US007355696B2
`
`(12)
`
`United States Patent
`Mueth et a].
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 7,355,696 B2
`Apr. 8, 2008
`
`(54) METHOD AND APPARATUS FOR SORTING
`CELLS
`
`3,960,449 A *
`4,395,397 A *
`
`6/1976 Carleton et a1. .......... .. 356/340
`7/1983 Shapiro .......... ..
`424/577
`
`4,660,971 A *
`
`4/1987 Sage et a1. . . . . .
`
`. . . .. 356/39
`
`(75) Inventors: Daniel Mueth, Chicago, IL (US); Amy
`$393???’ Plrl°Spl§CtI§e1%htS’IL
`.
`’ m "P "it
`'
`“'1 son’
`clncagos IL (Us), JosePh Plewa, Park
`Rldge, IL (US)
`_
`_
`(73) Asslgneei ArryX, II1¢,Ch1CagO,IL (Us)
`
`-
`
`,
`
`,
`
`i i
`5,138,181 A *
`5,180,065 A *
`5,194,909 A *
`5,483,469 A *
`5,707,808 A *
`5,879,625 A *
`
`. . . ..
`
`e
`
`.
`
`. . . . . . .
`
`230596037;
`giélkltztlal
`250/573
`8/1992 Lefevre et a1.
`209/577
`1/1993 Touge et a1. .... ..
`3/1993 Tycko ....................... .. 356/40
`1/1996 Van den Engh et a1. .... .. 702/21
`1/1998 Roslaniec et a1. ........... .. 435/6
`3/1999 Roslaniec et a1.
`422/50
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U_S_C_ 154(b) by 0 day,
`
`5,985,216 A * 11/1999 Rens et a1. . . . . . . .
`
`. . . .. 422/73
`
`2/2003 Seidel et a1. ................ .. 436/63
`6,524,860 B1 *
`2002/0176069 A1* 11/2002 Hansen et a1. .............. .. 356/73
`2006/0152707 Al* 7/2006 Kanda ....................... .. 356/73
`
`(21) Appl. NO.Z 11/046,896
`_
`(22) Flled?
`
`Feb- 13 2005
`
`(65)
`
`Prior Publication Data
`Us 2006/0170912 A1
`Aug. 3, 2006
`
`(51) Int CL
`(200601)
`G01N21/01
`(52) us. Cl. ...................... .. 356/244; 356/246; 356/39;
`209/31
`3 5 6 /2 4 4
`(58) Field of Classi?cation Search
`356/246 72 73 39 40 336'"éé'9"_"'ég0/222 PC’
`’_
`T ’
`T ’
`_
`T
`’
`_
`’
`250/461'2’ 436/10’ 63’ 18’ 4222/g92éiggg/s674li
`See a lication ?le for Com 1e te Search histo ’
`pp
`p
`ry'
`References Cited
`
`(56)
`
`US. PATENT DOCUMENTS
`
`* .
`
`.
`
`“ted by exammer
`Primary ExamineriSang H. Nguyen
`(74) Attorney, Agent, or Firmilean C. Edwards, ESQ;
`Akerman Senter?tt
`
`(57)
`
`ABSTRACT
`
`Apparatus for sorting and orienting sperm cells has a pair or
`Walls 1n confrontmg re1at1onsh1p formmg a How chamber
`haYing inlet’ 2,‘ downstr‘aiam Outlet’ and intermediate detector
`reglon. The mlet rece1ves ?rst and second spaced apart
`streams of input ?uid and a third stream of sample ?uid
`containing the cells to be sorted. The ?rst and second
`streams have respective flow rates relative to third stream,
`such that the third stream is constricted forming a relatively
`narroW sample stream, so that the cells are oriented parallel
`to the Walls. A detector detect desired cells and a sorter
`downstream of the detector for sorting the desired cells from
`the stream.
`
`3,649,829 A *
`
`3/1972 Randolph ................. .. 250/364
`
`22 Claims, 10 Drawing Sheets
`
`322 322 324
`324
`
`322
`
`a00\
`312
`
`322
`
`/
`%
`X
`
`)
`
`340
`
`318
`
`322
`
`346
`
`0 /Z'
`31422)
`____g :1 /‘3_28__
`316\_/—\l—w—:l\‘o
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`314
`
`33s\:/—\__<F — —
`326
`T3463 /34s
`A ___ 4:? __ A —i——-E .
