluid a4lll!!!l! 2015224429 09 Sep 2015 flow rate of at most 1900 microlitcrs per minute, and to adjust the volumetric ratio of the sheath fluid to the sample fluid by adjusting the volumetric ratio of the sheath fluid to the sample fluid to a ratio between about 3.8 to I and about 1.4 to 1. Furthermore, the computer readable medium may also control the apparatus to ensure that a transit time of the acoustically and hydrodynamically focused particles through the interrogation /.one exceeds about 20 microseconds, or to ensure that a transit lime of the acoustically and hydrodynamically focused particles through the interrogation zone exceeds about 40 microseconds. )0165) According to an embodiment of the present invention, there is provided an apparatus including (1) a capillary including a channel; (2) at least one vibration source coupled to the capillary, the at least one vibration source being configured to apply vibration to the channel: and (3) an interrogation source including a 405 nm laser, the interrogation source being -configured to have.an output that interacts with one or more particles flowing in tire capillary. The interrogation source may further include a 488 nm laser. The vibration source may include a piezoelectric material. The vibration source may be configured to produce an acoustic signal inducing acoustic radiation pressure within the channel, which may concentrate a plurality of selected particles within a fluid sample stream in die channel, and the capillary may include a sheath flow channel to hydrodynamically concentrate the selected particles within the fluid sample stream.

[0166] According to another embodiment of the present invention, there is provided a system including (T) a capillary having a channel; (2) at least one vibration producing transducer coupled to the capillary, the at least one vibration producing transducer being configured to produce an acoustic signal inducing acoustic radiation pressure within the channel, wherein the acoustic radiation pressure concentrates a plurality of selected particles Within a fluid sample stream in the channel; (3) an interrogation source including a 405 nm laser, the interrogation source being configured to have an output that interacts with at least some of the selected particles to produce an output signal; (4) an optical module to collect the output signal from the interrogation source; (5) a detector module to detect the output signal of the optical module; ami (6) a data acquisition module to process an output of the detector module. The vibration producing transducer may include a piezoelectric device. The interrogation source may further include a 488 nm laser, and both the 405 nm laser and the 488 mri laser may interrogate at least some of the selected particles. The capillary' may include at least One sheath flow channel, and the sheath flow channel may include a sheath fluid 1o hydrodynamically concentrate the selected particles within the fluid sample stream. 35 I.........,.,,, 2015224429 09 Sep 2015

The system may include a processor configured to control at least one of the vibration producing transducer, the detector module, and the data acquisition module, it may also include a blocker bar between the capillary and the optical module, :ah||the bloc|i|:|h|:mafi be attached to a substantially cylindricll|p|g, iVhibhlhihy be rotatable td:positioi|]|ilBidbkeb bar and adjust an output aperture of tIi^!:iiEi>iiiiitp'uliis:jj!j^^ interrogation sourceillilielbuipuf aperture of the output signal of the may be about I90|||i||?d|i|ioaf module may include a collection lensjji col|edj||hl|bifput signal from the interrogation source, and an output of the collectioitlens iip£i|||b||i|iit into two beamslpitila splial filtering pinhole device, wherein a first beam ikjbbtjpfllrqm the 405 nm la^|ihh|ia seedhd beam is output from the 488 nm laser. The dete|{d?fiip|lf|imay include detebfoikfo detect a forward scatter signal and a side scatter signal: nm laser.

The system may include a pump that moves a sample fluid front a reservoir to the capillary along a sample flow path, which may incIndie niapiibe configured to input the sample fluid into the capillary at a sample input rate of about 200 μΐ per minute to about 1000 ul per minute. The system may also include an imager for imaging the particles in the fluid sample stream. .... (0167 j According to another embodiment of the present invention, there is provided a flow cytometry system including (1) a first pump configured to flow a sample fluid including particles in a first channel in a capillar)'; (2) a piezoelectric device configured to produce acoustic radiation pressure in a planar direction to acoustically focus the particles in the first channel; (3) a second pump configured to flow a sheath fluid in a second planar direction in a second channel in the capillary to hydrodynamiealiy focus the particles in the second planar direction and further focus the particles: (4) art interrogation source, wherein an output of the interrogation source outputs a first light beam from a 405 nm laser and a second light beam from a 488 nm laser, and. wherein the first and the second light beams interact with at least some of the particles flowing in the capillary' to produce an output signal; (5) an optical module configured to collect the output signal from the interrogation source; (6) a detector module configured to detect an output signal of the opt ical module; and (7) a data acquisition module configured to process an output of the detector module.

