(12) United States Patent
`US 7,118,676 B2
`(10) Patent No.:
`Mueth et al.
`(45) Date of Patent:
`Oct. 10, 2006
`
`US007118676B2
`
`(54) MULTIPLE LAMINAR FLOW-BASED
`PARTICLE AND CELLULAR SEPARATION
`WITH LASER STEERING
`
`(75)
`
`Inventors: Daniel Mueth, Chicago, IL (US);
`Joseph Plewa, Park Ridge, IL (US);
`Jessica Shireman, Kansas City, MO
`(US); Amy Anderson, Prospect
`Heights, IL (US); Lewis Gruber,
`Chicago, IL (US); Neil Harris
`Rosenbaum, Chicago, IL (US)
`
`(73) Assignee: Arryx, Inc., Chicago, IL (US)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 10/934,597
`
`(22)
`
`Filed:
`
`Sep. 3, 2004
`
`(65)
`
`Prior Publication Data
`
`US 2005/0121604 A1
`
`Jun. 9, 2005
`
`Related US. Application Data
`
`(63) Continuation-in-part of application No. 10/867,328,
`filed on Jun. 13, 2004.
`
`(60) Provisional application No. 60/571,141, filed on May
`14, 2004, provisional application No. 60/511,458,
`filed on Oct. 15, 2003, provisional application No.
`60/499,957, filed on Sep. 4, 2003.
`
`(51)
`
`Int. Cl.
`(2006.01)
`B01D 21/01
`(52) U.S.Cl.
`...................... 210/732;210/800;210/802;
`210/927; 435/173.1; 422/72; 422/8205; 422/101;
`436/177
`
`(58) Field of Classification Search ................ 250/251;
`210/732, 800, 802, 927; 435/173.1, 72, 82.05,
`435/101; 436/177
`See application file for complete search history.
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`............... 210/732
`4,409,106 A * 10/1983 Furuta et al.
`4,424,132 A *
`1/1984 Iriguchi
`...................... 210/800
`5,849,178 A
`12/1998 Holm et al.
`
`* cited by examiner
`
`Primary ExamineriKiet T. Nguyen
`(74) Attorney, Agent, or Firmilean C. Edwards, Esq.;
`Akerman Senterfitt
`
`(57)
`
`ABSTRACT
`
`The invention provides a method, apparatus and system for
`separating blood and other types of cellular components, and
`can be combined with holographic optical trapping manipu-
`lation or other forms of optical
`tweezing. One of the
`exemplary methods includes providing a first flow having a
`plurality of blood components; providing a second flow;
`contacting the first flow with the second flow to provide a
`first separation region; and differentially sedimenting a first
`blood cellular component of the plurality of blood compo-
`nents into the second flow while concurrently maintaining a
`second blood cellular component of the plurality of blood
`components in the first flow. The second flow having the first
`blood cellular component
`is then differentially removed
`from the first flow having the second blood cellular com-
`ponent. Holographic optical traps may also be utilized in
`conjunction with the various flows to move selected com-
`ponents from one flow to another, as part of or in addition
`to a separation stage.
`
`1 Claim, 22 Drawing Sheets
`
`2630
`
`2M
`
`3535
`
`INPUT
`RESERVOIR (S)
`
`TEMPERATURE CONTROL
`AND MONITORING
`
`
`
`l
`
`
`PUMP (S)
`
`OUTPUT
`RESERVOIR(S)
`
`251°‘\—USER INPUT
`
`
`
`Exhibit No. 1018
`
`PGR of US. Patent 8,933,395
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 1 of 22
`
`US 7,118,676 B2
`
`
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 2 of 22
`
`US 7,118,676 B2
`
`._<QE%NUHE<ESDI m.mHm
`
`35:
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 3 of 22
`
`US 7,118,676 B2
`
`<zw<._n_
`
`.mmdzi m.
`
`.93
`
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 4 of 22
`
`US 7,118,676 B2
`
`FIG. 4
`
`THGIL
`1LENNAMn”C
`
`THGIL
`CHANNEL 3
`
`00000000000
`
`.0000000000.
`
`00000000000
`
`.0000000000.
`
`00000000000
`
`.0000000000.
`
`00000000000
`
`.0000000000I
`
`00000000000
`
`.0000000000.
`
`\..0000000000.
`
`00000000000
`
`ooooooooooo
`
`LIGHT
`CHANNEL 2
`
`
`
`