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`L )
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`342
`
`344
`
`01 T i W T
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`33a
`81
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`i :
`: f
`
`356
`
`332/ 33(4) \ 0
`
`334R 332
`
`3
`
`32°
`
`

`
`U.S. Patent
`
`Apr. 8, 2008
`
`Sheet 1 0f 10
`
`US 7,355,696 B2
`
`100 \/\
`
`Collection
`
`101 0 Extension
`
`Slow Cooling
`7
`102 w to~6°C
`
`103 w’ Gender Sorting
`
`'
`104 w Slow Cooling
`to 4°C and Settling
`‘
`105 \/\[ Final Extension
`
`.
`.
`106 \/\[ Packlng |n Straws
`
`\/\
`107
`
`Settllng
`'
`
`108 v1 Freezing
`
`Concentration
`109
`Testing TV
`
`Motility &
`f Morphology ~11 10
`Testing
`
`Loading into
`Disposable Chip
`
`“a 200
`
`7 Passive Filtration to
`Remove Large Yolk
`A
`ggregates
`
`Flow-Based
`% Alignment
`
`Parallelized Gender
`Detection
`
`Gender
`Discrimination
`
`Gender-Based
`Actuation
`
`l
`Passive
`Concentration
`Balancing
`
`Delivery into
`Output Reservoir
`
`’\1 207
`
`

`
`U.S. Patent
`
`Apr. 8,2008
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`Sheet 2 of 10
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`US 7,355,696 B2
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`

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`U.S. Patent
`
`Apr. 8,2008
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`Sheet 3 of 10
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`US 7,355,696 B2
`
`FIG. 3
`
`326
`
`328
`
`326
`
`
`Input
`Object
`
`Solution
`
`
`
`

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`U.S. Patent
`
`Apr. 8, 2008
`
`Sheet 4 0f 10
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`US 7,355,696 B2
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`? n [J This:
`
`FIG. 4A
`
`Input Sheath Fluid
`SF
`Input Sheath Fluid
`
`SF
`
`SF
`
`SF
`
`SF
`
`

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`U.S. Patent
`
`Apr. 8, 2008
`
`Sheet 5 0f 10
`
`US 7,355,696 B2
`
`680L
`
`6 6
`
`6|\A1
`
`6
`6 \
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`L\AI/v 6 I\
`
`0 6 6
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`L 6 4 6
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`llllll 4|
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`C 6 7 6
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`2 7 6/
`
`m 6 L eIA
`
`C 0 8 6
`
`FIG. 5A
`
`FIG. 5C
`
`FIG. 5D
`
`666R 674B
`
`2
`
`

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`U.S. Patent
`
`Apr. 8, 2008
`
`Sheet 6 6f 10
`
`US 7,355,696 B2
`
`“60%
`
`$8 968 ..... -- - - ..... -
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`08b
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`8% a8 8% Emma
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`V SQIV/
`88 \/\
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`com
`
`

`
`U.S. Patent
`
`Apr. 8,2008
`
`Sheet 7 of 10
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`US 7,355,696 B2
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`83
`
`______=________________________
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`___________=____________________r52
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`

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`U.S. Patent
`
`Apr. 8, 2008
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`Sheet 8 0f 10
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`US 7,355,696 B2
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`FIG. 8
`
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`FIG. 9
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`930
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`932
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`HE D
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`(9L U_
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`90C :1 H 0::
`
`

`
`U.S. Patent
`
`Apr. 8,2008
`
`US 7,355,696 B2
`
`FIG. 10A
`
`FIG. 108
`
`II
`
`934\\
`<5
`E)
`s1MWFs2
`Using Two Actuators on 81, S2
`
`Using Three Actuators on M, W, F
`
`FIG. 10C
`
`FIG. 10D
`
`6
`
`s1 M W F s2
`Using One Actuator on 81
`
`s1MWFs2
`Using Two Actuators on M, F
`
`

`
`U.S. Patent
`
`Apr. 8, 2008
`
`Sheet 10 0f 10
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`US 7,355,696 B2
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`F I G. 1 1 A
`
`940
`
`/’~- Optical Interrogation Region
`
`Laser
`
`—/l'-\\__/ 942
`
`Focusing Laser j
`
`[
`
`81 C S2
`Using Laser Killing or Activation
`
`/ Optical Interrogation Region
`/‘
`
`Electrode & Wire
`/ > (
`/\\
`
`944
`
`942
`
`l
`
`s1 0 32
`Using Electrical Killing or Activation
`
`

`
`1
`METHOD AND APPARATUS FOR SORTING
`CELLS
`
`2
`SEPARATION WITH LASER STEERING”, the teachings
`of both, which are herein incorporated by reference.