[0168] According to another embodiment of the present invention, there is provided a method for defecting a rare event using a flow cytometer including (1.) .flowing a sample including panicles in a flow channel at a flow rate between about 25 μΐ per minute to about l OOu ul per minute: (2) acoustically focusing at least some of the. particles in the sample in a firsl region contained within the flow channel; (3) hydrodynamiealiy focusing the sample 36 2015224429 09 Sep 2015 including the at least some acoustically focused particles in a second region downstream of the first region; and (4) detecting a rare event based on al least one signal delected at an interrogation; zone through which at [east some of the acoustically and hydrodynamicaily focused particles are allowed to flow. The method may include flowing the sample at a flow rate of at least 200 μΐ per minute, or at a How rate of at least 500 μΙ per minute. It may further include ensuring that a transit time of the acoustically and hydrodynainicaily focused particles through the interrogation zone exceeds about 20 microseconds, or exceeds about 40 microseconds. And it may include detecting a rare fluorescence event, detecting one or more cells of a rare cell type, and/or detecting one or more dead cells.

[0169] According to another exemplary embodiment of the invention, there is provided a computer loadable medium including computer readable instructions, which, when executed by a computer in or in communication with an acoustic flow' cytometry apparatus, control the apparatus to: (1) flow a sample including, particles in a flow channel at a flow rate between abou t 25 μϊ per minute to about 1000 μΐ per minute; (2) acoustically focus at least some of the particles in flic sample in a first region contained within the flow channel; (3) hydrodynamicaily focus the sample including the al least some acoustically focused particles in a second region downstream of the first, region; mid (4 ) detect a rare event based on al least one signal detected at an interrogation zone through which at least some of the acoustically and hydrodynamicaily focused particles are allowed to flow. The computer readable medium may also control the apparatus to flow the sample at a flow rate of at least 200 μϊ per minute, or al a flow rate of at. least 5O0 μϊ per minute. It may further control the apparatus to ensure that a transit time of the acoustically and hydrodynamicaily focused particles through the interrogation zone exceeds about 20 microseconds, or exceeds about 40: microseconds. And it may control the apparatus to defect a rare fluorescence event, detect One or more cells of a rare cell typb|ahd/b|:dllict one |0170j According to another embodiment of the present invention, there is provided a method for flow cytomelry. including (1) flowing a sample fluid including.particles into a fluid channel; (2) acoustically focusing the particles in a first region of the fluid channel; -(3) flowing a sheath fluid into a second region of the fluid channel downstream of the first region to hydrodynamicaily further focus the acoustically focused particles; (4) adjusting the volumetric ratio of sample fluid to sheath fluid to maintain a substantially - constant overall particle velocity' in the second region; and (5) analyzing the particles in the second region. [0171J According to another exemplary' embodiment of the invention, Lhorc is provided a computer readable medium including computer readable instructions, which* when executed 2015224429 09 Sep 2015 bllliComputeliidr in c0ii:^|:blbSt}:b|i:|V:ith an acoustic flow cytometry apparatus,:control:the:< appfratus tp;lp|::£iaw a particles into a fluid channel; (2) |c|«S|ica||f i focus the particles in a first region of the fluid channel; (3) flow a sheath fluid into a second region, of:t|dll|jd diandSlidf'ii^ first region to hydrodynamically further :fbc»s: the acoustically^ the volumetric ratio of sample flipdltdlslllatii::: fluid to maintain a substantially constant overall particle velocity in the second region; and (5) analyf$i;;t|§::))article$^ (0172] Examples of rare events or rare event particles that may be present or detected in or using one or more of tfrti::iaiBciyte:::::iitncnts of the present invention include:stem: Cells:.|ph|l:|ype), t-etramers, NET cells, Mdilltiilifi; hdi|bti:iii|ilcciis,. deaisCililllllslPth signatures, etc,, and mof|tp|di|liy: iliiilf. any idenii$||i§iiiiclll of particles or cells ha|ni|iC|ii£t:ii idehfifiC|:::§haracteristie^ihiif^ present only in a small fraction of the tiifiipes or celli^::;i|iiiii:iil^i:isi|ii|ii^ fraciiosn will, of course, d:epeii||bii|l|e particular cells or particles for a given problem or application, l or example, a rare identified population could represent particles or cells representing about 5% of the total number of particles or cells, or about 2,5%, or about 1%, or about 0.1%, or about 0.05%, or about 0.01%. These values arc exemplary only and other values between any (wo of them are also :|P|i|lcl|||f|:i|lsp smallebyaiues, , (0173] Examples of assaying suitable for use in or with one or more of the above exemplary embodiments of the present invention include antigen or ligand density measurement, apoptosis analysis, cell cycle studies, cell proliferation assaying, cell, sorting, chromosome analysis, DNA/RNA content analysis, drug uptake and efflux assaying, enzyme activity assaying, fluorescent protein detection, gene expression or transfection assaying, immunopbenotyping, membrane potential analysis, metabolic studies, multiplex bead analysis, nuclear staining detection, reticulocyte and platelet analysis, stem cell analysis, and viability and cytotoxicity assaying, (0174] Examples of media formulations suitable for use in or with one or more of the above exemplary embodiments- of the present invention include amidplnsioutc; cesium chloride with a non-ionic surfactant such as Pluronic® 1’68; compounds that contribute to high viscosity (e.g., glycerol, dextran, nanosilica coated with polyvinylpyrrolidone)- in some applications; diatrizoate; glycerol; heavy7 salts such as cesium chloride or potassium bromide; iodinaled compounds; iodixanol; iopamidol; ioxaglalc; mclrizamidc: metri/Oate: nanoparticulate material such as polymer coated silica; Nycodenz®; polydexlran; 38 2015224429 09 Sep 2015 polysucrosc; saline buffer; saline buffered with protein, detergents,, or other additives; sails and proteins combined with additives used to increase specific gravity without undue increase in salinity; and suctosc.