`

`Oct. 10, 2006
`
`Sheet 5 of 22
`
`US 7,118,676 B2
`
`MQMHmmmoz
`
`GZHH<HOE
`
`mom
`
`
`
`mzm;m>HHQmsmo
`
`mgmz<m
`
`UHzm<mwogoI
`
`
`
`QZHmm<mHA<QHH¢O
`
`kHz:
`
`“Hm
`
`mug
`
`<¢mz<u
`
`mwpzmon
`
`cam
`
`mom
`
`A<UHH¢D
`
`mmmHm
`
`5..U.mat
`
`Pgm
`«onmomm222223:m388%:
`
`l'r::.oom
`
`
`
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 6 of 22
`
`US 7,118,676 B2
`
`LIIIIIMDIIIP’
`
`500
`
`I: [13.
`
`till
`
`PWWMAFMMFwVMHMAPwmuw
`OF COMPONENTS
`
`PROVIDE A SECOND FLOM
`
`WWUMHWHWWM%flWHW
`TO PROVIDE A FIRST SEPARATION REGION
`
`DIFFERENTIALLV SEDIMENT A FIRST COMPONENT
`OF A PLURALITV 0F COMPONENTS FROM THE FIRST
`FLOW INTO THE SECOND FLOV
`
`CONCURRENTLY MAINTAIN A SECOND COMPONENT OF THE
`PLURALITV 0F COMPONENTS IN THE FIRST FLOV
`
`DIFFERENTIALLY REMOVE THE SECOND FLOW HAVING
`THE FIRST COMPONENT FROM THE FIRST FLOW HAVING
`THE SECOND COMPONENT
`
`5%
`
`510
`
`m
`
`520
`
`535
`
`530
`
`835
`
`
` YES
`ADDITIONAL
`SEPARATION
`
`
`
`
`PROVIDE A THIRD FLOW
`
`540
`
`N0
`
`0
`
`CONTACT THE FIRST FLOH MITH THE THIRD FLOW
`
`545
`
`TO PROVIDE A SECOND SEPARATION RECION
`
` HOLOGRAPHIC
`
`0 NO
`YES 0
`MANIPULATION 0R
`
`SEPARATION?
`
`
`
`550
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 7 of 22
`
`US 7,118,676 B2
`
`FIG. BB
`
`GENERATE A PLURALITY
`OF HOLOGRAPHIC TRAPS
`
`USING THE HOLOGRAPHIC
`TRAPS. DIFFERENTIALLY MOVING
`THE SECOND COMPONENT FROM
`
`THE FIRST FLOW INTO THE THIRD FLOW
`
`555
`
`EEO
`
`555
`
`DIFFERENTIALLY
`SEDIMENTING THE
`SECOND COMPONENT
`FROM THE FIRST
`FLOW INTO THE
`THIRD FLOW
`
`CONCURRENTLY MAINTAINING A THIRD COMPONENT
`OF THE PLURALITY OF COMPONENTS IN THE
`FIRST FLOW
`
`DIFFERENTIALLY REMOVING THE THIRD FLOW
`HAVING THE SECOND COMPONENT FROM THE
`FIRST FLOW HAVING THE THIRD COMPONENT
`
`570
`
`575
`
`RETURN
`
`530
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 8 of 22
`
`US 7,118,676 B2
`
`FIG.
`
`2A
`
`701
`
`700
`
`705
`
`FIG.
`
`7B
`
`701
`
`710
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 9 of 22
`
`US 7,118,676 B2
`
`FIG. 8
`
`PHOTO IMAGE
`
`FIG. 9
`
`PHO T0 IMAGE
`
`FIG. 10
`
`PHO T0 IMAGE
`
`

`

`U.S. Patent
`
`()ct 10,2006
`
`Sheet10 0f22
`
`US 7,118,676 B2
`
`g:
`
`mo:
`
`3:No:x\
`
`
`
`ma“.mHmqu.mHm
`
`EEK
`/
`
`0000.0fifififi§¢¢##
`
`g:g:
`
`g:.\\
`\booooooooo
`
`
`
`2:....fi,9A.»“.9
`
`«It!»
`fi
`if
`{(-
`{1-
`a
`
`00000000..
`
`0000000000
`
`0.00.00...
`
`oooooooooo
`
`oooooooooo
`
`oooooooooo
`
`fimmmfinaqun
`
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 11 0f 22
`
`US 7,118,676 B2
`
`FIG. 12
`
`IMAGING AND TRAPPING SYSTEM
`
`CONTROL AND
`ANALYSIS SYSTEM
`
`1202
`
`SAMPLE DISTRIBUTION DISK
`
`4/
`
`1204
`
`
`
`1200
`
`
`
`
`
`1203
`
`"
`
`
`
`SAMPLE
`COLLECTION 1305
`CHAMBERS
`
`1205
`
`SAMPLE DELIVERY SYSTEM
`
`SAMPLE INTAKE
`
`1201
`
`FIG. 13
`
`
`
`CHANNEL
`
`<:>
`
`