`
`US 7,355,696 B2
`
`20
`
`25
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`30
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`35
`
`The invention pertains to a ?ow sorter employing a
`multi-angular discriminating detection and imaging system,
`for sorting cells. Another aspect of the invention pertains to
`a method and apparatus for optical detection and for imaging
`of objects.
`Known imaging systems tend to be azimuthally symmet
`ric, accepting light within a certain range of angles estab
`lished by the numerical aperture, (NA) of the imaging
`system. All light coming from the object plane within the
`NA is ideally transferred to the imaging plane uniformly, in
`the absence of aberrations or vignetting by optics or aper
`tures which are too small. The reason for this design is that
`it is desirable to have a reasonably high light collection
`e?iciency (i.e. a high NA) and the angular variations in
`intensity often do not carry important information.
`An exemplary imaging system is a single round lens or a
`pair of round lenses. For cases where high collection e?i
`ciency is desired, such as in imaging systems which are dim,
`a high NA system is designed by using an optical element
`which is large compared to the size of the object, and which
`is close compared to its size. In this way, the lens captures
`a large fraction of the light. High NA is also important for
`maximizing resolution and for obtaining a narrow focal
`depth.
`Flow cytometers are devices which use optical scattering
`and ?uorescence to discriminate between cells or other small
`objects such as ?uorescent beads, and to sort them based
`upon the discriminated optical measurements. As objects
`stream through a narrow jet, input laser light scatters,
`impinging on the objects, and incites ?uorescence. Scattered
`and ?uorescent light signals are detected at varying angles to
`characterize and discriminate objects with differing proper
`ties.
`One di?iculty in gender sorting sperm is the very ?at
`shape of sperm, especially bovine sperm. The ?at shape,
`combined with the higher index of refraction of DNA
`relative to the aqueous environment, causes lensing of light
`40
`and internal re?ection, including ?uorescent light which
`originates in the sperm head. This lensing causes light to be
`emitted preferentially through the edges of the sperm, with
`much lower emission through the two ?at faces of the sperm
`head. Thus, detection of light intensity and determination of
`X orY chromosomal content of the sperm is dependent upon
`reliable alignment of the sperm and the ability to view the
`sperm ?uorescence from multiple angles.
`Known alignment systems employ a device in which
`sperm cells are oriented and sprayed into a detection zone by
`means of a nozzle such as illustrated in Rens et al., US. Pat.
`No. 5,985,216. In such a device, the sorting nozzle has an
`elliptical cross section for orienting ?attened cells. A disad
`vantage of Rens is that if the ?ow rate is above about 5000
`sperm cells per second, the cells can not be reliably imaged
`and characterized. A 5000 sperm cells per second sperm ?ow
`rate is ine?icient and time consuming. A more practical rate
`for sperm sorting is around 100,000 sperm cells per second
`or higher.
`One type of imaging system used to manipulate small
`particles is described in US. patent application Ser. No.
`10/974,976, entitled “SYSTEM AND METHOD FOR
`MANIPULATING AND PROCESSING NANOMATERI
`ALS USING HOLOGRAPHIC OPTICAL TRAPPING”,
`?led Oct. 28, 2004, and in US. patent application Ser. No.
`10/934,597, ?led Sep. 3, 2004, entitle “MULTIPLE LAMI
`NAR FLOW-BASED PARTICLE AND CELLULAR
`
`50
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`55
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`60
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`65
`
`SUMMARY OF THE INVENTION
`
`The present invention pertains to a ?ow sorter employing
`a multi-angular discriminating detection and imaging sys
`tem, for sorting cells. One aspect of the invention pertains to
`a method and apparatus for optical detection and for imaging
`of objects. In particular, the present invention is directed to
`a method and apparatus for characterizing and sorting
`bovine sperm by gender. However, it should be understood
`that other types of mammalian sperm cells and the like may
`be sorted by using the present invention.