[0175] Examples of probes suitable for use in or with one or more of the above exemplary embodiments of the present invention include dyes including BFP,· bioluminescent and/or chemiluminescent -substances, C>dots, Ca' Vaequorin, dye-loaded nanospheres, phycoerylhrin and fluorescein, fluorescein/terbiurn complex used in conjunction with plain fluorescein, fluorescent proteins, labels with extinction coefficient less than 25,000 cm'lM‘1 (e>g., Alexa Fluor® 4()5 and 430, APC-C7) and/or quantum efficiency less than 25% (6,g., ruthenium, Cy3), lanthanides, lanthanide chelates (especially those using europium and terbium), lanthanide tandem dves, l.RET probes, hicifcrm/lueilerasQ, metal-ligand complexes, microbe-specific probes, naturally occurring fluorescent species such as NAP(P)H, nucleic acid probes, phosphors, phoiobleach-suseeptiblc or triplet state prone dyes (c,g„ blue fluorescent protein), phycoery thrill tandem dyes, probes prone to non-radialive state excitation (e.g,, Rhodamine Atto532 and GFP), probes resistant to photobieaehing at laser power exceeding about 50,000/cm', probes with lifetimes greater than 10 nanoseconds, Qdol® products, •Qdot® tandem probes, Raman scattering probes, semiconductor nanocrystala, tandem probes, terbium complexes, terbium fluorescein complex, and up-convcrting phosphors.

[0176] Examples of secondary reagents suitable for use in or with one or more of the above exemplary embodiments of the present invention include secondary reagents using ligands such as biotin, prolcin A. and G, secondary antibodies, streptavidin, violet exciled dyes conjugated to antibodies or other iigand.s. (including such as Pacific Blue™ or Pacific Orange™ cory ugated to or profeiff and Qdot® products or semiconductor nanocrystals format (e.g., as streptavidin conj ugalea).