`

`U.S. Patent
`
`63%225%
`
`
`
`022%$3g32:052a? 522$.“‘W1.225%”ism5:22$;m,:2ngas;gamea225mg
`
`I.
`5%52;.22:38;
`
`22%”m53%mH525%xm20:38
`
`855mm53¢meEmmamSQZH
`
`
`
`20Hhagomonpsgcm
`
`was,as;
`
`
`
`.20:3875:3833n.5%5%
`
`Sheet 12 0f 22
`
`US 7,118,676 B2
`
`Eng288m
`
`a:was,
`
`2.:xE23oZHEomx.mzzfia
`Izmwwm£waxxzazmém3o:
`
`wo<¢mzm
`
`
`
`mzzsaI.-EZHas;E;1!gwas
`
`
`
`Im_mom
`
`
`
`£22.292235
`
`
`5;;3:22BEG@2585;;@2322
`
`
`
` E5?mzHEow NIIILJ\ll|[)
`
`
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 13 of 22
`
`US 7,118,676 B2
`
`FIG. 15
`
`fi 333%
`5:
`
`SOLUTION
`
`INPUT
`
`r —L--------
`sownou E --
`SELECTED
`
`WASTE
`
`—‘
`
`i:
`
`SOLUTION
`
`.AMULTI-CHANNEL
`PERISTALTIC PUMP
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 14 of 22
`
`US 7,118,676 B2
`
`FIG. 18
`
`DIVIDERS
`(e.g. COVER GLASS)
`
` WASTE
`
`SYRINGE
`NEEDLES
`0” TUBING
`
`1500
`
`WALL
`MATERIALS
`
`CHANuEEEB§§ENING
`
`EEENON
`DEPIH
`
`FIG. 17
`
`DIVIDERS
`
`
`2 TUBES OR
`_i
`NEEDLES
`
`(ABOVE EACH
`OTHER)
`
` WALL MATERIAL
`150°
`L+-—--REGION
`
`SORTING
`
`SORTING
`REGION
`WIDTH
`
`LENGTH
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 15 of 22
`
`US 7,118,676 B2
`
`F3113.
`
`.léi
`
`INPUT
`SOLUTION
`
`WASTE
`SOLUTION
`
`
`ONE OF MANY
`
`BUFFER
`HIGH-ASPECT-RATIO
`SOLUTION
`FLAT SORTERS
`IN DEVICE
`
`
`iillllli§lliiilllll"llllilllIIIIIIIEEEIIIE.
`‘TN—Tk
`
`BUFFER
`CHANNEL
`
`
`
`SELECTION
`CHANNEL
`
`
`
`BUFFER
`
`
`
`SELECTION
`CHANNEL
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 16 of 22
`
`US 7,118,676 B2
`
`FIG. 20
`
` —f
`
`SELECTION
`FLOW
`DEPTH
`
`WASTE FLOW
`REGION DEPTH
`
`
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 17 of 22
`
`US 7,118,676 B2
`
`FIG. 22
`
`2001
`
`2000
`
`
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 18 of 22
`
`US 7,118,676 B2
`
`FIG. 24A
`
`SUPPORT LAYER
`
`INPUTS
`
`LAYER 1
`
`LAYER 2
`
`LAYER 3
`
`4' OUTPUTS
`
`
`
`SUPPORT LAYER
`
`