`The present invention is based upon the discovery that a
`?ow sorter for sorting and orienting cells employs a ?ow
`channel having an inlet, an outlet, an intermediate detection
`zone, and optionally, a sorting region. The inlet receives one
`each of alternating spaced streams of input sheath ?uid and
`a sample stream containing the cells to be sorted between
`sheath streams. The sheath streams and the sample stream
`have respective ?ow rate or pressures in the ?ow chamber
`such that the sample stream is constricted thereby forming a
`relatively narrow sample stream in the detection region
`whereby the cells are oriented in a selected direction relative
`to the input light. A detector employing a multi angle or
`K-Vector imaging setup is focused in the detection zone for
`discriminating between desired and undesired cells.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a ?ow diagram of steps for sorting cells which
`have been dyed with a ?uorescent dye.
`FIG. 2 is a perspective schematic illustration of a ?ow
`device or cartridge according to the invention.
`FIG. 3 is a schematic illustration of a single channel ?ow
`device for sorting.
`FIG. 4 is a side-view of a multichannel ?ow device for
`sorting.
`FIG. 4A is a top plan (edge) view, facing the inputs, of the
`multichannel system shown in FIG. 4.
`FIG. 5 is a schematic representation of a single channel
`sensor for sensing scattered light (i.e., optical system for
`K-vector imaging).
`FIGS. 5A-5D illustrate alternative optical elements
`employed in the arrangement of FIG. 5.
`FIG. 6 is a schematic representation of a single channel
`sensor for detecting ?uorescent and scattered light using
`K-vector imaging.
`FIG. 7 is a multichannel channel device for K-vector
`imaging with excitation and detection of scattered and
`?uorescent light.
`FIG. 8 is a schematic representation of an external actua
`tor adjusting ?ow speeds in a channel or device for sorting
`cells.
`FIG. 9 is a schematic representation of a top view of a
`multichannel device or cartridge for detecting and sorting
`cells.
`FIGS. 10A-10D show various alternative ways of steering
`cells using three actuators on M, W, F (FIG. 10A), two
`actuators on S1, S2 (FIG. 10B), one actuator on S1 (FIG.
`10C), and two actuators on M, F (FIG. 10D).
`FIGS. 11A-11B show various alternative ways of killing
`cells by laser killing or activation (FIG. 11A), and electrical
`killing or activation (FIG. 11B).
`
`

`
`US 7,355,696 B2
`
`3
`DESCRIPTION OF THE INVENTION
`
`FIG. 1. illustrates a ?oW diagram setting forth the steps for
`characterizing, sorting and processing objects, for cryogenic
`preservation, particularly bovine sperm cells.
`The ?rst stage from collection 100, extension 101, to sloW
`cooling 102, is the subject of various procedures, some of
`Which are novel and others of Which are knoWn.
`A novel system for preparing cells for sorting is set forth
`in copending US. patent application Serial Number (to be
`assigned), entitled: “Novel Method For In Vivo Staining of
`Cells for Identi?cation and Sorting”, ?led on Feb. 1, 2005,
`the teachings of Which are incorporated herein by reference.
`The steps include loading a sample into a disposable chip
`200; ?ltering the sample to remove large aggregate material
`201, such as yolk aggregates; employing ?oW based align
`ment 202 as set forth hereinafter; employing parallelized
`gender detection 203, discrimination (i.e., gender discrimi
`nation 204) and actuation (i.e., gender actuation 205) steps;
`passive concentration and balancing 206, and delivery to an
`output reservoir 207. The method may also optionally elimi
`nate some steps and include a discrimination and killing step
`for removing unWanted live sperm.
`The gender sorting steps 103 Which includes the above,
`are then folloWed by sloW cooling to 40 C. and settling 104;
`?nal extension 105; packing in straWs 106; settling 107 and
`freezing 108 steps.
`FIGS. 2-3 illustrate in various forms a ?oW cytometer 300
`according to the invention. The device may be a single
`channel system. HoWever, the invention is Well suited for
`multichannel applications, particularly in sperm cell sorting
`applications, Where large numbers of sperm cells must be
`sorted in a reasonable amount of time.
`In FIGS. 2-3, the device 300 comprises a body or ?oW
`chamber 312 formed of a pair of confronting Walls 314 and
`end Walls 316 (only one of Which is shoWn in FIG. 1), an
`open top or input 318 and open bottom or output 320.