[1)177] One or more of the various exemplary li^|^^iidi|Si^Sj^i! present inventiorl described above may be used with many types samples: (especially when particles of interest are f£ij§jjjiitdf normai||lli||iiire significant concentrations). For example, they may be us|||t||i||pcess or'hMipejjniicrobes from;: /municipal waters, specific nucleic acid probes dji||bt|e|: microbe 11?/;beili/i:sifftiljap:i llierobelfesting in various food products indudil;|j|hii§, milk, beefliiflllli^^ §|p^td|di)yironmental and industrial 'analytes )||ijipeagents such. i^'iily^eltl||iiibape and size of particles where im|ibr|hpfih certain· i:ndiAS4.riiii:;:i: /iilclipbdhbbbn for copters and. printers an.dl|pili||: control in ch£t$i!i§f$i:| 391111111111 concentrate and/or remove particles from waste streams or feed streams; to extend the life of certain filters; to remove metal, ceramic, or other particulates from machining fluids or particulates from spent oils such as motor oils and cooking oils, etc. 2015224429 09 Sep 2015 [0178] Any of the methods above can be automated with a processor and a database. A computer readable medium containing instructions may cause a program in a data processing medium (e.g., a computing system) to perform any one or more steps described in the above exemplary embodiments.

[0179] The preceding exemplary embodiments may be repeated with similar success by adding or substituting the generically or specifically described components and/or substances and/or steps and/or operating conditions described above in the preceding exemplary embodiments. Although the invention has been described in detail with particular reference to the above exemplary embodiments, other embodiments are also possible and within the scope of the present invention. Variations and modifications of the present invention will be apparent to those skilled in the art from consideration of the specification and figures and practice of the invention described in the specification and figures.

[0180] Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof. 40

Claims (20)