`

`U.S. Patent
`
`US 7,118,676 B2
`
`m5::
`
`3%
`
`
`-_-"‘"‘ll
`_."‘”‘“I
`Alu‘u‘“.
`ilfl‘u‘“.7."..fl‘"-
`
`E“.
`
`m$5:Im5:;H$2..
`
`mvm.mHm
`
`
`
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 20 0f 22
`
`US 7,118,676 B2
`
`:E%omaflmm->:dm<§
`
`x8535‘;
`
`25%5222-85mdw$34362;
`
`fim”$355
`
`
`
`mm..mHm
`
`
`
`
`
`mfizfizmmmm#2826
`
`*2i322;
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 21 of 22
`
`US 7,118,676 B2
`
`FIG.
`
`25‘
`
`2830
`
`2535
`
`
`PUMP(S)
`
`
`OUTPUT
`RESERVDIH(S)
`
`
`251°\USER INPUT
`
`
`
`
`
`

`

`U.S. Patent
`
`Oct. 10, 2006
`
`Sheet 22 of 22
`
`US 7,118,676 B2
`
`3U1
`
`l‘I-u.
`CU
`
`ӎk-=r
`
`|"---
`CU
`
`mm.mHm
`
`$tz
`
`928%
`
`
`
`
`

`

`US 7,118,676 B2
`
`1
`MULTIPLE LAMINAR FLOW-BASED
`PARTICLE AND CELLULAR SEPARATION
`WITH LASER STEERING
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS AND PRIORITY CLAIMS
`
`The present invention is a continuation-in-part of Daniel
`M. Mueth et al., US. patent application Ser. No. 10/867,328,
`filed Jun. 13, 2004, entitled “Multiple Laminar Flow-Based
`Rate Zonal Or Isopycnic Separation With Holographic Opti-
`cal Trapping Of Blood Cells And Other Static Components”,
`commonly assigned herewith,
`the contents of which are
`incorporated by reference herein, with priority claimed for
`all commonly disclosed subject matter (the “first related
`application”).
`The present invention is related to Jessica Shireman et al.,
`US. Provisional Patent Application Ser. No. 60/571,141,
`filed May 14, 2004, entitled “System and Method of Sorting
`Blood Cells Using Holographic Laser Steering”, commonly
`assigned herewith, the contents of which are incorporated by
`reference herein, with priority claimed for all commonly
`disclosed subject matter (the “second related application”).
`The present invention is related to and a conversion to a
`full utility application of Daniel M. Mueth, US. patent
`application Ser. No. 60/499,957, filed Sep. 4, 2003, entitled
`“Passive Fluidic Sorter”, commonly assigned herewith, the
`contents of which are incorporated by reference herein, with
`priority claimed for all commonly disclosed subject matter
`(the “third related application”).
`The present invention is related to and a conversion to a
`full utility application of Daniel M. Mueth, US. patent
`application Ser. No. 60/511,458, filed Oct. 15, 2003, entitled
`“Passive Fluidic Sorter”, commonly assigned herewith, the
`contents of which are incorporated by reference herein, with
`priority claimed for all commonly disclosed subject matter
`(the “fourth related application”).
`The present invention is related to Lewis Gruber et al.,
`US. patent application Ser. No. 10/630,904, filed Jul. 31,
`2003, entitled “System and Method of Sorting Materials
`Using Holographic Laser Steering”, commonly assigned
`herewith, the contents of which are incorporated by refer-
`ence herein, with priority claimed for all commonly dis-
`closed subject matter (the “fifth related application”).
`
`FIELD OF THE INVENTION
`
`The present invention relates generally to techniques and
`systems for separation of particulate or cellular materials
`such as blood, semen and other particles or cells into their
`various components and fractions, using multiple laminar
`flows which further may be coupled with laser steering such
`as holographic optical trapping and manipulation.
`
`BACKGROUND OF THE INVENTION
`
`There are several categories of blood cells. Erythrocyte or
`red blood cell (RBC) counts are for women 4.8 million
`cells/pl and men 5.4 million cells/pl. RBCs make up 93% of
`the solid element in blood and about 42% of blood volume.
`
`Platelets are 2 um73 pm in size. They represent 7% of the
`solid elements in blood and about 3% of the blood volume,
`corresponding to about 1.5 to 4><10ll cells per liter. There are
`5 general types of white blood cells (WBCs) or leukocytes
`accounting for about 1.5 to 4><109 cells per liter. The WBCs