`The input 318 is divided into three sections including
`outboard inputs 322 and central or sample input 324. The
`outboard inputs 322 are for receiving a sheath ?uid 326
`therein and the central input 324 is for receiving a sample
`?uid 328 containing a liquid medium and cells 330 dispersed
`therein.
`The output 320 has outboard output sections 332 and
`central sample collection channels, namely left output
`sample channel 334L, central output sample channel 334C
`and right output sample channel 334R. Channel 334L is for
`a ?rst sorted sample, 334C is for a second sorted sample, and
`334L is for yet another sorted sample.
`Sheath ?uid 326 is input at outboard inputs 322 at a
`selected ?oW rate. Sample ?uid 326 is introduced in central
`input 324 at a selected ?oW rate or pressure relative to the
`sheath ?oW rate or pressure such that the sheath ?uids
`compress and constrict the sample ?oW to a relatively
`narroW sample ?oW path 336 as shoWn. In an exemplary
`embodiment, the Width of the sample ?oW path 336 is about
`10% or less of the Width of the sample ?uid at the central
`input 324, eg about 50 microns.
`The cells 330 are circular but ?attened. As a result,
`constriction of the sample ?uid causes the cells 330 to orient
`themselves so that their ?at sides are roughly parallel to the
`confronting Walls 314. The intensity of light radiated by a
`cell is different at different orientations. So to compare the
`intensity of tWo or more cells, they must have the same
`orientation. Thus, aligned cells reduce noise or systematic
`error caused by having anisotropic light emitter at random
`orientations.
`
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`4
`Alternating inputs of sheath ?uid 326 and input sample or
`object solution 328 (see FIG. 3) enter the system, creating a
`small amount of constriction (relative to the constriction in
`the orthogonal direction) 401, Which causes shear and the
`shear ?oW aligns the cells 400. This alternating ?oW pattern
`is squeezed more severely betWeen tWo long input sheath
`?uid ?oWs, Which achieves the necessary alignment. This
`arrangement may be combined With detection hereinafter to
`provide a parallel system Where multiple ?oWs may be
`interrogated.
`Speci?cally, the constricting ?oW moves objects into the
`focal plane 402, and accelerates movement through the
`detection region 403. The curve 404 in the system shoWs the
`?uid boundary 404, and the detection region 405 alloWs for
`characterization. The light cone 406 alloWs for interrogation,
`and the default stream position 407 can be steered betWeen
`multiple outlets.
`By varying the ?oW rate through the three output channels
`409-411, cells or other objects in the solution can be sorted
`into one of multiple output streams. The actuation may be
`done in various Ways, as enumerated above. High-speed
`?oW sWitching may be performed by piezo devices Which
`may be intrinsic to the machine, or intrinsic to the disposable
`?oW channel cartridge. FloW sWitching region 408 controls
`the precise ?oW rate, Which varies over time to sWitch
`betWeen output channels 409-411 (Where V2<V1, and
`V4~v2).
`The detector 340 (FIG. 2) comprises a laser 342 or other
`suitable source producing an output beam 344 directed
`toWards the sample ?oW path 336 in a detection zone 338
`intermediate the input and output. The beam 344 impinges
`on the cell 330 in the detection zone 338 and scatters
`forming an output beam 346. The cell contains a ?uophor
`and thus produces ?uorescent light as Well Which is con
`tained in the output beam 346. Respective scattered and
`?uorescent components 346S, 346E of the output beam 346
`are input to an optical detector containing an optical system
`348 and an electronic detector system 350. The optical
`system and electronic detector system are discussed herein
`after.
`The output beam 346 carries information to the detector
`340 Which discriminates among the cells 330 and produces
`an output 354 to a sorter 356. The sorter 356 controls drivers
`358 in operative relationship With the output channels 334 in
`order to vary the relative ?oW rates such that the each cell
`330 is sorted into a proper channel. Alternatively, the cells
`may be sorted as Wanted or unWanted, and the Wanted cells
`may be collected and the unWanted cells may be destroyed.
`FIG. 3 shoWs a single channel system having only tWo
`sheath ?oWs 326 and one sample ?oW 328. FIG. 4 shoWs a
`multi-channel system With four sample ?oWs 328 and shared
`sheath ?oWs 326. FIG. 4A shoWs the ?oW chamber and ?oW
`paths in top plan.