1. The claims defining the invention are as follows:

   1. A flow cytometer, comprising: a capillary comprising a sample channel; at least one vibration producing transducer coupled to the capillary, the at least one vibration producing transducer being configured to produce an acoustic signal inducing acoustic radiation pressure within the sample channel to acoustically concentrate particles flowing within a fluid sample stream in the sample channel; an interrogation source configured to interact with at least some of the acoustically concentrated particles to produce an output signal; a first pump configured to flow a fluid sample comprising particles in the sample channel; and a second pump configured to flow a sheath fluid in the sheath flow channel; wherein the first and second pumps are configured to maintain a total input rate of sample fluid and sheath fluid flowing in the capillary constant and flow the sample fluid into the capillary at a sample flow rate of at least 25 microliters per minute to 1000 microliters per minute.
2. 2. The flow cytometer of claim 1, wherein the sample flow rate is at least 200 microliters per minute to 1000 microliters per minute.
3. 3. The flow cytometer of claim 1 or 2, wherein the at least one vibration producing transducer comprises a piezoelectric device.
4. 4. The flow cytometer of any one of claims 1 to 3, wherein the sheath flow channel is configured to flow a sheath fluid around the fluid sample stream downstream of the acoustic concentration of the particles by the acoustic radiation pressure to hydrodynamically concentrate the acoustically concentrated particles within the fluid sample stream.
5. 5. The flow cytometer of any one of claims 1 to 4, comprising: an optical module to collect the output signal from the interrogation source; a detector module to detect an output signal of the optical module; and a data acquisition module to process an output of the detector module.
6. 6. The flow cytometer of claim 5, comprising a processor configured to control at least one of the at least one vibration producing transducer, the detector module, and the data acquisition module.
7. 7. The flow cytometer of claim 5 or 6, comprising a blocker bar between the capillary and the optical module.
8. 8. The flow cytometer of claim 7, wherein the blocker bar is attached to a substantially cylindrical peg that is rotatable to position the blocker bar and adjust an output aperture of the output signal of the interrogation source.
9. 9. The flow cytometer of claim 8, wherein the output aperture of the output signal of the interrogation source is between about 17 degrees and about 21 degrees.
10. 10. The flow cytometer of any one of claims 5 to 9, wherein the optical module comprises a collection lens to collect the output signal from the interrogation source, and wherein an output of the collection lens is split into two beams with a spatial filtering pinhole device.
11. 11. The flow cytometer of any one of claims 5 to 10, wherein the detector module comprises detectors to detect a forward scatter signal and a side scatter signal.
12. 12. A flow cytometer, comprising: a capillary configured to allow a sample fluid including particles to flow therein; a first focusing mechanism configured to acoustically focus at least some of the particles in the sample fluid in a first region within the capillary; a second focusing mechanism configured to hydrodynamically focus the sample fluid including the at least some acoustically focused particles in a second region within the capillary downstream of the first region; an interrogation zone in or downstream of the capillary through which at least some of the acoustically and hydrodynamically focused particles can flow; at least one detector configured to detect at least one signal obtained at the interrogation zone regarding at least some of the acoustically and hydrodynamically focused particles; and a sample fluid pump configured to flow a sample fluid into the capillary; and a sheath fluid pump configured to flow a sheath fluid into the capillary, wherein the first and second pumps are configured to maintain a total input rate of sample fluid and sheath fluid flowing in the capillary constant and flow the sample fluid into the capillary at a sample flow rate of at least 25 microliters per minute to 1000 microliters per minute.
13. 13. The flow cytometer of claim 12, wherein the first focusing mechanism is configured to focus at least some of the acoustically focused particles in the first region to a single file line flowing from the first region to the second region, and wherein the sample fluid and sheath fluid pumps are configured to maintain a total rate of sample fluid and sheath fluid flowing in the capillary constant to ensure that an interrogation time of the at least some of the acoustically and hydrodynamically focused particles through the interrogation zone remains constant regardless of the sample flow rate.
14. 14. The flow cytometer of claim 12, comprising a sample fluid pump configured to flow the sample fluid into the capillary at a sample flow rate between about 200 microliters per minute to about 1000 microliters per minute.
15. 15. The flow cytometer of any one of claims 12 to 14 comprises a sheath fluid pump configured to flow a sheath fluid into the capillary at a sheath flow rate between about 2200 microliters per minute to about 1400 microliters per minute.
16. 16. A method for detecting a rare event using a flow cytometer, comprising: flowing a sample fluid including particles into a channel; acoustically focusing at least some of the particles in the sample fluid in a first region contained within the channel by applying acoustic radiation pressure to the first region; hydrodynamically focusing the sample fluid comprising the at least some acoustically focused particles by flowing a sheath fluid around the sample fluid in a second region downstream of the first region; adjusting a volumetric ratio of the sheath fluid to the sample fluid to maintain a substantially constant overall particle velocity in an interrogation zone in or downstream of the second region; analyzing at least some of the acoustically and hydrodynamically focused particles in the interrogation zone; and detecting one or more rare events based on at least one signal detected at the interrogation zone, the one or more rare events being selected from the group consisting of one or more rare fluorescence events, one or more rare cell types, and one or more dead cells, wherein the system is configured to flow the sample fluid at a flow rate of 25 microliters per minute to 1000 microliters per minute.
17. 17. The method of claim 16 wherein the sample fluid flow rate is 200 microliters per minute to 1000 microliters per minute.
18. 18. The method of claim 16 or 17, comprising flowing the sample fluid at a sample flow rate of at least 200 microliters per minute and the sheath fluid at a sheath fluid flow rate of at most 2200 microliters per minute, and wherein adjusting the volumetric ratio of the sheath fluid to the sample fluid includes adjusting the volumetric ratio of the sheath fluid to the sample fluid to a ratio between about 1 1 to 1 and about 1.4 to 1.
19. 19. The method of any one of claims 16 to 18, comprising ensuring that a transit time of the acoustically and hydrodynamically focused particles through the interrogation zone exceeds about 20 microseconds.
20. 20. The method of any one of claims 16 to 18, comprising ensuring that a transit time of the acoustically and hydrodynamically focused particles through the interrogation zone exceeds about 40 microseconds.