`comprise: 50770% Neutrophils (12715 pm in size); 241%
`Eosinophils (12715 pm in size); 0.571% Basophils (9710
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`
`pm in size); 2(L40% Lymphocytes (25% B-cells and 75%
`T—cells) (8710 um in size); and 378% Monocytes (16720 pm
`in size). They comprise 0.16% of the solid elements in the
`blood, and approximately 0.1% of the blood volume corre-
`sponding to around 4 to 12><109 per liter. A subject with an
`infection might have a WBC count as high as 25><109 per
`liter.
`Platelets are the smallest cells in the blood and are
`
`important for releasing proteins into the blood that are
`involved in clotting. Patients with immune diseases that
`cause lower counts (such as cancer,
`leukemia and other
`chemotherapy patients) sometimes need platelet
`transfu-
`sions to prevent their counts from becoming too low. The
`platelet count in adults is normally between 140,0007440,
`000 cells/pl, and this number should not fall below 50,000
`cells/uL because platelets play an integral role in blood
`clotting.
`Blood separation techniques have traditionally employed
`discrete centrifugation processes. More particularly, a cer-
`tain volume of blood is removed from a donor at a particular
`time. That volume of blood is then subjected to different
`levels of centrifugation to provide corresponding blood
`fractions for blood components such as plasma, platelets, red
`blood cells, and white blood cells. This process is discrete,
`rather than continuous, such that if more blood from the
`donor is to be processed, another volume is removed from
`the donor, and the process is repeated.
`The steps in platelet collection are: collection of blood
`from donor: addition of anticoagulant; separation via cen-
`trifugation; return of red cells, leukocytes and plasma to the
`donor. A collection normally contains about 20(L400 ml of
`plasma, which is reduced to avoid incompatibility. This
`collection normally contains about 8 to 8.5><10lo platelets. A
`donor normally gives approximately 10% of his/her platelets
`with no loss in clotting ability, although a larger number of
`platelets could be separated from the blood. These platelets
`must be used within five days of collection.
`Plateletpheresis, called apheresis,
`is a state of the art
`process by which platelets are separated [Haemonetics Com-
`ponent Collection System (CCS) and Multi Component
`System (Multi)(Haemonetics, Braintree, Mass.)]. This auto-
`mated machine separates platelets from blood over a period
`of 1.5 to 2 hours (assuming 10% donation). This process is
`faster than traditional approaches and is completely auto-
`mated and can be used for single or double platelet doses.
`Nevertheless, the process is slow relative to the patience of
`donors and is capable of improvement for the purity of the
`separated platelet fraction.
`Other procedures are also time consuming, often taking
`several hours, particularly when unused blood fractions are
`to be returned to the donor. For example, platelet donation
`make take several hours, as whole blood is removed from
`the donor, fractionated through centrifugation to obtain the
`platelets, and the remaining blood components are then
`injected back into the donor. This centrifugation process is
`also comparatively harsh, also can result in damage to a
`proportion of the harvested cells, effectively reducing the
`usable yield of the blood fractions.
`Other types of separations are also either time consuming
`or cannot process large volumes of material in a timely
`fashion. For example, sperm sorting, in which viable and
`motile sperm are isolated from non-viable or non-motile
`sperm, is often a time-consuming task, with severe volume
`restrictions.
`
`As discussed below in greater detail in describing the
`present invention, manipulations of particles, such as that
`described in the second and fifth related applications, may
`
`