`FIGS. 4-4A shoW one possible Way of parallelizing the
`design to have many parallel streams 500 of input solution.
`FloW may constrict both in the plane and normal of the
`plane. HoWever, for cases Where alignment of cells is
`necessary, such as With bovine sperm, shear along the
`direction of the incoming light must be much larger to
`guarantee alignment normal to the incoming light. The
`optical investigation region 502 is Where laser scattering and
`?uorescence takes place.
`In the example, each sheath has a dedicated lens system,
`multiple PMT elements each of Which is dedicated to a
`corresponding stream. It should be understood that the
`system may have one lens detector system for all channels
`
`

`
`US 7,355,696 B2
`
`5
`and one laser optic system for all channels as Well. There are
`multiple possible output channels 503 for each ?oW stream.
`A simpli?ed plan vieW of the optical system of the
`detector 640 is shoWn in FIG. 5. A sample 660 lies in an
`object plane 662. In this illustration output beam or, light
`rays 646 emanate from the sample 660 as beamlets 646C,
`646L, 646R. The beamlets 646C represent centrally clus
`tered beams near the central optical axis C; and beamlets
`646L and 646R are clustered to the left and right of the
`central axis C. The beamlets provide different vieWs of the
`sample 660. A collection lens 664 (Which may be an
`objective lens from a microscope) is positioned a distance
`from the object plane 662 Which is equal to the e?fective
`focal length (EFL) of the lens. The lens 664 may be a
`compound lens if desired, but for simplicity it is described
`it as a single lens With an EFL. By positioning lens 664 thus,
`light 646 from the object exits the collection lens 664 is
`collimated as beamlets 666 likeWise divided as 666R, 666C,
`666L as shoWn. This creates an in?nity space in the imaging
`system. While the present invention does not require this
`in?nity space, it is a convenient arrangement. The lateral
`positioning of each collimated light ray is determined pre
`dominantly by its angle coming from the object plane.
`Beamlets 666L, 666R and 666C folloW respective beamlets
`6421, 642R and 642C.
`Central light beamlets 666C exit lens 664 along central
`axis C to focusing lens 676C Which focuses the light on
`forWard image plane 678C. Mirrors 672L, 672R separate
`o?-axis beamlets 666L and 666R exiting the collecting lens
`664. Note that this may also be done With the placement of
`detector 674A (FIG. 5A0 or optical ?ber 674B (FIG. 5B) in
`this region Which are small compared to the size of the beam
`in this space. Note also that additional optical elements may
`be inserted in this space, such as additional lenses 674C
`(FIG. 5C) or a pinhole 674D (FIG. 5D) to constrain the
`range of angles of light coming out of the collecting lens
`664, or to control (restart or expand) the focal depth from
`Which light is collected as in confocal microscopy measure
`ments. In some circumstances, a pair of additional lenses,
`With a pinhole, may be used. In other cases, a mask With
`controllable siZe shape, or position may be used to control
`the light reaching a given detector.
`The light from beamlets 666L is de?ected by mirror 672L
`to left focusing lens 676L; and from beamlet 666R light is
`directed by mirror 672R to lens 676R and right image plane
`678R. Light detectors 680L, 680R and 680C may be located
`in respective image focal planes 678L, 678R and 670C to
`detect the respective images. These light detectors may be
`CCD, photo diodes, photomultiplier tubes, multi-anode pho
`tomultiplier tubes, or other sensors.
`In many cases, it is desirable to collect both scattered light
`and ?uorescent light, Where at least one of the images or
`detections made require a reduced range of ray angles from
`the sample. FIG. 6 illustrates hoW this may be done, using
`the K Vector Imaging setup as described above, but With
`?lters and additional beam splitters as necessary. Similar
`elements have the same reference numerals.
`In FIG. 6, illumination and excitation light 800 passes
`through collecting lens 801, and is re?ected by mirrors 802,
`803 through emission ?lters 688EL, 688ER to focusing
`lenses 804, 805 and to photo detectors 690L, 690R, respec
`tively, Which form the left and right ?uorescent image
`planes.
`The beamsplitter in 686C redirects light in the central
`?eld 666C through an emission ?lter 688E to focusing lens
`676FC. The output 689E of emission ?lter 688E corresponds
`to ?uorescence emission from the cell. Laser line ?lter 688L
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6
`in the central optical axis ?lters scattered laser light to lens
`676C and photo detector 690C, Where is formed he forWard
`scattered image plane.