`

`US 7,118,676 B2
`
`3
`also be part of a novel separation technique. One conven-
`tional
`technique in manipulating microscopic objects is
`optical trapping. An accepted description of the effect of
`optical trapping is that tightly focused light, such as light
`focused by a high numerical aperture microscope lens, has
`a steep intensity gradient. Optical traps use the gradient
`forces of a beam of light to trap a particle based on its
`dielectric constant.
`
`To minimize its energy, a particle having a dielectric
`constant higher than the surrounding medium will move to
`the region of an optical trap where the electric field is the
`highest. Particles with at least a slight dielectric constant
`differential with their surroundings are sensitive to this
`gradient and are either attracted to or repelled from the point
`of highest light intensity, that is, to or from the light beam’s
`focal point. In constructing an optical trap, optical gradient
`forces from a single beam of light are employed to manipu-
`late the position of a dielectric particle immersed in a fluid
`medium with a refractive index smaller than that of the
`
`particle, but reflecting, absorbing and low dielectric constant
`particles may also be manipulated.
`The optical gradient force in an optical trap competes with
`radiation pressure which tends to displace the trapped par-
`ticle along the beam axis. An optical trap may be placed
`anywhere within the focal volume of an objective lens by
`appropriately selecting the input beam’s propagation direc-
`tion and degree of collimation. A collimated beam entering
`the back aperture of an objective lens comes to a focus in the
`center of the lens’ focal plane while another beam entering
`at an angle comes to a focus off-center. A slightly diverging
`beam focuses downstream of the focal plane while a con-
`verging beam focuses upstream. Multiple beams entering
`the input pupil of the lens simultaneously each form an
`optical trap in the focal volume at a location determined by
`its angle of incidence. The holographic optical
`trapping
`technique uses a phase modifying diffractive optical element
`to impose the phase pattern for multiple beams onto the
`wavefront of a single input beam, thereby transforming the
`single beam into multiple traps.
`Phase modulation of an input beam is preferred for
`creating optical traps because trapping relies on the inten-
`sities of beams and not on their relative phases. Amplitude
`modulations may divert light away from traps and diminish
`their effectiveness.
`
`When a particle is optically trapped, optical gradient
`forces exerted by the trap exceed other radiation pressures
`arising from scattering and absorption. For a Gaussian
`TEM00 input laser beam, this generally means that the beam
`diameter should substantially coincide with the diameter of
`the entrance pupil. A preferred minimum numerical aperture
`to form a trap is about 0.9 to about 1.0.
`One difliculty in implementing optical trapping technol-
`ogy is that each trap to be generated generally requires its
`own focused beam of light. Many systems of interest require
`multiple optical traps, and several methods have been devel-
`oped to achieve multiple trap configurations. One existing
`method uses a single light beam that is redirected between
`multiple trap locations to “time-share” the beam between
`various traps. However, as the number of traps increases, the
`intervals during which each trap is in its “off” state may
`become long for particles to diffuse away from the trap
`location before the trap is re-energized. All these concerns
`have limited implementations of this method to less than
`about 10 traps per system.
`Another traditional method of creating multi-trap systems
`relies on simultaneously passing multiple beams of light
`through a single high numerical aperture lens. This is done
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`
`by either using multiple lasers or by using one or more beam
`splitters in the beam of a single laser. One problem with this
`technique is that, as the number of traps increases,
`the
`optical system becomes progressively more and more com-
`plex. Because of these problems, the known implementa-
`tions of this method are limited to less than about 5 traps per
`system.
`In a third approach for achieving a multi-trap system, a
`diffractive optical element (DOE) (e.g., a phase shifting
`hologram utilizing either a transmission or a reflection
`geometry) is used to alter a single laser beam’s wavefront.
`This invention is disclosed in US. Pat. No. 6,055,106 to
`Grier et al. The wavefront is altered so that the downstream
`
`laser beam essentially becomes a large number of individual
`laser beams with relative positions and directions of travel
`fixed by the exact nature of the diffractive optical element.
`In effect, the Fourier transform of the DOE produces a set of
`intensity peaks each of which act as an individual trap or
`“tweezer.”
`
`Some implementations of the third approach have used a
`fixed transmission hologram to create between 16 and 400
`individual trapping centers.
`A fixed hologram has been used to demonstrate the
`principle of holographic optical trapping but using a liquid
`crystal grating as the hologram permitted ‘manufacture’ of a
`separate hologram for each new distribution of traps. The
`spatially varying phase modulation imposed on the trapping
`laser by the liquid crystal grating may be easily controlled in
`real
`time by a computer,
`thus permitting a variety of
`dynamic manipulations.
`Other types of traps that may be used to optically trap
`particles include, but are not limited to, optical vortices,
`optical bottles, optical rotators and light cages. An optical
`vortex produces a gradient surrounding an area of zero
`electric field which is useful to manipulate particles with
`dielectric constants lower than the surrounding medium or
`which are reflective, or other types of particles which are
`repelled by an optical trap. To minimize its energy, such a
`particle will move to the region where the electric field is the
`lowest, namely the zero electric field area at the focal point
`of an appropriately shaped laser beam. The optical vortex
`provides an area of zero electric field much like the hole in
`a doughnut (toroid). The optical gradient is radial with the
`highest electric field at the circumference of the doughnut.
`The optical vortex detains a small particle within the hole of
`the doughnut. The detention is accomplished by slipping the
`vortex over the small particle along the line of zero electric
`field.
`
`The optical bottle differs from an optical vortex in that it
`has a zero electric field only at the focus and a non-zero
`electric field in all other directions surrounding the focus, at
`an end of the vortex. An optical bottle may be useful in
`trapping atoms and nanoclusters which may be too small or
`too absorptive to trap with an optical vortex or optical
`tweezers. (See J. Arlt and M. J. Padgett. “Generation of a
`beam with a dark focus surrounded by regions of higher
`intensity: The optical bottle beam,” Opt. Lett. 25, 1917193,
`2000.)
`The light cage (US. Pat. No. 5,939,716) is loosely, a
`macroscopic cousin of the optical vortex. A light cage forms
`a time-averaged ring of optical traps to surround a particle
`too large or reflective to be trapped with dielectric constants
`lower than the surrounding medium. When the laser beam is
`directed through or reflected from the phase patterning
`optical element, the phase patterning optical element pro-
`duces a plurality of beamlets having an altered phase profile.
`Depending on the number and type of optical traps desired,
`
`