`To improve the throughput and overall capabilities of a
`device, paralleliZation is desired. FIG. 7 shoWs hoW this is
`accomplished. Laser 642 produces input laser beam 644,
`Which excites ?uorescence, is broken up into N multiple
`beams by beamsplitter 690 (e.g., diffraction grating or
`hologram). Beams 692A and 692N fan out and each are
`directed to the detection Zone of a corresponding sample
`sheath (see FIG. 4). The beamlets 692A-692N are colli
`mated into parallel beams by the collimating lens 694. The
`output 696 of the collimating lens 694, is directed through
`cylindrical lens 698 Which focuses individual beams 700A
`700N onto the sample streams in the object plane 701. As
`shoWn, (FIG. 4) the input stream is broken up into many
`streams. Light from one or all beamlets 700A-700N in the
`example shoWn and sample streams enters collecting lens
`702K and is handled as described in FIG. 6 above. Here,
`hoWever, detector array devices (such as multi-anode PMTs)
`device 720 (720C, 720R, 720L, 720CF .
`.
`. ) one for each
`group of detection beams are used. Detection is best accom
`plished not by placing a single detector in each imaging
`position, but by using array detectors, such as a 32-element
`linear PMT array 720.
`The throughput of a system Which images or make
`measurements on many objects Will depend, in part, upon
`the number of detectors and their speed. For many applica
`tions, a single detector such as photomultiplier tube (PMT)
`is used in each image plane. This is suitable for cases Where
`objects pass through the object plane in a single-?le line.
`The sorter 856 is hereinafter described in detail. A single
`channel sorter is shoWn in FIG. 8. The sorter 856 comprises
`a structural member 812; a channel de?ning layer 816
`formed With a slot 818 de?ning a ?oW channel 820; a
`?exible membrane layer 822 atop the channel de?ning layer
`816, and a structural member 824 completing the arrange
`ment. Piezoelectric or another type actuator 826 is coupled
`to a controller 828. A piston 860 driven by actuator 826
`engaged ?exible membrane 822 opposite the ?oW channel
`820 to change the ?oW pattern Within the channel in accor
`dance With the voltage supply to the pieZo-electric. A
`plurality of such structures may be miniaturiZed so as to be
`located in the actuator WindoW Whereby multiple streams of
`samples may be sorted.
`The one or more actuators 826 may include: a pieZo
`electric transducer for converting electrical signals into
`mechanical actuation of the ?oW rates; a thermal heater for
`heating a region to quickly expand a ?uid, material, or
`bubble; a thermal bubble generation for creation of a bubble
`to reduce the ?oW of the solution; a capacitive motion device
`for a membrane; an optical device for heating or moving
`material, Wall, membrane, bubble, or other material or object
`to impact the ?oW velocity into one or more of the output
`channels 934. The actuation may be intrinsic to the device or
`may be externally applied. For example, the actuator 826
`and piston 860 may be external equipment, separate from the
`disposable ?oW device 856 (i.e., disposable chip With non
`disposable/extemal actuator).
`FIG. 9 illustrates the parallel arrangement of multiple
`channels in Which input channels 900 feed the sheath
`streams to the ?oW channels and output channels 901
`receive the various sorted sperm cells. WindoW 930 is
`provided for coupling interrogation light to each of the
`parallel detector sample channels. Sorter WindoW 932
`
`

`
`US 7,355,696 B2
`
`7
`receives a plurality of actuators and disposable sorter ele
`ments. Chip registration pins of the device 100 are desig
`nated as 940.
`FIGS. 10A-10R illustrate alternative embodiments for
`steering cells, employing a variety of actuators 934 in a ?oW
`device for sorting sperm. In each case, one or more actuators
`are used, Where each actuator could be based off of pieZo
`electric devices (intrinsic or extrinsic to the cartridge),
`capacitive actuators, thermal expansion, or other technolo
`gies as described in the prior disclosures. In most cases, at
`least tWo actuators 934 are desired, and possibly more than
`tWo actuators, to control Which exit channel 936 a particular
`cell or object goes into. The actuators alloW one to control
`and vary the ?oWs at very high rates. Only minor perturba
`tions to the ?oW are necessary to cause a stream to tempo
`rarily move from one exit to another.