`

`US 7,118,676 B2
`
`5
`the alteration may include diffraction, wavefront shaping,
`phase shifting, steering, diverging and converging. Based
`upon the phase profile chosen, the phase patterning optical
`element may be used to generate optical traps in the form of
`optical traps, optical vortices, optical bottles, optical rota-
`tors, light cages, and combinations of two or more of these
`forms.
`
`Researchers have sought indirect methods for manipulat-
`ing cells, such as tagging the cells with diamond micro-
`particles and then tweezing the diamond particles. Cell
`manipulations have included cell orientation for micro-
`scopic analysis as well as stretching cells. Tissue cells have
`also been arranged with tweezers in vitro in the same spatial
`distribution as in vivo.
`
`In addition to the cells themselves, optical tweezers have
`been used to manipulate cellular organelles, such as vesicles
`transported along microtubules, chromosomes, or globular
`DNA. Objects have also been inserted into cells using
`optical tweezers.
`Accordingly, as an example of new types of sorting using
`laser steered optical traps, a method of cell sorting using a
`technique which isolates valuable cells from other cells,
`tissues, and contaminants is needed. Further, a way of
`achieving a unique contribution of optical trapping to the
`major industrial needs of blood cell sorting and purification
`is required. Still further, there is a need to separate sperm
`cells in the animal husbandry market.
`As a consequence, a need remains for a separation tech-
`nique and apparatus which is continuous, has high through-
`put, provides time saving, and which causes negligible or
`minimal damage to the various components for separation.
`In addition, such techniques should have further applicabil-
`ity to biological or medical areas, such as for separations of
`blood, sperm, other cellular materials, as well as viral, cell
`organelle, globular structures, colloidal suspensions, and
`other biological materials.
`
`SUMMARY OF THE INVENTION
`
`invention
`The exemplary embodiments of the present
`provide for separating components in a mixture, such as
`separating the various blood components of whole blood
`into corresponding fractions, such as a platelet fraction, a red
`blood cell fraction, a white blood cell fraction, and a plasma
`fraction. The various embodiments of the present invention
`provide separation of components on a continuous basis,
`such as within a continuous, closed system, without the
`potential damage and contamination of prior art methods,
`particularly for fractionation of blood components. The
`continuous process of the present invention also provides
`significant time savings and higher throughput for blood
`fractionation. In addition, the various embodiments may also
`include additional means for separating and manipulating
`the components, particularly holographic optical manipula-
`tion and separation. The various embodiments may also be
`applied to separations of other types of cellular and biologi-
`cal materials, such as sperm, viruses, bacteria, cell debris,
`cell organelles, globular structures, colloidal suspensions,
`cellular debris, and other biological materials.
`As used herein, “Particle” refers to a biological or other
`chemical material including, but not limited to, oligonucle-
`otides, polynucleotides, chemical compounds, proteins, lip-
`ids, polysaccharides,
`ligands, cells, antibodies, antigens,
`cellular organelles,
`lipids, blastomeres, aggregations of
`cells, microorganisms, peptides, cDNA, RNA and the like.
`An exemplary method of separating blood into compo-
`nents includes providing a first flow having a plurality of
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6
`blood components; providing a second flow; contacting the
`first flow with the second flow to provide a first separation
`region; and differentially sedimenting a first blood cellular
`component of the plurality of blood components into the
`second flow while concurrently maintaining a second blood
`cellular component of the plurality of blood components in
`the first flow. The second flow having the first blood cellular
`component is then differentially removed from the first flow
`having the second blood cellular component.
`The various sedimentation steps of the present invention
`may be rate zonal or isopycnic. In addition, the first flow and
`the second flow are substantially non-turbulent, and may
`also be substantially laminar.
`In a selected embodiment, the first blood cellular com-
`ponent is a plurality of red blood cells and a plurality of
`white blood cells, and the second blood cellular component
`is a plurality of platelets. For the first blood cellular com-
`ponent, the plurality of white blood cells may be holographi-
`cally separated (through laser steering) from the plurality of
`red blood cells. Other holographic manipulations of the
`present invention include holographically removing a plu-
`rality of contaminants from the first flow, holographically
`separating biological debris from the first flow, and holo-
`graphically separating a plurality of second blood cellular
`components from the first flow.
`Additional separation stages may also be included, with
`the exemplary method providing a third flow; contacting the
`first flow with the third flow to provide a second separation
`region; and differentially sedimenting the second blood
`cellular component of the plurality of blood components to
`sediment into the third flow while concurrently maintaining
`a third blood component of the plurality of blood compo-
`nents in the first flow. In selected embodiments, the second
`blood cellular component
`is a plurality of platelets and
`wherein the third blood component is plasma.
`A plurality of separation stages may also be combined to
`form more complicated structures having multiple separa-
`tion stages, connected in series, connected in parallel, or in
`combinations of both.
`
`A second exemplary method of separating a fluid mixture
`into constituent, non-motile components, in accordance with
`the present invention,
`includes: providing a substantially
`laminar first flow having the fluid mixture, the fluid mixture
`having a plurality of components, the plurality of compo-
`nents having a corresponding plurality of sedimentation
`rates; providing a substantially laminar second flow; con-
`tacting the first flow with the second flow to provide a first
`separation region, the first flow and the second flow having
`a substantially non-turbulent interface within the separation
`region; differentially sedimenting from the first flow a first
`component of the plurality of components into the second
`flow to form an enriched second flow and a depleted first
`flow, while concurrently maintaining a second component of
`the plurality of components in the first flow, the first com-
`ponent having a first sedimentation rate of the plurality of
`sedimentation rates and the second component having a
`second sedimentation rate of the plurality of sedimentation
`rates, wherein the first sedimentation rate is comparatively
`greater than the second sedimentation rate; differentially
`removing the enriched second flow from the depleted first
`flow; and holographically manipulating the second compo-
`nent in the depleted first flow.
`The second exemplary method may also include addi-
`tional separation stages, such as a holographic separation,
`including: providing a third flow; contacting the depleted
`first flow with the third flow to provide a second separation
`region; and holographically trapping the second component
`
`

`

`US 7,118,676 B2
`
`7
`and moving the second component from the depleted first
`flow into the third flow while concurrently maintaining a
`third component of the plurality of components in the
`depleted first flow.
`An exemplary apparatus embodiment of the invention for
`separating a fluid mixture into constituent, non-motile com-
`ponents includes: a first sorting channel having a first inlet
`for a first flow and a second inlet for a second flow; the first
`sorting channel further having a first outlet for the first flow
`and a second outlet for the second flow, the first sorting
`channel further having means to maintain the first flow and
`second flow substantially non-turbulent,
`the first sorting
`channel adapted to allow a first component in the first flow,
`of a plurality of components in the first flow, to sediment into
`the second flow to form an enriched second flow and a
`
`depleted first flow, while concurrently maintaining a second
`component of the plurality of components in the first flow;
`a second, optically transparent sorting channel having a first
`optical inlet coupled to the first outlet for the first flow and
`having a first optical outlet, the second, optically transparent
`sorting channel further having a second optical inlet for a
`third flow and a second optical outlet for the third flow; and
`a holographic optical trap coupled to the second, optically
`transparent sorting channel,
`the holographic optical
`trap
`adapted to generate a holographic optical trap to select and
`move the second component from the first flow into the third
`flow. The various components which are separated, for
`example, may be the various blood fractions or other bio-
`logical materials, such as separations of motile from non-
`motile sperm.
`Another apparatus or system for separating a plurality of
`components in a fluid comprises: an optically transparent
`sorting channel having a first inlet for a first flow and a
`second inlet for a second flow,
`the optically transparent
`sorting channel further having a first outlet for the first flow
`and a second outlet for the second flow; and a holographic
`optical
`trap system coupled to the optically transparent
`sorting channel, the holographic optical trap system adapted
`to generate a holographic optical trap to select and move a
`first component in the first flow, of a plurality of components
`in the first flow, into the second flow to form an enriched
`second flow and a depleted first
`flow, while a second
`component of the plurality of components is concurrently
`maintained in the