`FIGS. 11A-11B shoW examples of killing or activation
`setups Without sorting for achieving a desirable result. In the
`FIG. 11A, a laser 940 impinges the obj ect/ sperm stream 942.
`This laser is controlled to only impinge lethal energy on
`certain sperm or objects depending upon the result of the
`interrogation. For example, this laser may kill certain sperm
`or other cells as desired. For example, it may kill all sperm
`of a given or uncertain gender. Alternately, the laser may not
`directly kill the cells but may otherWise “activate” them. For
`example, it could activate some chemical Which has been
`previously introduced into the sperm, having the overall
`result of killing them or otherWise impairing fertilization. In
`more general applications, activation may have a much
`broader range of activities.
`FIG. 11B shoWs killing or activation using lethal electri
`cal pulses, introduced through electrodes 944 Which are
`inserted into the ?oW in order to locally access the central
`stream 942. As With the laser solution, the electrodes can be
`quickly pulsed on or off depending upon the result of the
`interrogation.
`Steering may also be achieved optically Where the cells
`are manipulated by an optical trapping apparatus. Alterna
`tively, the actuation process may be electroporation of the
`cells, Which may be lethal or have other effect on the cells.
`The folloWing technique aligns sperm cells using squeeZ
`ing ?oW:
`Three ?oWs Were fed into a ?oW chip using a peristaltic
`pump. Each ?oWs Were kept in laminar regime so that each
`?oW does not mix each other. The stream containing sperms
`?oWs betWeen top and bottom streams Which are Waters.
`While the velocity of top and bottom ?oW is kept same, by
`changing the ratio of those to the sperm ?oW, We could see
`the squeezing of sperm ?oW.
`
`sperm orientation (%)
`angle from ?oW direction (degree)
`
`.066
`1.35
`2.13
`
`51.35
`61.11
`66.67
`
`29.73
`14.81
`16.67
`
`13.51
`22.22
`16.67
`
`5.41
`1.85
`0
`
`20
`
`25
`
`35
`
`40
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`45
`
`50
`
`60
`
`It is expected that the squeeZed ?oW helps the sperm
`oriented to the ?oW direction.
`Images of the sperm in ?oW Were taken using a CCD
`camera equipped on our microscope.
`Above table shoWs the degree of sperm orientation in the
`?oW Where Re is the Reynolds number de?ned as Dur/m
`
`8
`Where D is diameter of ?oW channel, U is the speed, r is
`density of ?uid and m is the viscosity.
`Re indicates Whether the ?oW is laminar or not, even
`though Re beloW 1000 is considered laminar ?oW, in some
`applications, very small Re such as beloW 1 is required.
`As the speed of sperm ?oW increases the Re in the channel
`inlet increases but still remains in laminar region indicating
`the ?oW stream is not disturbed in our experimental region.
`The orientation of sperm Was quanti?ed by numbering of
`those as function of degree alignment of sperm head to ?oW
`direction.
`In the experiment range, about 80% of sperms imaged
`Were oriented in less than 15 degree to ?oW direction.
`Better alignment to ?oW direction Was shoWn at higher
`speed but more disturbed sperms Were also found.
`The results shoWs that the system could align sperms
`using this technique.
`It should be understood that temperature control of the
`sheath ?uid and sample ?uid can be employed to prolong
`sperm life. In an exemplary, embodiment the temperature of
`the ?uids in the ?oW device may be maintained around 2-10o
`C. in order immobiliZe the sperm cells and thereby extend
`their lifetime.
`
`The invention claimed is:
`1. A ?oW apparatus for orienting, examining and selec
`tively operating on cells comprising:
`a ?oW chamber having confronting Walls and an input
`region, a detector region, and a selective operation
`region;
`Wherein said ?oW chamber includes a plurality of inlets
`into said input region, each of said inlets adapted to
`receive an input stream of ?uid;
`Wherein said input streams of ?uid are one of an input
`sheath ?uid or an input sample ?uid containing cells to
`be processed;
`Wherein said input sample ?uid is in a contiguous rela
`tionship on all available sides With said input sheath
`?uid from at least said input region through to said
`selective operation region;
`Wherein at least one of ?oW rates or pressures of said input
`sheath ?uids are chosen such that said input sample
`?uid is constricted in tWo orthogonal directions,
`thereby alloWing said input sample ?uid to form a
`relatively narroW stream in at least